Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules of Natural and Synthetic Origin

*Mohammed Ali Al-Mamary and Ziad Moussa*

## **Abstract**

Polyhydroxylated natural phenolic compounds, especially those with low molecular weights, are characterized by their ability to eliminate free radicals as they act as strong antioxidants. The various types of phenolic compounds represent the most important natural antioxidants in addition to some vitamins. The chemical structures of these compounds is discussed in details with their action mechanisms to remove free radicals and prevent many incurable and malignant diseases. In addition to these natural compounds, the last two decades have witnessed increased attempts by many scientific groups and research centers to synthesize chemical compounds in large quantities to mimic these natural compounds, but at a lower cost and greater biological effectiveness. Herein, we conduct a chemical survey of relevant synthetic compounds containing the hydroxyl groups prepared in chemical laboratories and studied for their biological efficacies, such as their effectiveness as antioxidants, as well as the mechanism of elimination of free radicals.

**Keywords:** antioxidants, hydroxyl Groups, natural antioxidants, synthetic antioxidants, small-molecules antioxidants

## **1. Introduction**

## **1.1 Free radicals**

Free radicals are chemical species such as atoms or group of atoms with an odd (unpaired) number of electrons. They are produced due to splitting weak bonds. The biological free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), are usually produced in our bodies. It is known that free radicals are very reactive and may quickly react with other chemical entities (atoms or molecules) by capturing the required electron to gain stability. There are two types of biologically important reactive species. The first type contains oxygen and is known as reactive oxygen species (ROS), while the second type contains nitrogen and is known as reactive nitrogen species (RNS). Both ROS and RNS can be classified into radicals and non-radical species.

#### *1.1.1 Reactive oxygen species (ROS)*

ROS can be classified into two types, radical species and non-radical species. The most important ROS radicals are: superoxide anion radical (O2 .–), hydroxyl radical (. OH), alkoxyl radical (RO. ), lipid peroxide radical (ROO. ), and hydroperoxy radical (HOO. ). While the non-radicals ROS are: hydrogen peroxide (H2O2), singlet oxygen (1 O2), ozone (O3), organic peroxide (ROOH), and hypochlorous acid (HOCl).

#### *1.1.1.1 Superoxide anion radical*

It is important to emphasize that the mitochondria is the main source of the most active biological ROS [1–5] such as superoxide anion radical (O2 .−), hydrogen peroxide (H2O2) and hydroxyl radical (. OH). Thus, the initial reactive oxygen species (O2 .−) is produced due to the reduction of free oxygen by some electrons leaking out from the electron transport chain during the process of oxidative phosphorylation. This particle is relatively stable intermediate and considered as the precursor for most important ROS. The reduction of free oxygen by electrons in mitochondria can be illustrated as follows: O2 + e− → O2 .−. In addition, the superoxide anion radical may be produced in a process of oxygen reduction by enzymatic systems in mammalian cells as follows [6]:

. O e NADPD oxidase or xanthine oxidase or cy 2 2 tochrome P450 O . − − ++ − → (1)

The superoxide anion radical and hydrogen peroxide are formed in vivo, in the brain, and the central nervous system (CNS). It is known that several areas in the brain contain high amount of iron which stimulates free radical reactions.

#### *1.1.1.2 Hydroxyl radical (. OH)*

The superoxide anion and hydrogen peroxide can be converted rapidly to hydroxyl radical (. OH), which is known as the most reactive and destructive radical in biological system. This radical is quickly produced via Fenton [7] and Haber-Wiess reactions as follows [8, 9]:

$$\mathrm{H\_2O\_2 + Fe^{2+} \to \cdot OH + OH^- + Fe^{3+} \text{ (Fenton reaction)}}\tag{2}$$

$$\text{O}\_2\text{}^- + \text{H}\_2\text{O}\_2 \rightarrow \cdot \text{OH} + \text{ }^\cdot \text{OH} + \text{O}\_2 \text{ (Haber} - \text{Weiss reaction)}\tag{3}$$

**255**

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules…*

a molecule, a new free radical is generated. However, the new free radical usually has lower reactivity than the hydroxyl radical (·OH). The ·OH attacks all proteins, DNA, polyunsaturated fatty acids (PUFA) in membranes, and almost any biological molecule it encounters [10]. The hydroxyl radical (·OH) can be obtained by another reaction in neutrophils, where HOCl reacts with superoxide anion radical

. . HOCl O OH O Cl 2 2

systems. It reacts very rapidly and indiscriminately with most biological targets

) and alkoxy radicals (RO.

containing methylene groups (-CH2-) comprise the main targets [13]. This process

Then, peroxyl radical reacts with another polyunsaturated fatty acid (RH) to

Finally, to terminate lipid peroxidation, the following reaction takes place:

relatively stable, but in the presence of Fe and Cu ions, it causes the formation of

α-carbon. As a result, the presence of an electron-withdrawing group increases the reactivity, while the presence of an electron-donating group decreases it. Thus,

tion. These free radicals react with biomolecules by abstracting H-atom [16, 17].

It is clear that lipid peroxidation leads to the formation of alkyl (R.

and ROO.

oxidants. Lipid peroxidation starts with abstraction of H-atom by .

− − + → ++ (4)

*)*

) are moderately strong

OH, or by RO.

), peroxyl

OH) is the strongest oxidant produced in biological

), then oxygen (O2) is added to alkyl radical to generate

). Lipid peroxidation or the oxidative destruction of PUFA

+ →+ ( ) . . RH OH R H O Initiation step <sup>2</sup> (5)

+ →+ ( ) . . RH ROO R ROOH Propagation step (7)

+ → ++ ( ) . . . 1 ROO ROO ROH RO O Termination step <sup>2</sup> (8)

) radicals. Generally, lipid hydroperoxide (ROOH) is

+ + + →+ 3 .2 ROOH Fe RO Fe (9)

+ + +→ + 2 .3 ROOH Fe ROO Fe (10)

must be less reactive because of single electron delocaliza-

is related to the presence of substituents at the

R O ROO . . + →<sup>2</sup> (6)

*) and alkoxyl radical (RO.*

*DOI: http://dx.doi.org/10.5772/intechopen.95616*

[11, 12] as follows:

The hydroxyl radical (.

present at its site of formation.

Peroxy radicals (ROO.

to form alkyl radical (R.

peroxyl radical (ROO.

remove H-atom:

(ROO.

), and alkoxyl (RO.

The reactivity of RO.

aromatic ROO.

alkoxy and peroxy radicals [14, 15].

and RO.

*1.1.1.3 Lipid peroxide radical (ROO.*

can be illustrated in three steps as follows:

The reaction of H2O2 with Fe+2 and Cu+ metal ions which are typically complexed with certain intracellular proteins such as ferritin and ceruloplasmin, respectively [7], occurs due to stress conditions, which means an excess of superoxide anion radical (O2 .−). This phenomenon releases free ions (Fe+2) from ferritin which in turn reacts with H2O2 according to Fenton reaction to produce hydroxyl radical (. OH). This free radical can strongly react with biomolecules such as DNA, proteins, lipids, and carbohydrates and cause severe damage to the cells than any other ROS [10]. The. OH is the most destructive free radical and can more easily penetrate the phospholipid bilayer than O2 .−, which is negatively charged. When ·OH is generated by Fenton reaction, the extent of its formation is largely determined by the availability and location of the metal ion catalyst. One feature of ·OH is that it leads to the generation of another radical, so when it reacts with

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules… DOI: http://dx.doi.org/10.5772/intechopen.95616*

a molecule, a new free radical is generated. However, the new free radical usually has lower reactivity than the hydroxyl radical (·OH). The ·OH attacks all proteins, DNA, polyunsaturated fatty acids (PUFA) in membranes, and almost any biological molecule it encounters [10]. The hydroxyl radical (·OH) can be obtained by another reaction in neutrophils, where HOCl reacts with superoxide anion radical [11, 12] as follows:

$$\rm{HOCl} + \rm{O}\_2\rm{i}^- \rightarrow \rm{^{\cdot}OH} + \rm{O}\_2 + \rm{Cl}^- \tag{4}$$

The hydroxyl radical (. OH) is the strongest oxidant produced in biological systems. It reacts very rapidly and indiscriminately with most biological targets present at its site of formation.

#### *1.1.1.3 Lipid peroxide radical (ROO. ) and alkoxyl radical (RO. )*

Peroxy radicals (ROO. ) and alkoxy radicals (RO. ) are moderately strong oxidants. Lipid peroxidation starts with abstraction of H-atom by . OH, or by RO. to form alkyl radical (R. ), then oxygen (O2) is added to alkyl radical to generate peroxyl radical (ROO. ). Lipid peroxidation or the oxidative destruction of PUFA containing methylene groups (-CH2-) comprise the main targets [13]. This process can be illustrated in three steps as follows:

$$\text{RH} + \text{OH} \rightarrow \text{R}^\cdot + \text{H}\_2\text{O} \text{ (Inition step)}\tag{5}$$

$$\text{R}^\cdot + \text{O}\_2 \rightarrow \text{ROO}^\cdot \tag{6}$$

Then, peroxyl radical reacts with another polyunsaturated fatty acid (RH) to remove H-atom:

$$\text{RH} + \text{ROO}^{\cdot} \rightarrow \text{R}^{\cdot} + \text{ROOH} \text{(Propagation step)} \tag{7}$$

Finally, to terminate lipid peroxidation, the following reaction takes place:

$$\text{ROO}^{\cdot} + \text{ROO}^{\cdot} \rightarrow \text{ROH} + \text{RO}^{\cdot} + ^1\text{O}\_2 \text{ (Termination step)} \tag{8}$$

It is clear that lipid peroxidation leads to the formation of alkyl (R. ), peroxyl (ROO. ), and alkoxyl (RO. ) radicals. Generally, lipid hydroperoxide (ROOH) is relatively stable, but in the presence of Fe and Cu ions, it causes the formation of alkoxy and peroxy radicals [14, 15].

$$\text{ROOH} \,\,+\text{Fe}^{3+} \to \text{RO}^{\cdot} + \text{Fe}^{2+} \tag{9}$$

$$\text{ROOH} + \text{Fe}^{2+} \rightarrow \text{ROO}^{\cdot} + \text{Fe}^{3+} \tag{10}$$

The reactivity of RO. and ROO. is related to the presence of substituents at the α-carbon. As a result, the presence of an electron-withdrawing group increases the reactivity, while the presence of an electron-donating group decreases it. Thus, aromatic ROO. and RO. must be less reactive because of single electron delocalization. These free radicals react with biomolecules by abstracting H-atom [16, 17].

*Antioxidants - Benefits, Sources, Mechanisms of Action*

OH), alkoxyl radical (RO.

ROS can be classified into two types, radical species and non-radical species.

It is important to emphasize that the mitochondria is the main source of the

out from the electron transport chain during the process of oxidative phosphorylation. This particle is relatively stable intermediate and considered as the precursor for most important ROS. The reduction of free oxygen by electrons in mitochondria

radical may be produced in a process of oxygen reduction by enzymatic systems in

. O e NADPD oxidase or xanthine oxidase or cy 2 2 tochrome P450 O . − − ++ − → (1)

brain contain high amount of iron which stimulates free radical reactions.

The superoxide anion and hydrogen peroxide can be converted rapidly to

in biological system. This radical is quickly produced via Fenton [7] and Haber-

plexed with certain intracellular proteins such as ferritin and ceruloplasmin, respectively [7], occurs due to stress conditions, which means an excess of

ferritin which in turn reacts with H2O2 according to Fenton reaction to produce

as DNA, proteins, lipids, and carbohydrates and cause severe damage to the cells than any other ROS [10]. The. OH is the most destructive free radical and can more

When ·OH is generated by Fenton reaction, the extent of its formation is largely determined by the availability and location of the metal ion catalyst. One feature of ·OH is that it leads to the generation of another radical, so when it reacts with

( ) 2 . <sup>3</sup> H O Fe OH OH Fe Fenton reaction 2 2

( ) .

. O H O OH OH O Haber Weiss reaction − − + →+ + − (3)

OH), which is known as the most reactive and destructive radical

<sup>+</sup> − + +→+ + (2)

.−). This phenomenon releases free ions (Fe+2) from

OH). This free radical can strongly react with biomolecules such

metal ions which are typically com-

.−, which is negatively charged.

*OH)*

2 22 2

The reaction of H2O2 with Fe+2 and Cu+

easily penetrate the phospholipid bilayer than O2

The superoxide anion radical and hydrogen peroxide are formed in vivo, in the brain, and the central nervous system (CNS). It is known that several areas in the

.−) is produced due to the reduction of free oxygen by some electrons leaking

), lipid peroxide radical (ROO.

). While the non-radicals ROS are: hydrogen peroxide (H2O2),

OH). Thus, the initial reactive oxygen spe-

.−. In addition, the superoxide anion

O2), ozone (O3), organic peroxide (ROOH), and hypochlorous

.–), hydroxyl

), and hydroper-

.−), hydrogen

The most important ROS radicals are: superoxide anion radical (O2

most active biological ROS [1–5] such as superoxide anion radical (O2

*1.1.1 Reactive oxygen species (ROS)*

*1.1.1.1 Superoxide anion radical*

peroxide (H2O2) and hydroxyl radical (.

can be illustrated as follows: O2 + e− → O2

mammalian cells as follows [6]:

*1.1.1.2 Hydroxyl radical (.*

Wiess reactions as follows [8, 9]:

superoxide anion radical (O2

hydroxyl radical (.

hydroxyl radical (.

radical (.

cies (O2

oxy radical (HOO.

singlet oxygen (1

acid (HOCl).

**254**

#### *1.1.1.4 Hydroperoxyl radical (HOO. )*

Hydroperoxyl radical, also known as perhydroxyl radical (HOO. ), is formed due to the reversible reaction occurring between superoxide anion radical and proton. This reaction takes place in cells as follows:

$$\text{O}\_2\text{"}^\text{"} + \text{H}^\text{"} \leftrightarrow \text{HOO"}\text{"}\tag{11}$$

The pKa of this radical is 4*.*88 [18]. At pH 7.2 in the cytoplasm, a small amount of this radical (1% of O2 •−) exists as HOO. [19]. Perhaps for this reason, many researchers presumed that HOO• has little or no role in initiation of lipid peroxidation [20]. In comparison with other oxidants, HOO• shows high specificity in reaction with PUFA, linoleic (C18:2), and linolenic (C18:3) acids [21].

#### *1.1.2 Non-radicals of ROS*

#### *1.1.2.1 Singlet oxygen*

The singlet oxygen (1 O2) is a potent oxidizing agent, because it can react with different macromolecules such as DNA [22], and is responsible for lipid peroxidation of membrane and other tissues [23]. It is generated in cells, specifically in neutrophils and eosinophils [24, 23]. In addition, this particle can be formed by enzymatic reactions [25–27]. This reactive particle is produced due to the activation of molecular oxygen to two excited states. In the first excited state, oxygen has two electrons with opposite spins in the same ᴨ\* orbital, while in the second excited state oxygen has one electron in each of two degenerate ᴨ\* orbitals. However, singlet oxygen in the first excited state is extremely reactive in comparison with other excited states like the triplet state. Allen [28] suggested the mechanism for the production of singlet oxygen from H2O2 and Cl− in the presence of the myeloperoxidase (MPO) enzyme as follows:

$$\rm H\_2O\_2 + H^+ + Cl^- + MPO \to HOCl + H\_2O \tag{12}$$

$$\rm H\_2O\_2 + HOCl \rightarrow H\_2O + H^\* + Cl^- + ^1O\_2 \tag{13}$$

#### *1.1.2.2 Hydrogen peroxide (H2O2)*

Hydrogen peroxide is generated via an enzymatic reaction where the reactive superoxide anion radical is rapidly converted by an antioxidant enzyme called superoxide dismutase (SOD). The new formed oxygen species H2O2 is less reactive. Thus, hydrogen peroxide is formed as follows by SOD:

$$\text{2O}\_2\text{}^- + \text{2H}^+ + \text{SOD} \rightarrow \text{H}\_2\text{O}\_2 + \text{H}\_2\text{O} \tag{14}$$

**257**

follows:

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules…*

2H O cat O 2H O 2 2 +→+2 2 (15)

OH, NO.

−

). The in vivo

O2

with

, and HOCl. Thus,

2 H O 2GSH GP GSSG 2H O 2 2 + +→ + <sup>x</sup> <sup>2</sup> (16)

+ → →+ <sup>1</sup> H O O HOOOH O H O 2 2 3 22 (17)

− + +→ + . . O H HO O 3 2 (18)

<sup>−</sup> ++ → + (19)

As illustrated, glutathione peroxidase (GPx) removes hydrogen peroxide (H2O2)

destruction effects of hydrogen peroxide (H2O2) result due to the presence of transition metals or enzymes, such as heme-peroxidase. The destruction of H2O2 leads to

reaction of hydrogen peroxide with Cu1+ and Fe2+ leads to the production of. OH. On the other hand, in phagocytic cells, myeloperoxidase uses its substrate H2O2 to generate HOCl. The release of MPO during phagocytosis may play an important

Ozone gas (O3) exists in polluted atmosphere and the inhalation of this gas by human may lead to lung injury and inflammation. In living organisms, ozone is thought to be formed due to oxidation of H2O to H2O2 in the presence of antibodies [33]. Thus, antibodies use H2O as an electron source, facilitating its addition to <sup>1</sup>

Ozone reacts with fatty acids, cholesterol, amino acids and DNA. The lung is the most affected organ due to exposure to ozone. The effect of ozone on tissues occurs via free radical mechanisms [35–37]. The ozone radical anion then reacts with a

to generate dihydrogen trioxide (H2O3), which is converted to ozone [34].

This species (HOCl) is generated in neutrophils by the reaction of Cl <sup>−</sup>

H2O2, which is catalyzed by the enzyme myeloperoxidase [38]. It is illustrated as

. H O Cl MPO HOCl OH 2 2

The hypochlorous acid is considered to be a very reactive oxidizing agent. So, it may affect different biomolecules and may destroy phagocytized pathogens by causing oxidative damage to their biomolecules which include proteins [39], DNA [40], and lipids [41]. On the other hand, the overproduction of HOCl can lead to

Reactive nitrogen species (RNS) can be found in biological systems as free radical species and non-radical species. However, the most common RNS radical

proton to form the hydroxyl radical and oxygen as follows [36].

many health problems such as atherosclerosis and cancer [42, 38].

by oxidizing two glutathione molecules (GSH) to produce oxidized glutathione disulfide (GSSG). It is clear that the three SOD, CAT, and GPx enzymes show

synergistic effect in the scavenging of superoxide anion radical (O2˙

the formation of other more reactive oxidants such as.

role in microbial elimination [32].

*1.1.2.4 Hypochlorous acid (HOCl)*

*1.1.3 Reactive nitrogen species (RNS)*

*1.1.2.3 Ozone (O3)*

*DOI: http://dx.doi.org/10.5772/intechopen.95616*

It is clear that, in the dismutation reaction (an oxidation–reduction process), two superoxide anion radicals are involved. In this reaction, one superoxide anion radical is oxidized to oxygen while the other is reduced to hydrogen peroxide [29]. The latter (H2O2) is relatively stable and membrane permeable so this nonradical species can diffuse inside the cell and can be removed by mitochondrial antioxidant enzymatic systems such as catalase (CAT) and glutathione peroxidase (GPx) [30, 31].

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules… DOI: http://dx.doi.org/10.5772/intechopen.95616*

$$2\text{H}\_2\text{O}\_2 + \text{cat} \rightarrow \text{O}\_2 + 2\text{H}\_2\text{O} \tag{15}$$

$$2\,\mathrm{H}\_{\mathrm{2}}\mathrm{O}\_{\mathrm{2}} + 2\,\mathrm{GSH} + \mathrm{GP}\_{\mathrm{x}} \to \mathrm{GSSG} + 2\,\mathrm{H}\_{\mathrm{2}}\mathrm{O} \tag{16}$$

As illustrated, glutathione peroxidase (GPx) removes hydrogen peroxide (H2O2) by oxidizing two glutathione molecules (GSH) to produce oxidized glutathione disulfide (GSSG). It is clear that the three SOD, CAT, and GPx enzymes show synergistic effect in the scavenging of superoxide anion radical (O2˙ − ). The in vivo destruction effects of hydrogen peroxide (H2O2) result due to the presence of transition metals or enzymes, such as heme-peroxidase. The destruction of H2O2 leads to the formation of other more reactive oxidants such as. OH, NO. , and HOCl. Thus, reaction of hydrogen peroxide with Cu1+ and Fe2+ leads to the production of. OH. On the other hand, in phagocytic cells, myeloperoxidase uses its substrate H2O2 to generate HOCl. The release of MPO during phagocytosis may play an important role in microbial elimination [32].

## *1.1.2.3 Ozone (O3)*

*Antioxidants - Benefits, Sources, Mechanisms of Action*

This reaction takes place in cells as follows:

*)*

Hydroperoxyl radical, also known as perhydroxyl radical (HOO.

•−) exists as HOO.

reaction with PUFA, linoleic (C18:2), and linolenic (C18:3) acids [21].

dation [20]. In comparison with other oxidants, HOO•

to the reversible reaction occurring between superoxide anion radical and proton.

− + O H HOO <sup>2</sup>

The pKa of this radical is 4*.*88 [18]. At pH 7.2 in the cytoplasm, a small amount

researchers presumed that HOO• has little or no role in initiation of lipid peroxi-

different macromolecules such as DNA [22], and is responsible for lipid peroxidation of membrane and other tissues [23]. It is generated in cells, specifically in neutrophils and eosinophils [24, 23]. In addition, this particle can be formed by enzymatic reactions [25–27]. This reactive particle is produced due to the activation of molecular oxygen to two excited states. In the first excited state, oxygen has two electrons with opposite spins in the same ᴨ\* orbital, while in the second excited state oxygen has one electron in each of two degenerate ᴨ\* orbitals. However, singlet oxygen in the first excited state is extremely reactive in comparison with other excited states like the triplet state. Allen [28] suggested the mechanism for the pro-

• • + ↔ (11)

[19]. Perhaps for this reason, many

O2) is a potent oxidizing agent, because it can react with

+ − H O H Cl MPO HOCl H O 2 2 ++ + → + <sup>2</sup> (12)

+ − + → ++ + (13)

− + ++ → + (14)

<sup>1</sup> H O HOCl H O H Cl O 2 2 <sup>2</sup> <sup>2</sup>

Hydrogen peroxide is generated via an enzymatic reaction where the reactive superoxide anion radical is rapidly converted by an antioxidant enzyme called superoxide dismutase (SOD). The new formed oxygen species H2O2 is less reactive.

. 2O 2H SOD H O H O <sup>2</sup> 22 2

It is clear that, in the dismutation reaction (an oxidation–reduction process), two superoxide anion radicals are involved. In this reaction, one superoxide anion radical is oxidized to oxygen while the other is reduced to hydrogen peroxide [29]. The latter (H2O2) is relatively stable and membrane permeable so this nonradical species can diffuse inside the cell and can be removed by mitochondrial antioxidant enzymatic systems such as catalase (CAT) and glutathione peroxidase

shows high specificity in

in the presence of the myeloperoxidase

), is formed due

*1.1.1.4 Hydroperoxyl radical (HOO.*

of this radical (1% of O2

*1.1.2 Non-radicals of ROS*

The singlet oxygen (1

(MPO) enzyme as follows:

*1.1.2.2 Hydrogen peroxide (H2O2)*

duction of singlet oxygen from H2O2 and Cl−

Thus, hydrogen peroxide is formed as follows by SOD:

*1.1.2.1 Singlet oxygen*

**256**

(GPx) [30, 31].

Ozone gas (O3) exists in polluted atmosphere and the inhalation of this gas by human may lead to lung injury and inflammation. In living organisms, ozone is thought to be formed due to oxidation of H2O to H2O2 in the presence of antibodies [33]. Thus, antibodies use H2O as an electron source, facilitating its addition to <sup>1</sup> O2 to generate dihydrogen trioxide (H2O3), which is converted to ozone [34].

$$\rm H\_2O + \rm O\_2 \rightarrow \rm HOOOH \rightarrow \rm O\_3 + \rm H\_2O\_2 \tag{17}$$

Ozone reacts with fatty acids, cholesterol, amino acids and DNA. The lung is the most affected organ due to exposure to ozone. The effect of ozone on tissues occurs via free radical mechanisms [35–37]. The ozone radical anion then reacts with a proton to form the hydroxyl radical and oxygen as follows [36].

$$\text{O}\_{3}{}^{-} + \text{H}^{+} \rightarrow \text{HO}^{-} + \text{O}\_{2} \tag{18}$$

#### *1.1.2.4 Hypochlorous acid (HOCl)*

This species (HOCl) is generated in neutrophils by the reaction of Cl <sup>−</sup> with H2O2, which is catalyzed by the enzyme myeloperoxidase [38]. It is illustrated as follows:

$$\rm H\_2O\_2 + Cl^- + MPO \to HOCl + ^\cdot OH \tag{19}$$

The hypochlorous acid is considered to be a very reactive oxidizing agent. So, it may affect different biomolecules and may destroy phagocytized pathogens by causing oxidative damage to their biomolecules which include proteins [39], DNA [40], and lipids [41]. On the other hand, the overproduction of HOCl can lead to many health problems such as atherosclerosis and cancer [42, 38].

#### *1.1.3 Reactive nitrogen species (RNS)*

Reactive nitrogen species (RNS) can be found in biological systems as free radical species and non-radical species. However, the most common RNS radical is nitric oxide radical (NO. ) and nitrogen dioxide (NO2). On the other side, the important non-radical RNS is peroxynitrite ion (ONOO<sup>−</sup> ). Generations of these reactive species is discussed below.

#### *1.1.3.1 Nitric oxide (NO . )*

Nitric oxide free radical (NO. ) is an endogenous free radical synthesized in the presence of nitric oxide synthase (NOS) that oxidizes L-arginine to L-citrulline [43]. In this reaction, one of the guanidino nitrogen atoms is oxidized to form NO. . This process is shown below:

$$\text{L}-\text{Arginine} + \text{O}\_2 + \text{NADPH} + \text{NOS} \rightarrow \text{L}-\text{Citrullin} + \text{NO}^\cdot + \text{NADP}^+ \quad \text{(20)}$$

The NO. radical can diffuse easily and has the ability to reach many intracellular targets and cause biological damage [44]. The enzyme nitric oxide synthase (NOS) is found in different cells such as vascular endothelial cells, smooth muscle cells, platelets, neuronal cells, macrophages, and neutrophils [45]. In addition, this radical plays an important role in biological tissues such as vasodilation, memory, neuronal response, among others [46–50].

#### *1.1.3.2 Peroxynitrite (OONO<sup>−</sup> ) and Other Reactive Nitrogen Species*

This nitrogenous species is generated due to reaction of superoxide anion radical (O2 −.) with nitrogen oxide radical (NO. ) radical as follows:

$$\mathrm{O}\_{2}{}^{\cdot -} + \mathrm{NO}^{\cdot} \rightarrow \mathrm{ONOO}^{\cdot} \tag{21}$$

It is noted that at physiological pH (7.4), peroxynitrite exists in equilibrium with peroxynitrous acid, ONOOH [51].

$$\mathrm{ONOO}^{-} + \mathrm{H}^{\*} \leftrightarrow \mathrm{ONOOH} \tag{22}$$

Then, peroxynitrous acid (ONOOH) is subjected to homolysis to produce hydroxyl radical (OH• ) and nitrogen dioxide radical (NO2 . ), which may rearrange to form nitrate (NO3 − ).

$$\mathrm{ONOO}^{-} + \mathrm{H}^{+} \leftrightarrow \mathrm{ONOOH} \leftrightarrow \left[\mathrm{HO}^{\cdot} + \mathrm{NO}\_{2}^{\cdot}\right] \rightarrow \mathrm{NO}\_{3}^{-} + \mathrm{H}^{+} \tag{23}$$

The ONOO<sup>−</sup> is a very reactive anion, even more so than the particle (NO. and O2 •−) from which it is formed [52–54]. The peroxynitrite anion can cross biological membranes and interact with most critical biomolecules [55]. Thus, it can cause oxidation of lipids, and proteins via oxidation of methionine and tyrosine residues and can oxidize DNA to generate nitroguanine [56]. Under most biological conditions, ONOO- and ONOOH exist in equilibrium [57]:

$$\mathrm{ONOOO^{-}} + \mathrm{H^{\*}} \leftrightarrow \mathrm{ONOOH} \tag{24}$$

**259**

**Figure 1**.

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules…*

(ONOOCO2) that undergoes fast homolysis to NO2 and [58–60].

As a nucleophile, a central reaction of peroxynitrite in biology is the addition of the anion to carbon dioxide (CO2) to yield a nitrosoperoxocarboxylate adduct

. ONOO CO ONOOCO NO CO <sup>2</sup> 2 23

An antioxidant is any substance that has the ability to prevent, inhibit, or delay the oxidation of other substances. In biological systems, antioxidants play a very important roles in removing free radicals such as ROS and RNS, and consequently reduce oxidative stress. Antioxidant molecules can be classified based on the type of

These are endogenous antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). These enzymes efficiently suppress or prevent the formation of free radicals and other ROS in tissues. Thus, SOD

. 2O 2H SOD H O H O <sup>2</sup> 22 2

The GPx enzyme system detoxifies H2O2 by catalyzing its reduction using glutathione (GSH) as a sacrificial reductant to produce one molecule of oxidized glutathione (GSSG). Thus, the enzymes SOD, CAT, and GPx, work collectively to

In addition, Fe and Cu ions are included to this type of defense, since these ions bind proteins such as transferrin and caeruloplasmin and prevent them from free radical formation. Generally, any chemical compound having two or more of the following functional groups: –OH, –SH, –COOH, –PO3H2, C=O, –NR2, –S– and –O– may have chelating activity [61]. The mechanism of metal ion chelation with some natural phenolics such as protocatechuic acid and anthocyanins is shown in

of phenolic compounds such as flavonoids containing multiple hydroxyl groups (polyhydroxylated). The involvement of these ions in the formation of complexes prevents the Fenton reaction which leads to the formation of hydroxyl radical (.

( ) <sup>n</sup> . n1 H O M HO HO M M Fe orCu 2 2

On the other hand, CAT reduces formed H2O2 to water and oxygen:

→ + . ONOOH NO .OH <sup>2</sup> (25)

− −− +→ → + (26)

− + ++ → + (27)

2 H O cat O 2H O 2 2 +→+2 2 (28)

2 H O 2GSH GP GSSG 2H O 2 2 + +→ + <sup>x</sup> <sup>2</sup> (29)

+− + +→ ++ = (30)

) make complex species with different types

OH)

*DOI: http://dx.doi.org/10.5772/intechopen.95616*

**1.2 Antioxidants**

mechanistic defense they offer:

prevent the effect of O2˙

*1.2.1 Antioxidants suppressing formation of free radicals*

removes superoxide anion radical as follows:

− .

Transition metal ions (Fe+2 and Cu+

which is considered as the most dangerous ROS.

Indeed, protonation weakens the O–O bond in ONOOH and leads to homolytic cleavage to generate hydroxyl radicals (. OH) and nitrogen dioxide (. NO2), two strongly oxidizing/hydroxylating and nitrating species, respectively.

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules… DOI: http://dx.doi.org/10.5772/intechopen.95616*

$$\text{ONOOH} \rightarrow \text{NO}\_2 + \text{OH} \tag{25}$$

As a nucleophile, a central reaction of peroxynitrite in biology is the addition of the anion to carbon dioxide (CO2) to yield a nitrosoperoxocarboxylate adduct (ONOOCO2) that undergoes fast homolysis to NO2 and [58–60].

$$\text{CONOO}^{-} + \text{CO}\_{2} \rightarrow \text{ONOOCO}\_{2}^{-} \rightarrow \text{NO}\_{2} + \text{CO}\_{3}^{-} \tag{26}$$

#### **1.2 Antioxidants**

*Antioxidants - Benefits, Sources, Mechanisms of Action*

*)*

important non-radical RNS is peroxynitrite ion (ONOO<sup>−</sup>

presence of nitric oxide synthase (NOS) that oxidizes L-arginine to L-citrulline [43]. In this reaction, one of the guanidino nitrogen atoms is oxidized to form NO.

lular targets and cause biological damage [44]. The enzyme nitric oxide synthase (NOS) is found in different cells such as vascular endothelial cells, smooth muscle cells, platelets, neuronal cells, macrophages, and neutrophils [45]. In addition, this radical plays an important role in biological tissues such as vasodilation, memory,

<sup>+</sup> − + + + →− + +. L Arginine O NADPH NOS L Citrullin NO NADP <sup>2</sup> (20)

radical can diffuse easily and has the ability to reach many intracel-

*) and Other Reactive Nitrogen Species*

) radical as follows:

+ → (21)

− + ONOO H ONOOH + ↔ (22)

ONOO H ONOOH − + + ↔ (24)

OH) and nitrogen dioxide (.

.

), which may rearrange to

NO2), two

and

This nitrogenous species is generated due to reaction of superoxide anion radical

− − O NO ONOO <sup>2</sup> . .

Then, peroxynitrous acid (ONOOH) is subjected to homolysis to produce

) and nitrogen dioxide radical (NO2

It is noted that at physiological pH (7.4), peroxynitrite exists in equilibrium with

− + − + +↔ ↔ + → + . . ONOO H ONOOH HO NO NO H 2 3 (23)

is a very reactive anion, even more so than the particle (NO.

•−) from which it is formed [52–54]. The peroxynitrite anion can cross biological membranes and interact with most critical biomolecules [55]. Thus, it can cause oxidation of lipids, and proteins via oxidation of methionine and tyrosine residues and can oxidize DNA to generate nitroguanine [56]. Under most biological condi-

Indeed, protonation weakens the O–O bond in ONOOH and leads to homolytic

strongly oxidizing/hydroxylating and nitrating species, respectively.

) and nitrogen dioxide (NO2). On the other side, the

) is an endogenous free radical synthesized in the

). Generations of these

.

is nitric oxide radical (NO.

*1.1.3.1 Nitric oxide (NO .*

This process is shown below:

*1.1.3.2 Peroxynitrite (OONO<sup>−</sup>*

The NO.

(O2

reactive species is discussed below.

Nitric oxide free radical (NO.

neuronal response, among others [46–50].

−.) with nitrogen oxide radical (NO.

peroxynitrous acid, ONOOH [51].

− ).

tions, ONOO- and ONOOH exist in equilibrium [57]:

cleavage to generate hydroxyl radicals (.

hydroxyl radical (OH•

form nitrate (NO3

The ONOO<sup>−</sup>

**258**

O2

An antioxidant is any substance that has the ability to prevent, inhibit, or delay the oxidation of other substances. In biological systems, antioxidants play a very important roles in removing free radicals such as ROS and RNS, and consequently reduce oxidative stress. Antioxidant molecules can be classified based on the type of mechanistic defense they offer:

### *1.2.1 Antioxidants suppressing formation of free radicals*

These are endogenous antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). These enzymes efficiently suppress or prevent the formation of free radicals and other ROS in tissues. Thus, SOD removes superoxide anion radical as follows:

$$\text{2DO}\_2^{\cdot-} + \text{2H}^+ + \text{SOD} \rightarrow \text{H}\_2\text{O}\_2 + \text{H}\_2\text{O} \tag{27}$$

On the other hand, CAT reduces formed H2O2 to water and oxygen:

$$2\,\mathrm{H}\_{2}\mathrm{O}\_{2} + \mathrm{cat} \rightarrow \mathrm{O}\_{2} + 2\mathrm{H}\_{2}\mathrm{O} \tag{28}$$

The GPx enzyme system detoxifies H2O2 by catalyzing its reduction using glutathione (GSH) as a sacrificial reductant to produce one molecule of oxidized glutathione (GSSG). Thus, the enzymes SOD, CAT, and GPx, work collectively to prevent the effect of O2˙ − .

$$2\,\mathrm{H}\_{2}\mathrm{O}\_{2} + 2\mathrm{GSH} + \mathrm{GP}\_{x} \rightarrow \mathrm{GSSG} + 2\mathrm{H}\_{2}\mathrm{O} \tag{29}$$

In addition, Fe and Cu ions are included to this type of defense, since these ions bind proteins such as transferrin and caeruloplasmin and prevent them from free radical formation. Generally, any chemical compound having two or more of the following functional groups: –OH, –SH, –COOH, –PO3H2, C=O, –NR2, –S– and –O– may have chelating activity [61]. The mechanism of metal ion chelation with some natural phenolics such as protocatechuic acid and anthocyanins is shown in **Figure 1**.

Transition metal ions (Fe+2 and Cu+ ) make complex species with different types of phenolic compounds such as flavonoids containing multiple hydroxyl groups (polyhydroxylated). The involvement of these ions in the formation of complexes prevents the Fenton reaction which leads to the formation of hydroxyl radical (. OH) which is considered as the most dangerous ROS.

$$\mathrm{H\_2O\_2 + M^{\*n} \to HO^- + HO^- + M^{n+1}} \left(\mathrm{M = Fe \, or \, Cu}\right) \tag{30}$$

**Figure 1.** *Mechanism of metal ion chelation with some natural phenolics.*

#### *1.2.2 Antioxidants that repair damage resulting from the action of free radicals*

This type of antioxidants are enzymes which are involved in repairing damage due to the effects of free radicals on biomolecules (DNA, proteins, lipids and carbohydrates). These enzymes prevent the accumulation of toxic substances resulting from destruction of biomolecules in body tissues. Examples of this type of enzymatic antioxidants include the DNA repair enzyme systems (polymerases, glycosylases and nucleases), and proteolytic enzymes (proteinases, proteases and peptidases) located in both, cytosol and mitochondria of mammalian cells.

#### *1.2.3 Antioxidants that utilize signals for the formation of free radicals*

This type of antioxidants use the signals, which are required for the formation of free radicals. As a result, the signal generated from the formed free radical causes the formation and transport of the appropriate antioxidant to the appropriate and required site [62].

#### *1.2.4 Antioxidants scavenging free radicals*

This type of scavenging antioxidants can directly neutralize free radicals by two mechanisms, either by donating a hydrogen free radical (H. ) or donating an electron (e− ). These mechanisms can be illustrated as follows:

$$\text{Ar}-\text{OH} + \text{R}^{\cdot} \rightarrow \text{Ar}-\text{O}^{\cdot} + \text{RH} \tag{31}$$

**261**

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules…*

**2. Small antioxidant molecules containing hydroxyl groups**

natural and synthetic phenolic compounds acting as antioxidants.

ity of catechol and hydroquinone is illustrated as shown in **Figure 3**.

Phenolic acids are also known as phenol carboxylic acids (**Figure 4**). There are two important groups of natural phenolic acids which are hydroxybenzoic acids and hydroxycinnamic acids. These are derived from benzoic and cinnamic acid, respectively. The molecular structural features of phenolic acids, such as the numbers and positions of the hydroxyl groups in relation to the carboxyl functional group, esterification, and glycosylation great impacts their antioxidant properties. Many studies [68, 69] have shown that the antioxidant activity of phenolic acids and their esters was enhanced substantially when the number of hydroxyl (-OH) and methoxy (-OCH3) groups increased. On the other hand, the carboxyl group has an electron

*2.1.2 Phenolic acids: hydroxybenzoic and hydroxycinnamic acids*

**2.1 Natural antioxidants containing hydroxyl groups**

*2.1.1 Phenols*

harmless and removed easily from biological systems. Many antioxidants such as ascorbic acid, uric acid, glutathione, vitamin E, and other natural compounds like polyhydroxyphenolic compounds belong to this class. This type of antioxidants are usually small molecules containing hydroxyl groups either of natural or synthetic origin. The importance of these compounds prompted us to review them in details.

There are many studies that have shown the biological effectiveness of phenolic compounds as natural antioxidants. They play very important roles in the prevention of dangerous diseases such as cancers, heart diseases, diabetes and others. There is a need for simple molecules capable of neutralizing free radicals responsible for what is known as oxidative stress, the lead cause of dangerous diseases like cancers, heart disease, diabetes and others. Antioxidants play a critical role in biological systems in getting rid of free radicals and work to prevent the phenomenon of oxidative stress. The most available natural antioxidants exist in plants such as fruits, vegetables, and medicinal plants. Herein, we present an overview of the

Simple phenols are known as compounds containing at least one hydroxyl group attached to an aromatic ring which comprises the basic skeleton. The most important compounds under this class are: phenol, catechol, resorcinol, and phloroglucinol. Generally, phenols are widely distributed in plants and play very important roles in human health because of their ability to neutralize free radicals due to their hydroxyl groups. It is considered that these simple phenols along with other phenolic compounds can inhibit and prevent cancer diseases in humans (**Figure 2**) [63]. The study by Spiegel et al. [64] has shown that the most active of simple natural phenols as antioxidants were those containing more than one hydroxyl group in the *ortho* position of the aromatic ring. This suggests that the most active antioxidant compound is catechol since it contains two hydroxyl groups in the ortho position. This could be attributed to the bond dissociation energy (BDE) of O-H which is typically used to evaluate the activity of an antioxidant to neutralize free radicals [65–67]. Thus, the weaker the O-H BDE, the faster the reaction of antioxidant with the free radical. In other words, the weaker the BDE of O-H in phenols, the easier it will be to transfer an H-radical to deactivate the free radical. The antioxidant activ-

*DOI: http://dx.doi.org/10.5772/intechopen.95616*

In the preceding mechanism, the antioxidant donates a hydrogen free radical (H. ) to scavenge free radicals, and the antioxidant (Ar-OH) itself becomes a free radical, though not as biologically harmful.

$$\text{Ar}-\text{OH} + \text{R}^{\cdot} \rightarrow \text{Ar}-\text{OH}^{\cdot \cdot \cdot} + \text{R}^{\cdot \cdot} \rightarrow \text{Ar}-\text{O}^{\cdot} + \text{H}^{\cdot} \tag{32}$$

The second mechanism involves one-electron transfer where the antioxidant donates an electron to the free radical and becomes itself a radical cation. Generally, the new radicals are more stable and can be easily neutralized and made completely

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules… DOI: http://dx.doi.org/10.5772/intechopen.95616*

harmless and removed easily from biological systems. Many antioxidants such as ascorbic acid, uric acid, glutathione, vitamin E, and other natural compounds like polyhydroxyphenolic compounds belong to this class. This type of antioxidants are usually small molecules containing hydroxyl groups either of natural or synthetic origin. The importance of these compounds prompted us to review them in details.

## **2. Small antioxidant molecules containing hydroxyl groups**

There are many studies that have shown the biological effectiveness of phenolic compounds as natural antioxidants. They play very important roles in the prevention of dangerous diseases such as cancers, heart diseases, diabetes and others. There is a need for simple molecules capable of neutralizing free radicals responsible for what is known as oxidative stress, the lead cause of dangerous diseases like cancers, heart disease, diabetes and others. Antioxidants play a critical role in biological systems in getting rid of free radicals and work to prevent the phenomenon of oxidative stress. The most available natural antioxidants exist in plants such as fruits, vegetables, and medicinal plants. Herein, we present an overview of the natural and synthetic phenolic compounds acting as antioxidants.

#### **2.1 Natural antioxidants containing hydroxyl groups**

#### *2.1.1 Phenols*

*Antioxidants - Benefits, Sources, Mechanisms of Action*

*Mechanism of metal ion chelation with some natural phenolics.*

*1.2.2 Antioxidants that repair damage resulting from the action of free radicals*

*1.2.3 Antioxidants that utilize signals for the formation of free radicals*

mechanisms, either by donating a hydrogen free radical (H.

). These mechanisms can be illustrated as follows:

This type of antioxidants are enzymes which are involved in repairing damage due to the effects of free radicals on biomolecules (DNA, proteins, lipids and carbohydrates). These enzymes prevent the accumulation of toxic substances resulting from destruction of biomolecules in body tissues. Examples of this type of enzymatic antioxidants include the DNA repair enzyme systems (polymerases, glycosylases and nucleases), and proteolytic enzymes (proteinases, proteases and peptidases) located in both, cytosol and mitochondria of mammalian cells.

This type of antioxidants use the signals, which are required for the formation of free radicals. As a result, the signal generated from the formed free radical causes the formation and transport of the appropriate antioxidant to the appropriate and

This type of scavenging antioxidants can directly neutralize free radicals by two

In the preceding mechanism, the antioxidant donates a hydrogen free radical

) to scavenge free radicals, and the antioxidant (Ar-OH) itself becomes a free

The second mechanism involves one-electron transfer where the antioxidant donates an electron to the free radical and becomes itself a radical cation. Generally, the new radicals are more stable and can be easily neutralized and made completely

− +→ −+ . . Ar OH R Ar O RH (31)

+ − <sup>+</sup> − +→ − + → −+ .. . Ar OH R Ar OH R Ar O H (32)

) or donating an elec-

**260**

required site [62].

tron (e−

**Figure 1.**

(H.

*1.2.4 Antioxidants scavenging free radicals*

radical, though not as biologically harmful.

Simple phenols are known as compounds containing at least one hydroxyl group attached to an aromatic ring which comprises the basic skeleton. The most important compounds under this class are: phenol, catechol, resorcinol, and phloroglucinol. Generally, phenols are widely distributed in plants and play very important roles in human health because of their ability to neutralize free radicals due to their hydroxyl groups. It is considered that these simple phenols along with other phenolic compounds can inhibit and prevent cancer diseases in humans (**Figure 2**) [63].

The study by Spiegel et al. [64] has shown that the most active of simple natural phenols as antioxidants were those containing more than one hydroxyl group in the *ortho* position of the aromatic ring. This suggests that the most active antioxidant compound is catechol since it contains two hydroxyl groups in the ortho position. This could be attributed to the bond dissociation energy (BDE) of O-H which is typically used to evaluate the activity of an antioxidant to neutralize free radicals [65–67]. Thus, the weaker the O-H BDE, the faster the reaction of antioxidant with the free radical. In other words, the weaker the BDE of O-H in phenols, the easier it will be to transfer an H-radical to deactivate the free radical. The antioxidant activity of catechol and hydroquinone is illustrated as shown in **Figure 3**.

#### *2.1.2 Phenolic acids: hydroxybenzoic and hydroxycinnamic acids*

Phenolic acids are also known as phenol carboxylic acids (**Figure 4**). There are two important groups of natural phenolic acids which are hydroxybenzoic acids and hydroxycinnamic acids. These are derived from benzoic and cinnamic acid, respectively. The molecular structural features of phenolic acids, such as the numbers and positions of the hydroxyl groups in relation to the carboxyl functional group, esterification, and glycosylation great impacts their antioxidant properties. Many studies [68, 69] have shown that the antioxidant activity of phenolic acids and their esters was enhanced substantially when the number of hydroxyl (-OH) and methoxy (-OCH3) groups increased. On the other hand, the carboxyl group has an electron

**Figure 2.**

*Natural phenolic antioxidants containing hydroxyl groups.*

#### **Figure 3.**

*Mechanism of action of natural phenolic antioxidants by transfer of hydrogen free radical (H•).*

**263**

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules…*

withdrawing effect, making the H-atom less available to be donated. However, the antioxidant activity of hydroxylated cinnamates are greater than that of benzoates [70–72]. The antioxidant activities of different hydroxybenzoic acids such as 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, and 3,4,5-trihydroxybenzoic acid were shown to be dependent on the number and position of attached hydroxyl groups to the aromatic ring [73]. Based on bond dissociation energy of O-H group, the dihydroxybenzoic acid has greater antioxidant activity than monohydroxybenzoic acid. It was observed that the BDE for -OH at 3-position is greater than the BDE of -OH at 4-position, as a result the abstraction of H-atom from the 4-position becomes easier than abstraction from the 3-position. Thus, it can be concluded that in 3,4-dihydroxybenzoic acid, the ability to abstract H-atom from the 4-position is easier than the 3-position. On the other hand, gallic acid (3,4,5-trihydroxybenzoic acid) showed lower antioxidant activity than that of 3,4-dihydroxybenzoic acid. This phenomenon could be attributed to the formation of a weak intramolecular H-bond between the -OH at 4-position and -OH at 5-position [74]. The obtained theoretical BDE of the -OH groups in gallic acid were in the order 4-OH ≤ 5-OH ˂ 3-OH, which indicates that the removal of H-atom is easier from 4-OH and 5-OH. Both of these values in gallic acid become lower than that of 4-hydroxybenzoic acid. Thus, the introduction of two hydroxyl groups at 3-position and 5-position signifi-

Similarly, the antioxidant activities of hydroxycinnamic acids (**Figure 5**) are related to their hydroxyl groups. The study of relationship between antioxidant activities and structures of hydroxycinnamic acids was carried out by Chen and Ho [74]. The BDE value of O-H group is a good indicator to evaluate the antioxidant activity of an antioxidant. Thus, the weaker the O-H bond, the greater the ability of an antioxidant to neutralize free radicals. In addition, phenolic molecules bearing two hydroxyl groups in *o*-position relative to one another showed high antioxidant activities [75–77] as observed with caffeic acid. On the other hand, replacement of one hydroxyl group by methoxy group as in ferulic acid leads to lower antioxidant activity [65–67, 75–80]. Therefore, the BDE value of O-H would be expected to follow the following order: caffeic acid ˂ ferulic acid ˂ p-coumaric acid. As a result, the antioxidant activities of these acids will be in the order: caffeic acid ˃ ferulic acid ˃ p-coumaric acid. However, it is important to remember that the removal of H-atom from caffeic acid could arise from *m*-OH and *p*-OH to form free radicals. Consequently, the resulting free radical due to removal of the H-atom from *p*-OH would be more stable because of resonance where the electron is delocalized over the whole molecule, but in the case of removal of the H-atom from *m*-OH, the unpaired electron cannot be delocalized over the whole molecule since it cannot

The flavonoids consist of a large group of low-molecular weight polyphenolic substances, benzo-γ-pyrone derivatives (**Figure 5**). The basic structural feature of all flavonoids is the flavane (2-phenyl-benzo-γ-pyran) nucleus, a system of two benzene rings (A and B) linked by an oxygen-containing pyran ring (C). According to the degree of oxidation of the C ring, the hydroxylation pattern of the nucleus, and the substituent at carbon 3, flavonoids can be categorized into the following subclasses: flavones, isoflavones, flavanols (catechins), flavonols, flavanones, anthocyanins, and proanthocyanidins. Flavonols differ from flavanones by a hydroxyl group at the C3 position and by a C2–C3 double bond. Anthocyanidins differ from the other flavonoids by possessing a charged oxygen atom in the C ring (**Table 1**).

*DOI: http://dx.doi.org/10.5772/intechopen.95616*

cantly increases the antioxidant activity [73].

cross the propenoic tether [81].

*2.1.3 Flavonoids*

**Figure 4.**

*Benzoic acid and the related hydroxybenzoic acids.*

#### *Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules… DOI: http://dx.doi.org/10.5772/intechopen.95616*

withdrawing effect, making the H-atom less available to be donated. However, the antioxidant activity of hydroxylated cinnamates are greater than that of benzoates [70–72]. The antioxidant activities of different hydroxybenzoic acids such as 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, and 3,4,5-trihydroxybenzoic acid were shown to be dependent on the number and position of attached hydroxyl groups to the aromatic ring [73]. Based on bond dissociation energy of O-H group, the dihydroxybenzoic acid has greater antioxidant activity than monohydroxybenzoic acid. It was observed that the BDE for -OH at 3-position is greater than the BDE of -OH at 4-position, as a result the abstraction of H-atom from the 4-position becomes easier than abstraction from the 3-position. Thus, it can be concluded that in 3,4-dihydroxybenzoic acid, the ability to abstract H-atom from the 4-position is easier than the 3-position. On the other hand, gallic acid (3,4,5-trihydroxybenzoic acid) showed lower antioxidant activity than that of 3,4-dihydroxybenzoic acid. This phenomenon could be attributed to the formation of a weak intramolecular H-bond between the -OH at 4-position and -OH at 5-position [74]. The obtained theoretical BDE of the -OH groups in gallic acid were in the order 4-OH ≤ 5-OH ˂ 3-OH, which indicates that the removal of H-atom is easier from 4-OH and 5-OH. Both of these values in gallic acid become lower than that of 4-hydroxybenzoic acid. Thus, the introduction of two hydroxyl groups at 3-position and 5-position significantly increases the antioxidant activity [73].

Similarly, the antioxidant activities of hydroxycinnamic acids (**Figure 5**) are related to their hydroxyl groups. The study of relationship between antioxidant activities and structures of hydroxycinnamic acids was carried out by Chen and Ho [74]. The BDE value of O-H group is a good indicator to evaluate the antioxidant activity of an antioxidant. Thus, the weaker the O-H bond, the greater the ability of an antioxidant to neutralize free radicals. In addition, phenolic molecules bearing two hydroxyl groups in *o*-position relative to one another showed high antioxidant activities [75–77] as observed with caffeic acid. On the other hand, replacement of one hydroxyl group by methoxy group as in ferulic acid leads to lower antioxidant activity [65–67, 75–80]. Therefore, the BDE value of O-H would be expected to follow the following order: caffeic acid ˂ ferulic acid ˂ p-coumaric acid. As a result, the antioxidant activities of these acids will be in the order: caffeic acid ˃ ferulic acid ˃ p-coumaric acid. However, it is important to remember that the removal of H-atom from caffeic acid could arise from *m*-OH and *p*-OH to form free radicals. Consequently, the resulting free radical due to removal of the H-atom from *p*-OH would be more stable because of resonance where the electron is delocalized over the whole molecule, but in the case of removal of the H-atom from *m*-OH, the unpaired electron cannot be delocalized over the whole molecule since it cannot cross the propenoic tether [81].

### *2.1.3 Flavonoids*

*Antioxidants - Benefits, Sources, Mechanisms of Action*

*Natural phenolic antioxidants containing hydroxyl groups.*

**262**

**Figure 4.**

*Benzoic acid and the related hydroxybenzoic acids.*

**Figure 3.**

**Figure 2.**

*Mechanism of action of natural phenolic antioxidants by transfer of hydrogen free radical (H•).*

The flavonoids consist of a large group of low-molecular weight polyphenolic substances, benzo-γ-pyrone derivatives (**Figure 5**). The basic structural feature of all flavonoids is the flavane (2-phenyl-benzo-γ-pyran) nucleus, a system of two benzene rings (A and B) linked by an oxygen-containing pyran ring (C). According to the degree of oxidation of the C ring, the hydroxylation pattern of the nucleus, and the substituent at carbon 3, flavonoids can be categorized into the following subclasses: flavones, isoflavones, flavanols (catechins), flavonols, flavanones, anthocyanins, and proanthocyanidins. Flavonols differ from flavanones by a hydroxyl group at the C3 position and by a C2–C3 double bond. Anthocyanidins differ from the other flavonoids by possessing a charged oxygen atom in the C ring (**Table 1**).

#### **Table 1.**

*Types of flavonoids.*

**265**

**Figure 6.**

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules…*

Flavonoids are secondary metabolites and mainly distributed in the plant kingdom such as green and black tea, coffee, vegetables, fruits, olive oil, red wine, white wines, and chocolate [82–92]. They are consumed in milligrams per serving of these plant sources. Many researchers have shown that flavonoids possess different biological activities which include vasodilating, anti-allergenic, antiviral, and anti-inflammatory actions [93–95]. However, the antioxidant activity of these compounds attracted the most interest because, in addition to their ability to scavenge free radicals, they also reduce or prevent free radical formation.

The capability of antioxidant activities of flavonoids is mainly related to their chemical structures. Many previous investigations attributed the high antioxidant activities of these compounds to the presence and positions of hydroxyl groups attached to the A and B rings and/or to the C2 = C3 double bond in conjugation with the carbonyl group at 4-position, and the -OH group at 3-position [93, 94, 96]. On the other hand, the replacement of hydrogen atom by a saccharide at 3-position to form a glycosidic bond, the antioxidant activity decreases. The radical scavenging efficiency of flavonoids is related to their phenolic hydroxyl groups which follow the mechanism of H-atom transfer or the single electron transfer followed by sequential electron transfer-proton transfer (SETPT) [97–100]. As in the case of phenolic acids, the antioxidant activity of flavonoids, is based on the value of the dissociation energy of the O-H bond [67, 97, 101]. The study by Quan et al. [102] showed that the dissociation energy of C-H at 3-position in some flavonoids appeared to be lower than that of the dissociation energy of O-H. As a result, the antioxidant activity might be due the donation of H-atom from C-H at 3-position. However, the mechanism of antioxidant activity via H-atom transfer from the -OH group appeared to be the most significant [102]. Generally, flavonoids as antioxidants may act by different mechanisms such as hydrogen atom transfer, single electron transfer, and transition metal chelation. These mechanisms are shown below in **Figures 6**–**9**. **Figure 6** shows the proposed mechanism of radical scavenging activity of cyanidin by Nimse

The flavonoid quercetin is found in many plants and foods and in notable quantities especially in onions, red wine, green tea, apples, berries, and others.

*Proposed mechanism of radical scavenging activity of cyanidin by Nimse and Palb [103].*

*DOI: http://dx.doi.org/10.5772/intechopen.95616*

and Palb [103] following HAT mechanism.

*2.1.3.1 -Hydrogen atom transfer (HAT)*

#### **Figure 5.**

*Cinnamic acid and hydroxycinnamic acids.*

#### *Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules… DOI: http://dx.doi.org/10.5772/intechopen.95616*

Flavonoids are secondary metabolites and mainly distributed in the plant kingdom such as green and black tea, coffee, vegetables, fruits, olive oil, red wine, white wines, and chocolate [82–92]. They are consumed in milligrams per serving of these plant sources. Many researchers have shown that flavonoids possess different biological activities which include vasodilating, anti-allergenic, antiviral, and anti-inflammatory actions [93–95]. However, the antioxidant activity of these compounds attracted the most interest because, in addition to their ability to scavenge free radicals, they also reduce or prevent free radical formation.

The capability of antioxidant activities of flavonoids is mainly related to their chemical structures. Many previous investigations attributed the high antioxidant activities of these compounds to the presence and positions of hydroxyl groups attached to the A and B rings and/or to the C2 = C3 double bond in conjugation with the carbonyl group at 4-position, and the -OH group at 3-position [93, 94, 96]. On the other hand, the replacement of hydrogen atom by a saccharide at 3-position to form a glycosidic bond, the antioxidant activity decreases. The radical scavenging efficiency of flavonoids is related to their phenolic hydroxyl groups which follow the mechanism of H-atom transfer or the single electron transfer followed by sequential electron transfer-proton transfer (SETPT) [97–100]. As in the case of phenolic acids, the antioxidant activity of flavonoids, is based on the value of the dissociation energy of the O-H bond [67, 97, 101]. The study by Quan et al. [102] showed that the dissociation energy of C-H at 3-position in some flavonoids appeared to be lower than that of the dissociation energy of O-H. As a result, the antioxidant activity might be due the donation of H-atom from C-H at 3-position. However, the mechanism of antioxidant activity via H-atom transfer from the -OH group appeared to be the most significant [102]. Generally, flavonoids as antioxidants may act by different mechanisms such as hydrogen atom transfer, single electron transfer, and transition metal chelation. These mechanisms are shown below in **Figures 6**–**9**. **Figure 6** shows the proposed mechanism of radical scavenging activity of cyanidin by Nimse and Palb [103] following HAT mechanism.

## *2.1.3.1 -Hydrogen atom transfer (HAT)*

The flavonoid quercetin is found in many plants and foods and in notable quantities especially in onions, red wine, green tea, apples, berries, and others.

#### **Figure 6.** *Proposed mechanism of radical scavenging activity of cyanidin by Nimse and Palb [103].*

*Antioxidants - Benefits, Sources, Mechanisms of Action*

**Entry Types of flavonoids Examples**

1 **Flavanone**

2 **Flavan-3-ol**

3 **Flavone**

4 **Flavonol**

5 **Anthocyanidin**

6 **Isoflavone**

**264**

**Figure 5.**

**Table 1.**

*Types of flavonoids.*

*Cinnamic acid and hydroxycinnamic acids.*

#### **Figure 7.**

*Proposed mechanism of superoxide anion radical scavenging activity of quercetin by Nimse and Palb [103].*

#### **Figure 8.**

*Proposed mechanism of single electron transfer by Leopoldini et al. [104].*

**Figure 9.**

*Proposed metal–quercetin chelation by Leopoldinia et al. [104].*

The proposed mechanism of superoxide anion radical scavenging activity of quercetin by Nimse and Palb [103] is shown in **Figure 7**.

The proposed mechanism of single electron transfer by Leopoldini et al. [104] for single electron transfer (SET) and transition metal chelation (TMC) are shown in **Figures 8** and **9**.

#### *2.1.3.2 Single electron transfer (SET)*

#### *2.1.3.3 Transition metal chelation (TMC)*

Flavonoids with their multiple hydroxyl groups and the carbonyl group at the 4 position on ring C may offer several available sites for metal chelation. The ability of flavonoids to chelate Fe and Cu ions is related to their indirect antioxidant activities. This property of flavonoids is attributed to their multiple hydroxyl groups and the carbonyl group at 4-position [104].

**267**

**Figure 11.**

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules…*

The Stilbene family includes several compounds [105] among which resveratrol, pterostilbene, and piceatannol are the main representatives, characterized by a *trans*

Stilbene compounds are part of a group of natural polyphenols occurring in plant kingdom such as grapes [106], peanuts [107], and berries [108]. Resveratrol (3, 5, 4′-trihydroxy-trans-stilbene), which is found in grapes, showed different biological activities including antidiabetic, antiobesity, and neuroprotective properties against Alzheimer's disease (AD) [109]. In addition, other stilbenes have shown additional activities as antimicrobials and antioxidants [110]. Different studies have shown that piceatannol (4′, 5′, 3, 5-tetrahydroxystilbene) expresses a wide spectrum of biological activities: anti-inflammatory, anticarcinogenic, antiviral, antioxidative, neuroprotective and estrogenic properties, and antioxidant activities [111–117]. A study by Hussein [118] demonstrated the strong ability of resveratrol to scavenge free radicals using different tests. The mechanism of antioxidant activity of resveratrol was proposed to be as follows

Synthetic antioxidants are usually used as food preservatives to prevent lipid oxidation [119]. The well-known synthetic antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and *t*-butyl-hydroxyquinone

*DOI: http://dx.doi.org/10.5772/intechopen.95616*

double bond connecting the phenolic rings (**Figure 10**).

**2.2 Synthetic antioxidants containing hydroxyl groups**

*2.1.4 Stilbenes*

(**Figure 11**).

**Figure 10.**

*Stilbene and its related polyphenolic derivatives.*

*Proposed mechanism of resveratrol antioxidant activity [118].*

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules… DOI: http://dx.doi.org/10.5772/intechopen.95616*

#### *2.1.4 Stilbenes*

*Antioxidants - Benefits, Sources, Mechanisms of Action*

*Proposed mechanism of single electron transfer by Leopoldini et al. [104].*

quercetin by Nimse and Palb [103] is shown in **Figure 7**.

*Proposed metal–quercetin chelation by Leopoldinia et al. [104].*

The proposed mechanism of superoxide anion radical scavenging activity of

*Proposed mechanism of superoxide anion radical scavenging activity of quercetin by Nimse and Palb [103].*

The proposed mechanism of single electron transfer by Leopoldini et al. [104] for single electron transfer (SET) and transition metal chelation (TMC) are shown

Flavonoids with their multiple hydroxyl groups and the carbonyl group at the 4 position on ring C may offer several available sites for metal chelation. The ability of flavonoids to chelate Fe and Cu ions is related to their indirect antioxidant activities. This property of flavonoids is attributed to their multiple hydroxyl groups and the

**266**

in **Figures 8** and **9**.

*2.1.3.2 Single electron transfer (SET)*

carbonyl group at 4-position [104].

*2.1.3.3 Transition metal chelation (TMC)*

**Figure 8.**

**Figure 7.**

**Figure 9.**

The Stilbene family includes several compounds [105] among which resveratrol, pterostilbene, and piceatannol are the main representatives, characterized by a *trans* double bond connecting the phenolic rings (**Figure 10**).

Stilbene compounds are part of a group of natural polyphenols occurring in plant kingdom such as grapes [106], peanuts [107], and berries [108]. Resveratrol (3, 5, 4′-trihydroxy-trans-stilbene), which is found in grapes, showed different biological activities including antidiabetic, antiobesity, and neuroprotective properties against Alzheimer's disease (AD) [109]. In addition, other stilbenes have shown additional activities as antimicrobials and antioxidants [110]. Different studies have shown that piceatannol (4′, 5′, 3, 5-tetrahydroxystilbene) expresses a wide spectrum of biological activities: anti-inflammatory, anticarcinogenic, antiviral, antioxidative, neuroprotective and estrogenic properties, and antioxidant activities [111–117]. A study by Hussein [118] demonstrated the strong ability of resveratrol to scavenge free radicals using different tests. The mechanism of antioxidant activity of resveratrol was proposed to be as follows (**Figure 11**).

#### **2.2 Synthetic antioxidants containing hydroxyl groups**

Synthetic antioxidants are usually used as food preservatives to prevent lipid oxidation [119]. The well-known synthetic antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and *t*-butyl-hydroxyquinone

**Figure 11.** *Proposed mechanism of resveratrol antioxidant activity [118].*

(TBHQ ). These antioxidants stop the free radical chain of oxidative reactions via the donation of an H-atom radical from the phenolic -OH attached to the aromatic rings (**Figure 12**). The new formed radicals become stable and do not initiate or propagate further oxidation of lipids [120].

The progressively more sterically hindered BHT and the related BHA operated as radical terminators in a similar fashion to TBHQ (**Figure 13**).

Another type of radical quencher is shown in **Figure 14** where the generated phenoxy radical is stabilized by intramolecular hydrogen bond.

The presence of a bulky group introduces steric hindrance in proximity to the radical center, decreasing the rate of further propagation reactions. Another example which illustrates the increase in antioxidant activity is the presence of an extra hydroxyl group at the ortho or para position of the hydroxyl group of phenol. The stability of the phenoxy radical in this case is enhanced by the formation of an intramolecular hydrogen bond. Other studies [121–123] described the synthesis of different compounds like aromatic Schiff bases and aromatic hydrazones containing hydroxyl groups attached to different positions in the aromatic rings. These compounds were designed to mimic as much as possible natural phenolic compounds such as stilbene and chalcones. The number of hydroxyl groups and their locations in the aromatic rings play an important role in the antioxidant activity. The mechanism of antioxidant activity can be illustrated as follows and involves the donation of hydrogen radical (**Figure 15**).

**Figure 12.**

*Antioxidant action of t-butyl-hydroxyquinone as a radical terminator via the donation of a hydrogen radical and subsequent radical delocalization by resonance.*

**269**

antioxidants [129–133].

**4. Conclusion**

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules…*

Oxidative stress is a phenomenon occurring in living systems and is related to the presence of free radicals (oxidants) and antioxidants (reductants). When we talk about free radicals in biological systems, we mean two types: reactive oxygen species (ROS) and reactive nitrogen species (RNS). Imbalance between free radicals and antioxidants (endogenous and exogenous) in biological systems creates a state know as oxidative stress. In this case, the present antioxidants cannot remove the ROS and RNS from living species. As a result, excess free radicals can negatively impact different biological processes, leading to the destruction of cell membrane, blocking pathways of major enzymes, stopping cell division, destruction of DNA, and halting energy production [124–126]. On the other hand, free radicals appear to be necessary for some processes in living organisms since they destroy bacteria by phagocytes (granulocytes and macrophages). In addition, ROS can be beneficial for the maintenance of homeostasis as well as other cellular functions [125, 127]. Again, it is important to remember

OH which are derived from molecular oxygen (O2). High levels of these radicals may cause different biological problems which may lead to cancer, stroke (Reuter et al., 2010) [126], myocardial infarction, diabetes, and other significant conditions [128]. It is not easy to avoid the exposure of free radicals and consequently oxidative stress. However, the increase of consumption of natural antioxidants through diet may help to decrease the production of free radicals. In other words, to prevent oxidative stress, it is highly recommended to consume enough amounts of vegetables, fruits, medicinal plants, and honey to ensure sufficient supplementation of natural

To maintain normal health and avoid incurable diseases such as cardiovascular disease, cancer diseases, diabetes, among other, it is necessary to protect the

. − and hydroxyl radical.

that the primary free radicals are superoxide anion radicals O2

*DOI: http://dx.doi.org/10.5772/intechopen.95616*

*Generation of a phenoxy radical with intramolecular hydrogen bond shown.*

*Proposed mechanism for the action of aromatic hydrazones via H radical donation.*

**3. Oxidative stress**

**Figure 14.**

**Figure 15.**

**Figure 13.**

*Oxidation of BHT and BHA via donation of a hydrogen radical from a phenolic hydroxyl group.*

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules… DOI: http://dx.doi.org/10.5772/intechopen.95616*

#### **Figure 14.**

*Antioxidants - Benefits, Sources, Mechanisms of Action*

propagate further oxidation of lipids [120].

donation of hydrogen radical (**Figure 15**).

*and subsequent radical delocalization by resonance.*

radical terminators in a similar fashion to TBHQ (**Figure 13**).

phenoxy radical is stabilized by intramolecular hydrogen bond.

(TBHQ ). These antioxidants stop the free radical chain of oxidative reactions via the donation of an H-atom radical from the phenolic -OH attached to the aromatic rings (**Figure 12**). The new formed radicals become stable and do not initiate or

The progressively more sterically hindered BHT and the related BHA operated as

Another type of radical quencher is shown in **Figure 14** where the generated

The presence of a bulky group introduces steric hindrance in proximity to the radical center, decreasing the rate of further propagation reactions. Another example which illustrates the increase in antioxidant activity is the presence of an extra hydroxyl group at the ortho or para position of the hydroxyl group of phenol. The stability of the phenoxy radical in this case is enhanced by the formation of an intramolecular hydrogen bond. Other studies [121–123] described the synthesis of different compounds like aromatic Schiff bases and aromatic hydrazones containing hydroxyl groups attached to different positions in the aromatic rings. These compounds were designed to mimic as much as possible natural phenolic compounds such as stilbene and chalcones. The number of hydroxyl groups and their locations in the aromatic rings play an important role in the antioxidant activity. The mechanism of antioxidant activity can be illustrated as follows and involves the

*Oxidation of BHT and BHA via donation of a hydrogen radical from a phenolic hydroxyl group.*

*Antioxidant action of t-butyl-hydroxyquinone as a radical terminator via the donation of a hydrogen radical* 

**268**

**Figure 13.**

**Figure 12.**

*Generation of a phenoxy radical with intramolecular hydrogen bond shown.*

**Figure 15.** *Proposed mechanism for the action of aromatic hydrazones via H radical donation.*

### **3. Oxidative stress**

Oxidative stress is a phenomenon occurring in living systems and is related to the presence of free radicals (oxidants) and antioxidants (reductants). When we talk about free radicals in biological systems, we mean two types: reactive oxygen species (ROS) and reactive nitrogen species (RNS). Imbalance between free radicals and antioxidants (endogenous and exogenous) in biological systems creates a state know as oxidative stress. In this case, the present antioxidants cannot remove the ROS and RNS from living species. As a result, excess free radicals can negatively impact different biological processes, leading to the destruction of cell membrane, blocking pathways of major enzymes, stopping cell division, destruction of DNA, and halting energy production [124–126]. On the other hand, free radicals appear to be necessary for some processes in living organisms since they destroy bacteria by phagocytes (granulocytes and macrophages). In addition, ROS can be beneficial for the maintenance of homeostasis as well as other cellular functions [125, 127]. Again, it is important to remember that the primary free radicals are superoxide anion radicals O2 . − and hydroxyl radical. OH which are derived from molecular oxygen (O2). High levels of these radicals may cause different biological problems which may lead to cancer, stroke (Reuter et al., 2010) [126], myocardial infarction, diabetes, and other significant conditions [128].

It is not easy to avoid the exposure of free radicals and consequently oxidative stress. However, the increase of consumption of natural antioxidants through diet may help to decrease the production of free radicals. In other words, to prevent oxidative stress, it is highly recommended to consume enough amounts of vegetables, fruits, medicinal plants, and honey to ensure sufficient supplementation of natural antioxidants [129–133].

#### **4. Conclusion**

To maintain normal health and avoid incurable diseases such as cardiovascular disease, cancer diseases, diabetes, among other, it is necessary to protect the

existing balance between free radicals and antioxidants in biological systems. Naturally the human body has means of internal defense to neutralize free radicals. These means of defense are represented by a group of biological molecules known as antioxidant enzymes. In addition, there are a number of small molecules such as urea, bilirubin, vitamin E, vitamin A, and others. These simple molecules play a positive role in eliminating free radicals. However, when the internal system fails to get rid of free radicals, a supply of external antioxidants, especially those from natural sources, is needed to remove excess free radicals. There are many antioxidants in nature especially those that contain hydroxyl groups such as phenolic compounds, such as phenolic acids (derivatives of hydroxybenzoic and hydroxy cinnamic acids), flavonoids, stilbenes, chalcones and others. These compounds are found in fruits, vegetables and medicinal herbs. There are some chemically prepared antioxidants in laboratories which use is almost limited to the food and pharmaceutical industries. However, there are many attempts to manufacture antioxidants that mimic those found in nature, especially those containing hydroxyl groups, in the hope of obtaining compounds at the lowest cost, safe to use, and in large quantities.

## **Acknowledgements**

Dr. Ziad Moussa is grateful to the United Arab Emirates University (UAEU) of Al-Ain and to the Research Office for supporting the research developed in his laboratory (Grant no. G00003291/Fund no.31S401/Project #852).

## **Author details**

Mohammed Ali Al-Mamary1 and Ziad Moussa<sup>2</sup> \*

1 Department of Chemistry, Faculty of Applied Science, Taiz University, Republic of Yemen-Taiz

2 Department of Chemistry, College of Science, United Arab Emirates University, Al Ain, United Arab Emirates

\*Address all correspondence to: zmoussa@uaeu.ac.ae

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**271**

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules…*

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[10] Halliwell B. Oxidants and human disease: some new concepts. FASEB J. 1987;1(5):358-64. https://doi. org/10.1096/fasebj.1.5.2824268

[11] Cohen MS, Britigan BE, Hassett DJ, Rosen GM: Do human neutrophils form hydroxyl radical? Evaluation of an unresolved controversy. Free Radic Biol Med. 1988; 5:81. doi: 10.1016/0891-5849(88)90033-0

[12] Kettle AJ, Winterbourn CC: Superoxide-dependent hydroxylation by myeloperoxidase. J Biol Chem. 1994;

[13] Halliwell B, and Gutteridge JM. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J. 1984; 219(1): 1-14. doi:

[14] Repetto MG, Ferrarotti N,

[15] Repetto MG, Semprine J,

Boveris A. The involvement of transition metal ions on iron-dependent lipid peroxidation. Archives of Toxicology. 2010; 84(4):255-62. doi: 10.1007/

Boveris A. Lipid Peroxidation: Chemical Mechanism, Biological Implications and Analytical Determination. D.A. Catala (Ed.) Lipid Peroxidation (2012). InTechopen 2012: 1-30. DOI:

[16] Antunes F, Salvador A, Marinho HS, Alves R, Pinto RE. Lipid peroxidation in mitochondrial inner membranes.1.

Press, Washington, 1999.

rspa.1934.0221

269:17146.

10.1042/bj2190001

s00204-009-0487-y

10.5772/45943

antioxidants, aging and disease, AACC

*DOI: http://dx.doi.org/10.5772/intechopen.95616*

[1] Li X, Fang P, Mai J, Choi ET, Wang H, Yang XF. Targeting mitochondrial reactive oxygen species as novel therapy for inflammatory diseases and cancers. J Hematol Oncol. 2013; 6 (19): 19. DOI:

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e13214. DOI: 10.1111/apha.13214

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*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules… DOI: http://dx.doi.org/10.5772/intechopen.95616*

## **References**

*Antioxidants - Benefits, Sources, Mechanisms of Action*

existing balance between free radicals and antioxidants in biological systems. Naturally the human body has means of internal defense to neutralize free radicals. These means of defense are represented by a group of biological molecules known as antioxidant enzymes. In addition, there are a number of small molecules such as urea, bilirubin, vitamin E, vitamin A, and others. These simple molecules play a positive role in eliminating free radicals. However, when the internal system fails to get rid of free radicals, a supply of external antioxidants, especially those from natural sources, is needed to remove excess free radicals. There are many antioxidants in nature especially those that contain hydroxyl groups such as phenolic compounds, such as phenolic acids (derivatives of hydroxybenzoic and hydroxy cinnamic acids), flavonoids, stilbenes, chalcones and others. These compounds are found in fruits, vegetables and medicinal herbs. There are some chemically prepared antioxidants in laboratories which use is almost limited to the food and pharmaceutical industries. However, there are many attempts to manufacture antioxidants that mimic those found in nature, especially those containing hydroxyl groups, in the hope of obtaining compounds at the lowest cost, safe to use, and in

**270**

**Author details**

large quantities.

**Acknowledgements**

Mohammed Ali Al-Mamary1

Al Ain, United Arab Emirates

\*Address all correspondence to: zmoussa@uaeu.ac.ae

provided the original work is properly cited.

Republic of Yemen-Taiz

and Ziad Moussa<sup>2</sup>

2 Department of Chemistry, College of Science, United Arab Emirates University,

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Dr. Ziad Moussa is grateful to the United Arab Emirates University (UAEU) of Al-Ain and to the Research Office for supporting the research developed in his

laboratory (Grant no. G00003291/Fund no.31S401/Project #852).

1 Department of Chemistry, Faculty of Applied Science, Taiz University,

\*

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[93] Wang TY, Li Q, Bi KS. Bioactive flavonoids in medicinal plants: Structure, activity and biological fate. Asian J. Pharm. Sci. 2018; 13: 12-23. doi: 10.1016/j.ajps.2017.08.004

[94] Heim KE, Tagliaferro AR, Bobilya DJ. Flavonoid antioxidants: chemistry, metabolism and structureactivity relationships. J. Nutr. Biochem. 2002; 13:572-584. doi: 10.1016/ s0955-2863(02)00208-5

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[106] Bavaresco L, Fregoni M, Trevisan M, Mattivi F, Vrhovsek U, Falchetti R . The occurrence of the stilbene piceatannol in grapes.Vitis. 2002; 41: 133-136.

[107] Ku KL, Chang PS, Cheng YC, Lien CY. Production of stilbenoids from the callusof *Arachis hypogaea*: A novel source of the anticancer compound piceatannol. Journal of Agricultural and Food Chemistry. 2005; 5: 33877-3881. https://doi. org/10.1021/jf050242o

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[109] Chang J, Rimando A, M. Pallas M, Camins A, Porquet D, Reeves J, Shukitt-Hale B, Smith MA, Joseph JA, Gemma Casadesus G. Lowdose pterostilbene, but not resveratrol, is a potent neuromodulator in aging and Alzheimer's disease, Neurobiology of Aging. 2012; 33 (9): 2062-2071. DOI: 10.1016/j. neurobiolaging.2011.08.015

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[111] Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CWW, Fong HH, Farnsworth NR, Kinghorn AD, Mehta RG, Moon RC, Pezzuto JM. (1997) Cancer chemo-preventive activity of resveratrol, a natural product derived from grapes. Science. 1997; 275: 218-220. DOI: 10.1126/ science.275.5297.218

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[113] Djoko B, Chiou RYY, Shee JJ, Liu YW. Characterization of immunological activities of peanut stilbenoids, arachidin-1, piceatannol, and resveratrol on lipopolysaccharideinduced inflammation of RAW264.7 macrophages. Journal of Agricultural and Food Chemistry. 2007; 55: 2376- 2383. https://doi.org/10.1021/jf900612n

[114] Bastianetto S, Dumont Y, Han Y, Quirion R. Comparative neuroprotective properties of stilbene and catechin analogs: action via a plasma membrane receptor site? CNS Neuroscience & Therapeutics. 2009; 15: 76-83. DOI: 10.1111/j.1755-5949.2008.00074.x

[115] Murias M, Jager W, Handler N, Erker T, Horvath Z, Szekeres T, Nohl H, Gille L. (2005) Antioxidant, prooxidant and cytotoxic activity of hydroxylated resveratrol analogues: structureactivity relationship. Biochemical Pharmacology. 2005; 69: 903-912. DOI: 10.1016/j.bcp.2004.12.001

[116] Rhayem Y, Therond P, Camont L, Couturier M, Beaudeux JL, Legrand A, Jore D, Gardés-Albert M, Bonnefront-Rousselot D. Chainbreaking activity of resveratrol and piceatannol in a linoleate micellar model. Chemistry and Physics of Lipids. 2008; 155: 48-56. DOI: 10.1016/j. chemphyslip.2008.06.001

[117] Yang Lu, AiHua Wang, Peng Shi, Hui Zhang. A Theoretical Study on the Antioxidant Activity of Piceatannol and Isorhapontigenin Scavenging Nitric Oxide and Nitrogen Dioxide Radicals. PLOS ONE. 2017; 12(1):e0169773.

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[125] Finkel T and N J Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature. 2000; 408(6809):239-47. doi:

Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked?. Free Radic Biol Med. 2010; 49(11):1603-16. doi: 10.1016/j.

Chattopadhyay R, Mitra S, Crowe SE. Oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases. Physiol Rev. 2014; 94(2):329-54. doi: 10.1152/

10.1038/35041687

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freeradbiomed.2010.09.006

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physrev.00040.2012

10.3390/jcm8111815

10.1079/phn2003543

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10.2174/1381612054065783

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[130] Cherubini A, Vigna GB, Zuliani G, Ruggiero C, Senin U, Fellin R. Role of antioxidants in atherosclerosis: epidemiological and clinical update, Current Pharmaceutical Design. 2005; 11 (16): 2017-2032, 2005. doi:

[131] Lotito SB and Frei B. Consumption of flavonoid-rich foods and increased

[128] Padureanu R, Albu CV, Mititelu RR, Bacanoiu MV,

Docea AO, Calina D, Padureanu V, Olaru G, Sandu RE, Malin RD, Buga AM. Oxidative Stress and Inflammation Interdependence in Multiple Sclerosis. J Clin Med. 2019; 8(11):1815. doi:

*DOI: http://dx.doi.org/10.5772/intechopen.95616*

https://doi.org/10.1371/journal.

[118] Hussein MA. A Convenient Mechanism for the Free Radical Scavenging Activity of Resveratrol. International Journal of Phytomedicine.

[119] Shahidi, F, Janitha, P.K,

Wanasundara, P.D. (1992). Phenolic antioxidants. Critical Reviews in Food Science and Nutrition. 1992; 32: 67—103. https://doi. org/10.1080/10408399209527581

[120] Anderson K, Domingos I, Emir B, Saad I, Wellington W, D. Vechiatto D, Helena M. Wilhelm HM, Luiz P. Ramos LP.

[121] Al-Mamary MA, Abdelwahab SI, Ali HM, Salma Ismail, Abdulla MA, Darvish P. Synthesis of Some Schiff Bases Containing Hydroxyl and

Methoxy Groups: thier Antioxidant and Antibacterial Activities. Asian Journal of Chemistry. 2012; 24(10):4335-4339.

[122] Said MA, Hughes DL, Al-Mamary MA , Al-Kaff NS , Al-Harbi WS. Different Chemical Behaviors and Antioxidant Activity of Three Novel Schiff bases Containing Hydroxyl Groups. X-ray structure of CH2{cyclo-C6H10-NH=CH-(2-O-naphth)}2. H2O. Journal of Molecular Structure. 2018; 1165: 305-311. DOI: 10.1016/j.

molstruc.2018.03.089

heliyon.2020.e05019

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The influence of BHA, BHT and TBHQ on the oxidation stability of soybean oil ethyl esters (biodiesel). J. Braz. Chem. Soc. 2007; 18 (2): 416-423. https://doi.org/10.1590/ S0103-50532007000200026

pone.0169773

2011; 3 (4): 459-469.

*Antioxidant Activity: The Presence and Impact of Hydroxyl Groups in Small Molecules… DOI: http://dx.doi.org/10.5772/intechopen.95616*

https://doi.org/10.1371/journal. pone.0169773

*Antioxidants - Benefits, Sources, Mechanisms of Action*

[112] Stivala LA, Savio M, Carafoli F, Perucca P, Bianchi L, Maga G, Forti L, Pagnoni UM, Albini A, Prosperi E, Vannini V. (2001) Specific structural determinants are responsible for the antioxidant activity and the cell cycle effects of resveratrol. Journal of Biological Chemistry. 2001; 276: 22586- 22594. DOI: 10.1074/jbc.M101846200

[113] Djoko B, Chiou RYY,

Shee JJ, Liu YW. Characterization of immunological activities of peanut stilbenoids, arachidin-1, piceatannol, and resveratrol on lipopolysaccharideinduced inflammation of RAW264.7 macrophages. Journal of Agricultural and Food Chemistry. 2007; 55: 2376- 2383. https://doi.org/10.1021/jf900612n

[114] Bastianetto S, Dumont Y, Han Y, Quirion R. Comparative neuroprotective properties of stilbene and catechin analogs: action via a plasma membrane receptor site? CNS Neuroscience & Therapeutics. 2009; 15: 76-83. DOI: 10.1111/j.1755-5949.2008.00074.x

[115] Murias M, Jager W, Handler N, Erker T, Horvath Z, Szekeres T, Nohl H, Gille L. (2005) Antioxidant, prooxidant and cytotoxic activity of hydroxylated resveratrol analogues: structureactivity relationship. Biochemical Pharmacology. 2005; 69: 903-912. DOI:

10.1016/j.bcp.2004.12.001

[116] Rhayem Y, Therond P,

chemphyslip.2008.06.001

Camont L, Couturier M, Beaudeux JL, Legrand A, Jore D, Gardés-Albert M, Bonnefront-Rousselot D. Chainbreaking activity of resveratrol and piceatannol in a linoleate micellar model. Chemistry and Physics of Lipids. 2008; 155: 48-56. DOI: 10.1016/j.

[117] Yang Lu, AiHua Wang, Peng Shi, Hui Zhang. A Theoretical Study on the Antioxidant Activity of Piceatannol and Isorhapontigenin Scavenging Nitric Oxide and Nitrogen Dioxide Radicals. PLOS ONE. 2017; 12(1):e0169773.

whose time has come? And gone?. Clin Biochem. 1997; 30(2):91-113. doi: 10.1016/s0009-9120(96)00155-5

[107] Ku KL, Chang PS, Cheng YC, Lien CY. Production of stilbenoids from the callusof *Arachis hypogaea*: A novel source of the anticancer compound piceatannol. Journal of Agricultural and Food Chemistry. 2005; 5: 33877-3881. https://doi.

[106] Bavaresco L, Fregoni M, Trevisan M, Mattivi F, Vrhovsek U, Falchetti R . The occurrence of the stilbene piceatannol in grapes.Vitis.

2002; 41: 133-136.

org/10.1021/jf050242o

10.1021/jf040095e

plantsci.2009.05.012

science.275.5297.218

[111] Jang M, Cai L, Udeani GO,

[108] Rimando AM, Kalt W,

Resveratrol, pterostilbene, and piceatannol in vaccinium berries. Journal of Agricultural and Food Chemistry. 2004; 52: 4713-4719. doi:

[109] Chang J, Rimando A, M. Pallas M, Camins A, Porquet D, Reeves J, Shukitt-Hale B, Smith MA, Joseph JA, Gemma Casadesus G. Lowdose pterostilbene, but not resveratrol,

is a potent neuromodulator in aging and Alzheimer's disease, Neurobiology of Aging. 2012; 33 (9): 2062-2071. DOI: 10.1016/j. neurobiolaging.2011.08.015

[110] Chong J, Poutaraud A, Hugueney P. Metabolism and roles of stilbenes in plants. Plant Science. 2009; 177 (3): 143-155, 2009. https://doi.org/10.1016/j.

Slowing KV, Thomas CF, Beecher CWW, Fong HH, Farnsworth NR, Kinghorn AD, Mehta RG, Moon RC, Pezzuto JM. (1997) Cancer chemo-preventive activity of resveratrol, a natural product derived from grapes. Science. 1997; 275: 218-220. DOI: 10.1126/

Magee JB, Dewey J, Ballington JR (2004)

**278**

[118] Hussein MA. A Convenient Mechanism for the Free Radical Scavenging Activity of Resveratrol. International Journal of Phytomedicine. 2011; 3 (4): 459-469.

[119] Shahidi, F, Janitha, P.K, Wanasundara, P.D. (1992). Phenolic antioxidants. Critical Reviews in Food Science and Nutrition. 1992; 32: 67—103. https://doi. org/10.1080/10408399209527581

[120] Anderson K, Domingos I, Emir B, Saad I, Wellington W, D. Vechiatto D, Helena M. Wilhelm HM, Luiz P. Ramos LP. The influence of BHA, BHT and TBHQ on the oxidation stability of soybean oil ethyl esters (biodiesel). J. Braz. Chem. Soc. 2007; 18 (2): 416-423. https://doi.org/10.1590/ S0103-50532007000200026

[121] Al-Mamary MA, Abdelwahab SI, Ali HM, Salma Ismail, Abdulla MA, Darvish P. Synthesis of Some Schiff Bases Containing Hydroxyl and Methoxy Groups: thier Antioxidant and Antibacterial Activities. Asian Journal of Chemistry. 2012; 24(10):4335-4339.

[122] Said MA, Hughes DL, Al-Mamary MA , Al-Kaff NS , Al-Harbi WS. Different Chemical Behaviors and Antioxidant Activity of Three Novel Schiff bases Containing Hydroxyl Groups. X-ray structure of CH2{cyclo-C6H10-NH=CH-(2-O-naphth)}2. H2O. Journal of Molecular Structure. 2018; 1165: 305-311. DOI: 10.1016/j. molstruc.2018.03.089

[123] Moussa Z, Al-Mamary MA, Al-Juhani S, Ahmed SA. Preparation and biological assessment of some aromatic hydrazones derived from hydrazides of phenolic acids and aromatic aldehydes. Heliyon. 2020; 6 (9): e05019. https://doi.org/10.1016/j. heliyon.2020.e05019

[124] Kurutas EB. The importance of antioxidants which play the role in cellular response against oxidative/ nitrosative stress: current state. Nutrition Journal. 2016; 15:71. https:// doi.org/10.1186/s12937-016-0186-5

[125] Finkel T and N J Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature. 2000; 408(6809):239-47. doi: 10.1038/35041687

[126] Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked?. Free Radic Biol Med. 2010; 49(11):1603-16. doi: 10.1016/j. freeradbiomed.2010.09.006

[127] Bhattacharyya A, Chattopadhyay R, Mitra S, Crowe SE. Oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases. Physiol Rev. 2014; 94(2):329-54. doi: 10.1152/ physrev.00040.2012

[128] Padureanu R, Albu CV, Mititelu RR, Bacanoiu MV, Docea AO, Calina D, Padureanu V, Olaru G, Sandu RE, Malin RD, Buga AM. Oxidative Stress and Inflammation Interdependence in Multiple Sclerosis. J Clin Med. 2019; 8(11):1815. doi: 10.3390/jcm8111815

[129] Stanner SA, Hughes J, Kelly CNM, Buttriss J. A review of the epidemiological evidence for the 'antioxidant hypothesis'. Public Health Nutrition. 2004; 7 (3): 407-422. doi: 10.1079/phn2003543

[130] Cherubini A, Vigna GB, Zuliani G, Ruggiero C, Senin U, Fellin R. Role of antioxidants in atherosclerosis: epidemiological and clinical update, Current Pharmaceutical Design. 2005; 11 (16): 2017-2032, 2005. doi: 10.2174/1381612054065783

[131] Lotito SB and Frei B. Consumption of flavonoid-rich foods and increased

plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon? Free Radical Biology and Medicine. 2006; 41 (12): 1727-1746, 2006. doi: 10.2174/1381612054065783

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**281**

**Chapter 14**

**Abstract**

**1. Introduction**

Vitamin C and Sepsis

*and Mauricio Homem-de-Mello*

*Adriana Françozo de Melo, Giulia Oliveira Timo* 

supportive treatment that could help septic patients recover.

**Keywords:** vitamin C, sepsis, emergency, Intensive Care Unit

since it scavenges those oxygen-free radicals.

Vitamin C is a supplement used orally by several people globally. It may help in many other conditions, like sepsis, which is caused by an infection that leads to an imbalanced immune response involving pro (e.g., TNF-α, IL-1, IL-2, IL-6) and antiinflammatory (e.g., IL-10, IL-4, IL-7) cytokines. Ascorbic acid is an antioxidant and acts against reactive oxygen species. At the same time, this vitamin influences cellular immune signaling, avoiding exacerbated transcription of pro-inflammatory cytokines. Very high intravenous doses have already shown to be beneficial in septic patients. Some clinical trials are still running to evaluate the real impact of vitamin C in this condition. To the moment, the combination of low-dose corticosteroids, high-dose parenteral ascorbate, and thiamine seems to be the most effective

Vitamin C is a well-known potent antioxidant essential to various biological processes such as carnitine synthesis, neurotransmitter synthesis, hormone synthesis, and tyrosine metabolism. Furthermore, it stabilizes collagen and acts in iron absorption on the intestinal tract. Nevertheless, much is still discussed on its role in common cold, pneumonia, stress-related disorders, metabolic syndrome, and sepsis. Sepsis is a dysregulated host response to an infection that triggers the release of both pro and anti-inflammatory cytokines throughout its course. This "cytokine storm" is responsible for systemic septic symptoms such as vasodilatation, which leads to hypotension and hypoxia. Also, there is the activation of the clotting cascade leading to disseminated intravascular coagulation (DIC). This hemodynamic instability associated with high immune response makes sepsis a deadly disease. Having such nonspecific symptoms, treating sepsis is also problematic. However, the great majority of protocols include antimicrobial and fluid therapy, vasopressors, and inotropic agents. Using anticoagulants and corticosteroids is debated and varies according to symptoms and local protocols. The use of vitamin C in sepsis treatment is also a highly discussed subject, and there are many clinical trials ongoing trying to associate a better outcome with the help of vitamin C in high doses. Considering that sepsis leads to a depletion in vitamin C because of the increased need for reactive oxygen species (ROS) and the elevated cytokine release, it is fair to assume that supplementing it in high doses might help improve septic symptoms

## **Chapter 14** Vitamin C and Sepsis

*Adriana Françozo de Melo, Giulia Oliveira Timo and Mauricio Homem-de-Mello*

## **Abstract**

*Antioxidants - Benefits, Sources, Mechanisms of Action*

plasma antioxidant capacity in humans: cause, consequence, or epiphenomenon? Free Radical Biology and Medicine. 2006; 41 (12): 1727-1746, 2006. doi: 10.2174/1381612054065783

[132] Boeing H, Bechthold A, Bub A, Ellinger S, Haller D,

s00394-012-0380-y

10.1093/eurheartj/ehq465

Kroke A, Leschik-Bonnet E, Müller MJ, Oberritter H, Schulze M, Stehle P, Watzl B. Critical review: vegetables and fruit in the prevention of chronic diseases,European Journal of Nutrition. 2012; 51 (6): 637-663. doi: 10.1007/

[133] Crowe FL, Roddam AW, T. J. Key TJ et al. Fruit and vegetable intake and mortality from ischaemic heart disease: results from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Heart study. European Heart Journal. 2011; 32 (10):1235-1243. doi:

**280**

Vitamin C is a supplement used orally by several people globally. It may help in many other conditions, like sepsis, which is caused by an infection that leads to an imbalanced immune response involving pro (e.g., TNF-α, IL-1, IL-2, IL-6) and antiinflammatory (e.g., IL-10, IL-4, IL-7) cytokines. Ascorbic acid is an antioxidant and acts against reactive oxygen species. At the same time, this vitamin influences cellular immune signaling, avoiding exacerbated transcription of pro-inflammatory cytokines. Very high intravenous doses have already shown to be beneficial in septic patients. Some clinical trials are still running to evaluate the real impact of vitamin C in this condition. To the moment, the combination of low-dose corticosteroids, high-dose parenteral ascorbate, and thiamine seems to be the most effective supportive treatment that could help septic patients recover.

**Keywords:** vitamin C, sepsis, emergency, Intensive Care Unit

## **1. Introduction**

Vitamin C is a well-known potent antioxidant essential to various biological processes such as carnitine synthesis, neurotransmitter synthesis, hormone synthesis, and tyrosine metabolism. Furthermore, it stabilizes collagen and acts in iron absorption on the intestinal tract. Nevertheless, much is still discussed on its role in common cold, pneumonia, stress-related disorders, metabolic syndrome, and sepsis. Sepsis is a dysregulated host response to an infection that triggers the release of both pro and anti-inflammatory cytokines throughout its course. This "cytokine storm" is responsible for systemic septic symptoms such as vasodilatation, which leads to hypotension and hypoxia. Also, there is the activation of the clotting cascade leading to disseminated intravascular coagulation (DIC). This hemodynamic instability associated with high immune response makes sepsis a deadly disease. Having such nonspecific symptoms, treating sepsis is also problematic. However, the great majority of protocols include antimicrobial and fluid therapy, vasopressors, and inotropic agents. Using anticoagulants and corticosteroids is debated and varies according to symptoms and local protocols. The use of vitamin C in sepsis treatment is also a highly discussed subject, and there are many clinical trials ongoing trying to associate a better outcome with the help of vitamin C in high doses. Considering that sepsis leads to a depletion in vitamin C because of the increased need for reactive oxygen species (ROS) and the elevated cytokine release, it is fair to assume that supplementing it in high doses might help improve septic symptoms since it scavenges those oxygen-free radicals.

All things considered, this chapter intends to shed light on the pathophysiology of sepsis, and its current treatments, vitamin C's biochemical and therapeutic properties, and the pieces of evidence from clinical trials that applied vitamin C to treat sepsis and its outcomes.

## **2. Sepsis**

#### **2.1 Pathophysiology, molecular pathways, and mediators of sepsis**

Sepsis is an overreaction to infections, resulting in multiple organ failure and septic shock, frequently leading to death [1]. Sepsis is commonly associated with a super systemic inflammatory condition followed by an immunosuppression phase in which secondary infections typically occur [2]. First sepsis models were developed using animal experiments after confirmation in human volunteers. Bacterial debris can stimulate an acute rise of pro-inflammatory cytokines, implying that this was the cause of the sepsis-associated organ failure. Guided by these results, several different proposed therapies failed to achieve a substantial positive outcome [3, 4]. Following the overwhelming inflammatory process, an intense anti-inflammatory reply leads to a lack of immune response, lymphopenia, and a high propensity for developing infections [5]. This information had provided the basis of the suppositions that the initial hyperinflammatory condition advances to a following immunosuppression [2]. Pro-inflammatory (as IL-6 and TNF) and anti-inflammatory (as IL-10) cytokines are elevated and death-related in septic patients [6].

After innate recognition of conserved microbial patterns, a substantial inflammatory response begins. The recognition, usually through Toll-Like receptors, leads to the activation of cytokines, growth factors and chemokines [7]. After CD4 T cells activation (**Figure 1**), both pro and anti-inflammatory cytokines are released [4]. The reason why CD4 T cells response is pro (Th1) or anti (Th2) inflammatory is supposed to be related to the size of the bacterial inoculum, pathogen type, and

#### **Figure 1.**

*Immune activation following microbial exposition or cellular damage. NF-*κ*B: Nuclear Factor-*κ*B; PAMP: Pathogen-associated molecular pattern; DAMP: Damage-associated molecular pattern; TLR: Toll-like Receptor; IL: Interleukin; TNF-*α*: Tumor Necrosis Factor-*α*; IFN-*γ*: Interferon-*γ*; PD-1: Programmed cell death protein 1.*

**283**

**3. Vitamin C**

**3.1 Redox potential**

*Vitamin C and Sepsis*

failure [7].

**2.2 Therapeutics of sepsis**

the patient's improvement [2, 12].

common, effective, and widespread therapies [13].

hypernatremia) are the clinical guidance in those cases [12].

studies are still needed to evaluate its effectiveness [12, 29].

then discuss what is already known and what still needs investigation.

*DOI: http://dx.doi.org/10.5772/intechopen.95623*

the infected organ [8–11]. A more intense inflammatory response with higher cytokine levels is associated with severe sepsis situations. The spectrum of organic reactions is more intense, as well. General vasodilation, capillary leak, and lessened circulating fluid volume lead to blood clotting and multiple organ malfunction or

The earlier the sepsis or septic shock diagnosis is achieved, the higher are the recovery chances. Broad-spectrum antibiotics (piperacillin/tazobactam, vancomycin, anidulafungin) are initiated while culture and antibiogram results are not available. Clindamycin associated with a β-lactam scheme can be recommended to avoid streptococcal toxic shock. Once the pathogen is identified and its susceptibility to antibiotics is defined, early interruption should be performed, depending on

Supportive therapy is needed in virtually all cases. Fluid resuscitation, inotropic

Other therapies have been studied over the years, but few or controversial results were obtained. Corticosteroids are frequently associated with septic shock therapy. Several randomized controlled trials focused on this issue, and some meta-analysis evaluated the outcomes. Considering all observed flaws of the trials (heterogeneity across studies, doses, the uncertainty of the statistical approach, time of observation, among others), the meta-analysis showed a small benefit using low doses of corticosteroids for a more extended period [14–20]. International Guidelines for Management of Sepsis and Septic Shock recommend corticosteroid therapy only if fluid resuscitation and vasopressor administration are not enough to restore patients' stability. Intravenous hydrocortisone (200 mg/day) and continuous evaluation of blood glucose and sodium (corticosteroids may induce hyperglycemia and

Anticoagulant therapy would be beneficial to oppose the disseminated intravascular coagulation that happens in sepsis conditions. However, antithrombin use did not show evidence to lower the mortality rate and was more prone to bleeding development [12, 21, 22]. On the other hand, thrombomodulin and heparin showed some positive effects on the mortality rate and reduced bleeding risk [23, 24]. Immunoglobulins are still controversial in sepsis. Studies using intravenous immunoglobulins could not show benefits on septic shock or sepsis conditions [25–29]. However, the majority of studies use a small sample size, so more extensive

To the present, numerous researches are trying to achieve a satisfactory result for septic shock or sepsis. However, a long list of failures is along with all the tries. There is a rationale behind Vitamin C usage in these cases, and this chapter will

Vitamin C (VitC, ascorbate) is an antioxidant vitamin. This classification is based on the emission of solvated electrons in aqueous media. In organisms, this process can be enzymatically induced. VitC quickly loses electrons in aqueous media, forming ascorbate free radicals. This is why ascorbate is classified as a very

(e.g., dobutamine), and vasopressor agents (e.g., norepinephrine) are the most

#### *Vitamin C and Sepsis DOI: http://dx.doi.org/10.5772/intechopen.95623*

the infected organ [8–11]. A more intense inflammatory response with higher cytokine levels is associated with severe sepsis situations. The spectrum of organic reactions is more intense, as well. General vasodilation, capillary leak, and lessened circulating fluid volume lead to blood clotting and multiple organ malfunction or failure [7].

## **2.2 Therapeutics of sepsis**

*Antioxidants - Benefits, Sources, Mechanisms of Action*

treat sepsis and its outcomes.

**2. Sepsis**

All things considered, this chapter intends to shed light on the pathophysiology of sepsis, and its current treatments, vitamin C's biochemical and therapeutic properties, and the pieces of evidence from clinical trials that applied vitamin C to

Sepsis is an overreaction to infections, resulting in multiple organ failure and septic shock, frequently leading to death [1]. Sepsis is commonly associated with a super systemic inflammatory condition followed by an immunosuppression phase in which secondary infections typically occur [2]. First sepsis models were developed using animal experiments after confirmation in human volunteers. Bacterial debris can stimulate an acute rise of pro-inflammatory cytokines, implying that this was the cause of the sepsis-associated organ failure. Guided by these results, several different proposed therapies failed to achieve a substantial positive outcome [3, 4]. Following the overwhelming inflammatory process, an intense anti-inflammatory reply leads to a lack of immune response, lymphopenia, and a high propensity for developing infections [5]. This information had provided the basis of the suppositions that the initial hyperinflammatory condition advances to a following immunosuppression [2]. Pro-inflammatory (as IL-6 and TNF) and anti-inflammatory (as

After innate recognition of conserved microbial patterns, a substantial inflammatory response begins. The recognition, usually through Toll-Like receptors, leads to the activation of cytokines, growth factors and chemokines [7]. After CD4 T cells activation (**Figure 1**), both pro and anti-inflammatory cytokines are released [4]. The reason why CD4 T cells response is pro (Th1) or anti (Th2) inflammatory is supposed to be related to the size of the bacterial inoculum, pathogen type, and

*Immune activation following microbial exposition or cellular damage. NF-*κ*B: Nuclear Factor-*κ*B; PAMP: Pathogen-associated molecular pattern; DAMP: Damage-associated molecular pattern; TLR: Toll-like Receptor; IL: Interleukin; TNF-*α*: Tumor Necrosis Factor-*α*; IFN-*γ*: Interferon-*γ*; PD-1: Programmed cell death* 

**2.1 Pathophysiology, molecular pathways, and mediators of sepsis**

IL-10) cytokines are elevated and death-related in septic patients [6].

**282**

**Figure 1.**

*protein 1.*

The earlier the sepsis or septic shock diagnosis is achieved, the higher are the recovery chances. Broad-spectrum antibiotics (piperacillin/tazobactam, vancomycin, anidulafungin) are initiated while culture and antibiogram results are not available. Clindamycin associated with a β-lactam scheme can be recommended to avoid streptococcal toxic shock. Once the pathogen is identified and its susceptibility to antibiotics is defined, early interruption should be performed, depending on the patient's improvement [2, 12].

Supportive therapy is needed in virtually all cases. Fluid resuscitation, inotropic (e.g., dobutamine), and vasopressor agents (e.g., norepinephrine) are the most common, effective, and widespread therapies [13].

Other therapies have been studied over the years, but few or controversial results were obtained. Corticosteroids are frequently associated with septic shock therapy. Several randomized controlled trials focused on this issue, and some meta-analysis evaluated the outcomes. Considering all observed flaws of the trials (heterogeneity across studies, doses, the uncertainty of the statistical approach, time of observation, among others), the meta-analysis showed a small benefit using low doses of corticosteroids for a more extended period [14–20]. International Guidelines for Management of Sepsis and Septic Shock recommend corticosteroid therapy only if fluid resuscitation and vasopressor administration are not enough to restore patients' stability. Intravenous hydrocortisone (200 mg/day) and continuous evaluation of blood glucose and sodium (corticosteroids may induce hyperglycemia and hypernatremia) are the clinical guidance in those cases [12].

Anticoagulant therapy would be beneficial to oppose the disseminated intravascular coagulation that happens in sepsis conditions. However, antithrombin use did not show evidence to lower the mortality rate and was more prone to bleeding development [12, 21, 22]. On the other hand, thrombomodulin and heparin showed some positive effects on the mortality rate and reduced bleeding risk [23, 24].

Immunoglobulins are still controversial in sepsis. Studies using intravenous immunoglobulins could not show benefits on septic shock or sepsis conditions [25–29]. However, the majority of studies use a small sample size, so more extensive studies are still needed to evaluate its effectiveness [12, 29].

To the present, numerous researches are trying to achieve a satisfactory result for septic shock or sepsis. However, a long list of failures is along with all the tries. There is a rationale behind Vitamin C usage in these cases, and this chapter will then discuss what is already known and what still needs investigation.

## **3. Vitamin C**

## **3.1 Redox potential**

Vitamin C (VitC, ascorbate) is an antioxidant vitamin. This classification is based on the emission of solvated electrons in aqueous media. In organisms, this process can be enzymatically induced. VitC quickly loses electrons in aqueous media, forming ascorbate free radicals. This is why ascorbate is classified as a very potent electron donor. Peroxyl radicals may be formed under oxygenated conditions, by the reaction of solvated electrons with oxygen in aerated solutions [30].

Ascorbate can directly scavenge free radicals or restore other redox systems like α-tocopherol or glutathione (**Figure 2**). Simultaneously, it is vital to the activity of several iron and copper-dependent enzymes [31]. VitC and monodehydroascorbate radicals have low electron reduction potentials [32] to reduce more common radicals present in metabolic conditions.

It is well established that a severe dietary undersupply of vitamin C will result in scurvy. But vitamin C has also a role as a cofactor in several enzymes. It takes part in carnitine synthesis, which is essential for the transport of fatty acids into mitochondria for ATP generation [33, 34]; in the biosynthesis of norepinephrine from dopamine [35, 36], peptide hormones [37, 38], and tyrosine metabolism [39, 40]; in collagen synthesis, increasing its stability [41–43]; finally, it acts in nonheme iron absorption on the intestinal tract [44].

However, ascorbic acid's role in preventing or treating common and complex diseases is still uncertain. Even the widely held assumption that ascorbic acid is a significant biological antioxidant and has a prominent role in disease prevention has not been definitively validated [45, 46].

#### **3.2 Evaluation of vitamin C therapeutic efficacy**

Hundreds of studies have been published over the years on vitamin C's effects and its roles in preventing or treating several diseases. There have been many controversial outcomes from this association, whether they are positive or negative. **Table 1** presents the reviews that summarize those outcomes.

Nevertheless, when they are critically analyzed, one can realize they show many inconsistencies regarding the methodology. Recently, Lykkesfeldt interestingly analyzed some more expressive clinical trials and unraveled many of the study's limitations and flaws, as described below, which should be avoided in future researches in this field [59] (**Table 2**).

#### **Figure 2.**

*Antioxidant network. Ascorbate plays a central role in the human antioxidant system. ROS: Reactive Oxygen Species; R·: Free Radical; DHAR: Dehydroascorbate Reductase; GPX: Glutathione Peroxidase; GSSG: Glutathione Disulfide; GR: Glutathione Reductase.*

**285**

**Table 1.**

sepsis is discussed in the rest of this chapter.

Considering the lack of high-quality data to evaluate the efficacy of ascorbate in less severe or more chronic conditions, it is fair to assume that an acute and severe disease such as sepsis is a hard-to-evaluate condition. Vitamin C in sepsis has some particularities such as a diverse route of administration and a peculiar dose– response relationship. The scientific rationale behind this therapeutic proposal to

*Vitamin C and Sepsis*

Cardiovascular protection

Neurologic protection

Common cold (CC) treatment and prevention

Pneumonia treatment and prevention

*DOI: http://dx.doi.org/10.5772/intechopen.95623*

diabetes.

trials.

developing CVD.

**Effect Conclusion of the study Reference**

Vitamin C deficiency is associated with a higher risk of

High vitamin C intake from supplements is associated with an increased risk of CVD mortality in postmenopausal women with

Population with optimal plasma levels of VitC has no benefit from supplementation. People with VitC deficiency have a higher risk of

Antiexcitotoxic, neuromodulator, and neurotrophic effects of ascorbic acid over the CNS are critical for neuroprotective strategies. Clinical trials have demonstrated that ascorbate supplementation produces beneficial results for depression and anxiety. More controlled clinical trials are still necessary to better understand the action mechanisms in stress-related disorders.

needs to be confirmed in animals and human populations. Combination of vitamin C with other antioxidants may be

In adults, the duration of colds was reduced by 8% and in children by 14%. The severity of colds was also reduced by vitamin C administration during the cold process. No reliable effect of vitC was seen on the duration or severity of colds in the therapeutic

Regular supplementation has shown that ascorbate reduces the

Supplementation with vitamin C appears to be able to both prevent

Due to the small number of included studies and the low quality of the existing evidence, data is uncertain about the effect of vitamin C supplementation on preventing and treating pneumonia.

peroxidation) and inflammatory response (IL-6) to a single exercise bout. No effects of vitamin C supplementation were found on creatine kinase (CK), C-reactive protein (CRP), cortisol levels,

would promote the removal of 8-Oxo-2′-deoxyguanosine from DNA by upregulation of repair enzymes due to pro-oxidative properties. Vitamin C showed protection against radiation-induced

No clinically relevant positive effect of vitamin C in cancer patients on the overall survival, clinical status, quality of life, and performance status. The quality of the evaluated studies, however, is low. Small advantages were more associated to intravenous than

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

cardiovascular disease (CVD) mortality.

Metabolic syndrome A direct positive effect of vitamin C alone on Metabolic syndrome

duration and severity of CC.

worthwhile in managing Metabolic syndrome.

and treat respiratory and systemic infections.

Exercise recovery Vitamin C supplementation attenuates the oxidative stress (lipid

muscle soreness, and muscle strength.

Cancer treatment Ascorbate can be positive as a pro-oxidative factor as well. VitC

cell damage.

*Summary of reviews about therapeutic evidence associated with VitC.*

oral administration.

*Antioxidants - Benefits, Sources, Mechanisms of Action*

present in metabolic conditions.

absorption on the intestinal tract [44].

not been definitively validated [45, 46].

researches in this field [59] (**Table 2**).

*Glutathione Disulfide; GR: Glutathione Reductase.*

**3.2 Evaluation of vitamin C therapeutic efficacy**

**Table 1** presents the reviews that summarize those outcomes.

potent electron donor. Peroxyl radicals may be formed under oxygenated conditions, by the reaction of solvated electrons with oxygen in aerated solutions [30]. Ascorbate can directly scavenge free radicals or restore other redox systems like α-tocopherol or glutathione (**Figure 2**). Simultaneously, it is vital to the activity of several iron and copper-dependent enzymes [31]. VitC and monodehydroascorbate radicals have low electron reduction potentials [32] to reduce more common radicals

It is well established that a severe dietary undersupply of vitamin C will result in scurvy. But vitamin C has also a role as a cofactor in several enzymes. It takes part in carnitine synthesis, which is essential for the transport of fatty acids into mitochondria for ATP generation [33, 34]; in the biosynthesis of norepinephrine from dopamine [35, 36], peptide hormones [37, 38], and tyrosine metabolism [39, 40]; in collagen synthesis, increasing its stability [41–43]; finally, it acts in nonheme iron

However, ascorbic acid's role in preventing or treating common and complex diseases is still uncertain. Even the widely held assumption that ascorbic acid is a significant biological antioxidant and has a prominent role in disease prevention has

Hundreds of studies have been published over the years on vitamin C's effects and its roles in preventing or treating several diseases. There have been many controversial outcomes from this association, whether they are positive or negative.

Nevertheless, when they are critically analyzed, one can realize they show many

inconsistencies regarding the methodology. Recently, Lykkesfeldt interestingly analyzed some more expressive clinical trials and unraveled many of the study's limitations and flaws, as described below, which should be avoided in future

*Antioxidant network. Ascorbate plays a central role in the human antioxidant system. ROS: Reactive Oxygen Species; R·: Free Radical; DHAR: Dehydroascorbate Reductase; GPX: Glutathione Peroxidase; GSSG:* 

**284**

**Figure 2.**


#### **Table 1.**

*Summary of reviews about therapeutic evidence associated with VitC.*

Considering the lack of high-quality data to evaluate the efficacy of ascorbate in less severe or more chronic conditions, it is fair to assume that an acute and severe disease such as sepsis is a hard-to-evaluate condition. Vitamin C in sepsis has some particularities such as a diverse route of administration and a peculiar dose– response relationship. The scientific rationale behind this therapeutic proposal to sepsis is discussed in the rest of this chapter.


#### **Table 2.**

*Concerns about clinical trials performed to evaluate ascorbate efficacy on diseases, according to Lykkesfeldt [59] (modified by the authors).*

#### **3.3 Dose-response in supplementation versus high dose**

Sepsis is a condition associated with VitC deficiency because of its high consumption due to enhanced reactive oxygen species (ROS) production. Ascorbate supplementation is thus necessary, and the best results are thought to be achieved through high intravenous doses [60, 61]. The absorption, distribution, metabolism, and excretion (ADME) of vitC in humans are distinct from other small molecules.

#### *3.3.1 Pharmacokinetics of vitamin C*

#### *3.3.1.1 Oral and intravenous administration*

In biological systems, VitC exists in two main chemical forms (**Figure 3**), the reduced and predominant ascorbate anion and the oxidized dehydroascorbate (DHA). Due to the DHA reductase activity (**Figure 2**), virtually every cell can recycle DHA. Therefore, total ascorbate is considered the sum of VitC and DHA. The membrane transport can be performed by three possible mechanisms: passive or facilitated diffusion and active transport, the most relevant of the three [62].

Orally, VitC is absorbed by the saturable mucosal sodium-dependent Vitamin C transporter 1 (SVCT1) [60]. Ascorbate oral absorption is limited, achieving a plateau after 200-300 mg (**Figure 4**). SVCT transporters are widely distributed throughout organs and are responsible for most VitC passage across membranes, even against a concentration gradient [63–65].

**287**

**Figure 4.**

concentrations [62].

*transporter 1 and 2, DHA: Dehydroascorbate.*

*Vitamin C and Sepsis*

**Figure 3.**

*Dehydroascorbate.*

*DOI: http://dx.doi.org/10.5772/intechopen.95623*

Some SVCT polymorphisms have already been identified, which may be associated with a critical pharmacokinetic variation. Some of those SVCT alleles are supposed to

lead to permanent ascorbate deficiency (plasma concentrations <23 μM) [62]. Humans do not synthesize vitC, so the oral ingestion of food is the primary source of vitC. There is enough ascorbate for healthy individuals in the average diets that contain food rich in it. However, pathologic conditions associated with low ascorbate levels may need supplementation to achieve the minimum plasma

*VitC oral absorption and distribution. ASC: Ascorbate, SVCT1 and 2: sodium-dependent Vitamin C* 

*VitC chemical forms in biological systems – Redox cycle. MDHA: Monodehydroascorbate radical. DHA:* 

Intravenously, plasma levels of ascorbic acid continuously increase, producing plasma levels up to 70-fold higher than the maximum oral doses, achieving the millimolar concentration [66]. A linear relationship between dose and Cmax

#### **Figure 3.**

*Antioxidants - Benefits, Sources, Mechanisms of Action*

Measurement of Vit C intake vs. status

Healthy Enrolee Effect

*[59] (modified by the authors).*

**Table 2.**

**Concern Trouble Resolution**

are significant challenges in correlating vitamin C status to disease risk. This is due to the lability of ascorbate. Ascorbate is quickly oxidized ex vivo, and the resulting oxidation products are quickly

intervention periods to accumulate sufficient disease endpoints. This perspective is needed to observe an accumulated preventive potential of a lifelong VitC intake of both placebo and intervention groups up to the trial. This issue has been completely neglected in the available

A tendency towards recruiting health-conscious, self-motivated subjects eating a healthy diet already rich in micronutrients, with higher exercise frequency and lower disease rate than the Retrieving blood samples from fasted individuals.

Process samples in a cold (4 °C) environment. Avoid hemolysis. Choose HPLC with electrochemical

Perform multicentric randomized follow-up clinical trials.

Work with more significant samples, baseline adjustment among groups, previous genetic evaluation

detection.

Focus on VitC intake rather than its status. Even large cohort studies used estimates of micronutrient intakes from self-reported questionnaires or diaries. Lack of precision due to recall error, loss of vitamin from storage and preparation, diet change over time, and possible

different polymorphisms.

Lack of stability Fasted blood samples can be obtained, but there

degraded or metabolized

Study Design Random Controlled Trials may require very long

literature.

**3.3 Dose-response in supplementation versus high dose**

background population.

*3.3.1 Pharmacokinetics of vitamin C*

*3.3.1.1 Oral and intravenous administration*

even against a concentration gradient [63–65].

Sepsis is a condition associated with VitC deficiency because of its high consumption due to enhanced reactive oxygen species (ROS) production. Ascorbate supplementation is thus necessary, and the best results are thought to be achieved through high intravenous doses [60, 61]. The absorption, distribution, metabolism, and excretion (ADME) of vitC in humans are distinct from other small molecules.

*Concerns about clinical trials performed to evaluate ascorbate efficacy on diseases, according to Lykkesfeldt* 

In biological systems, VitC exists in two main chemical forms (**Figure 3**), the reduced and predominant ascorbate anion and the oxidized dehydroascorbate (DHA). Due to the DHA reductase activity (**Figure 2**), virtually every cell can recycle DHA. Therefore, total ascorbate is considered the sum of VitC and DHA. The membrane transport can be performed by three possible mechanisms: passive or facilitated diffusion and active transport, the most relevant of the three [62]. Orally, VitC is absorbed by the saturable mucosal sodium-dependent Vitamin C transporter 1 (SVCT1) [60]. Ascorbate oral absorption is limited, achieving a plateau after 200-300 mg (**Figure 4**). SVCT transporters are widely distributed throughout organs and are responsible for most VitC passage across membranes,

**286**

*VitC chemical forms in biological systems – Redox cycle. MDHA: Monodehydroascorbate radical. DHA: Dehydroascorbate.*

#### **Figure 4.**

*VitC oral absorption and distribution. ASC: Ascorbate, SVCT1 and 2: sodium-dependent Vitamin C transporter 1 and 2, DHA: Dehydroascorbate.*

Some SVCT polymorphisms have already been identified, which may be associated with a critical pharmacokinetic variation. Some of those SVCT alleles are supposed to lead to permanent ascorbate deficiency (plasma concentrations <23 μM) [62].

Humans do not synthesize vitC, so the oral ingestion of food is the primary source of vitC. There is enough ascorbate for healthy individuals in the average diets that contain food rich in it. However, pathologic conditions associated with low ascorbate levels may need supplementation to achieve the minimum plasma concentrations [62].

Intravenously, plasma levels of ascorbic acid continuously increase, producing plasma levels up to 70-fold higher than the maximum oral doses, achieving the millimolar concentration [66]. A linear relationship between dose and Cmax (maximum concentration plasma level) was observed in doses up to about 70 g/m2 , leading to nearly 50 mM plasma levels. Apparently, the pharmacokinetic of vitC changes from zero to first-order after high-dose intravenous administration [62].

#### *3.3.1.2 Distribution*

Intracellular levels of ascorbate vary between 0.5 to 10 mM, which is much higher than the 50–80 μM usually found in healthy individuals' plasma. Simultaneously, human erythrocytes can turn DHA to VitC and keep an intracellular ascorbate level similar to that of plasma. This recycling ability of the red blood cells is essential as an antioxidant reserve [62].

As it happens at the absorption phase, distribution depends on active transport as well. Ascorbate exits the bloodstream and crosses the organ's cell membranes through SVCT2 carriers (**Figure 4**). Yet, even in the steady-state achieved concentration after regular ascorbate dosage, different tissues present highly diverse concentrations. This may happen because of distinct levels of SVCT2 expression [62].

#### *3.3.1.3 Metabolism*

Metabolism of VitC is essentially associated with the redox cycle involved with the antioxidant function (**Figure 3**). As previously cited, ascorbate is an electron donor, and it can reduce free radicals (**Figure 2**) by oxidizing itself to the stable radical monodehydroascorbate (MDHA). This radical can react to another equal, providing an ascorbate molecule and the DHA metabolite that can be reduced, as mentioned before, to ascorbate through DHA reductase activity [62].

#### *3.3.1.4 Excretion*

VitC is a highly water-soluble (about 330 g/L) small molecule (about 8 Å large, 176.1 g/mol), it has a pKa of 4.2, and is almost insoluble in hydrophobic organic solvents [67]. Like other molecules with similar solubility, ascorbate is filtered through the glomerulus and is concentrated after water resorption. At this time, local pH drops to five, leading to an increase of the non-ionized ascorbic acid fraction. However, passive reabsorption does not occur because of the highly hydrophilic characteristic of the molecule. In the proximal tubules, the reuptake of ascorbate is controlled by the saturable active transporter SVCT1. In individuals with saturated plasma levels, supplemental vitC is excreted quantitatively [68].

After high-dose intravenous administration, vitC is rapidly eliminated through glomerular filtration. Reuptake is non-significant under this condition, and the half-life is constant, about two hours (after discontinuation of infusion), and first-order kinetic applies to this case. In about 16 h, physiological levels are back to normal [62, 69–71].

#### *3.3.1.5 Pharmacokinetics in critically ill patients*

Critically ill patients, such as those in septic shock conditions, have an increased ascorbate turnover, needing a dose many folds higher (oral or intravenous) than would be expected to saturate a healthy person. Systemic inflammation and severe pressure due to oxidative stress increase VitC consumption [61, 72, 73]. Mathematically predicted plasmatic ascorbate values are much higher than what is achieved in critically ill patients, suggesting that pharmacokinetics in this group of patients is changed.

**289**

**Figure 5.**

*HMGB1 - High Mobility Group Box 1.*

*Vitamin C and Sepsis*

disorder [74].

fatty acids [77].

calcium profusion [79].

(neutrophil-extracellular-trap) formation [61, 80].

*DOI: http://dx.doi.org/10.5772/intechopen.95623*

In sepsis conditions, the mitochondrial impairment may be a relevant route to cell death and organ collapse. Anomalies in the citric acid cycle and reduction of the fatty acid's beta-oxidation seem to be a characteristic aspect of this mitochondrial

While ascorbate is transported across membranes through SVCT's proteins, DHA can be transported by glucose transporters GLUT1, 3, and 4 [75]. DHA is transported into the mitochondria by GLUT1 and converted to ascorbate (**Figure 5**), where it works as an antioxidant, avoiding damage to the organelle [76]. Ascorbate can also act as a cofactor to the mitochondrial Trimethyllysine dioxygenase (TMLD) enzyme, responsible for the first L-carnitine synthesis, needed for the β-oxidation of

The heart is a vital organ that may be affected by sepsis. Proteolysis, mitochondrial injury, and calcium homeostasis dysfunction are expected consequences of the oxidative myocyte damage. Experimental models show that supplementation of the redox scavengers can diminish cardiac disorder [78]. Ascorbate can decrease apoptosis and improve mitochondrial integrity in myocytes through the blockage of the mitochondrial permeability transition pore opening, limiting

VitC can achieve high concentrations in leukocytes, especially lymphocytes and macrophages. In other defense cells, VitC acts to improve chemotaxis, stimulating interferon expression, and promoting lymphocyte proliferation. In neutrophils, ascorbate increases phagocytic capacity and oxidative burst, and decreases NET

VitC can mediate immune modulation. VitC inhibits nuclear Factor Kappa-B (NF-κB) activation. The mechanism that underlies this suppression involves the blockade of the TNFα-induced activation of NIK (NFκB-inducing kinase) and

*Vitamin C multiple anti-inflammatory mechanisms. DHA: Dehydroascorbate, ASC: Ascorbate, GLUT: Glucose Transporter, (H)TML(D): (Hydroxy) Trimethyllysine (Dioxygenase), CPT1: Carnitine Palmitoyltransferase 1, LCFA: Long-Chain Fatty Acids; LACS: Long-Chain Acyl-CoA Synthetase, TNFR: Tumor Necrosis Factor Receptor, NF-*κ*B: Nuclear Factor Kappa-light-chain-enhancer of activated B cells, NIK: NF-*κ*B-Inducing Kinase, NEMO: NF-*κ*B Essential Modulator, IKK*α *and β: I*κ*B*α *and β kinases, PI3K: Phosphoinositide 3-Kinase, Nrf2: Nuclear Factor Erythroid 2-Related Factor 2, HO-1: Heme Oxygenase 1,* 

**3.4 Vitamin C in septic conditions**

## **3.4 Vitamin C in septic conditions**

*Antioxidants - Benefits, Sources, Mechanisms of Action*

cells is essential as an antioxidant reserve [62].

*3.3.1.2 Distribution*

expression [62].

*3.3.1.3 Metabolism*

*3.3.1.4 Excretion*

normal [62, 69–71].

patients is changed.

(maximum concentration plasma level) was observed in doses up to about 70 g/m2

As it happens at the absorption phase, distribution depends on active transport as well. Ascorbate exits the bloodstream and crosses the organ's cell membranes through SVCT2 carriers (**Figure 4**). Yet, even in the steady-state achieved concentration after regular ascorbate dosage, different tissues present highly diverse concentrations. This may happen because of distinct levels of SVCT2

Metabolism of VitC is essentially associated with the redox cycle involved with the antioxidant function (**Figure 3**). As previously cited, ascorbate is an electron donor, and it can reduce free radicals (**Figure 2**) by oxidizing itself to the stable radical monodehydroascorbate (MDHA). This radical can react to another equal, providing an ascorbate molecule and the DHA metabolite that can be reduced, as

VitC is a highly water-soluble (about 330 g/L) small molecule (about 8 Å large, 176.1 g/mol), it has a pKa of 4.2, and is almost insoluble in hydrophobic organic solvents [67]. Like other molecules with similar solubility, ascorbate is filtered through the glomerulus and is concentrated after water resorption. At this time, local pH drops to five, leading to an increase of the non-ionized ascorbic acid fraction. However, passive reabsorption does not occur because of the highly hydrophilic characteristic of the molecule. In the proximal tubules, the reuptake of ascorbate is controlled by the saturable active transporter SVCT1. In individuals with saturated

After high-dose intravenous administration, vitC is rapidly eliminated through glomerular filtration. Reuptake is non-significant under this condition, and the half-life is constant, about two hours (after discontinuation of infusion), and first-order kinetic applies to this case. In about 16 h, physiological levels are back to

Critically ill patients, such as those in septic shock conditions, have an increased ascorbate turnover, needing a dose many folds higher (oral or intravenous) than would be expected to saturate a healthy person. Systemic inflammation and severe pressure due to oxidative stress increase VitC consumption [61, 72, 73]. Mathematically predicted plasmatic ascorbate values are much higher than what is achieved in critically ill patients, suggesting that pharmacokinetics in this group of

mentioned before, to ascorbate through DHA reductase activity [62].

plasma levels, supplemental vitC is excreted quantitatively [68].

*3.3.1.5 Pharmacokinetics in critically ill patients*

leading to nearly 50 mM plasma levels. Apparently, the pharmacokinetic of vitC changes from zero to first-order after high-dose intravenous administration [62].

Intracellular levels of ascorbate vary between 0.5 to 10 mM, which is much higher than the 50–80 μM usually found in healthy individuals' plasma. Simultaneously, human erythrocytes can turn DHA to VitC and keep an intracellular ascorbate level similar to that of plasma. This recycling ability of the red blood

,

**288**

In sepsis conditions, the mitochondrial impairment may be a relevant route to cell death and organ collapse. Anomalies in the citric acid cycle and reduction of the fatty acid's beta-oxidation seem to be a characteristic aspect of this mitochondrial disorder [74].

While ascorbate is transported across membranes through SVCT's proteins, DHA can be transported by glucose transporters GLUT1, 3, and 4 [75]. DHA is transported into the mitochondria by GLUT1 and converted to ascorbate (**Figure 5**), where it works as an antioxidant, avoiding damage to the organelle [76]. Ascorbate can also act as a cofactor to the mitochondrial Trimethyllysine dioxygenase (TMLD) enzyme, responsible for the first L-carnitine synthesis, needed for the β-oxidation of fatty acids [77].

The heart is a vital organ that may be affected by sepsis. Proteolysis, mitochondrial injury, and calcium homeostasis dysfunction are expected consequences of the oxidative myocyte damage. Experimental models show that supplementation of the redox scavengers can diminish cardiac disorder [78]. Ascorbate can decrease apoptosis and improve mitochondrial integrity in myocytes through the blockage of the mitochondrial permeability transition pore opening, limiting calcium profusion [79].

VitC can achieve high concentrations in leukocytes, especially lymphocytes and macrophages. In other defense cells, VitC acts to improve chemotaxis, stimulating interferon expression, and promoting lymphocyte proliferation. In neutrophils, ascorbate increases phagocytic capacity and oxidative burst, and decreases NET (neutrophil-extracellular-trap) formation [61, 80].

VitC can mediate immune modulation. VitC inhibits nuclear Factor Kappa-B (NF-κB) activation. The mechanism that underlies this suppression involves the blockade of the TNFα-induced activation of NIK (NFκB-inducing kinase) and

#### **Figure 5.**

*Vitamin C multiple anti-inflammatory mechanisms. DHA: Dehydroascorbate, ASC: Ascorbate, GLUT: Glucose Transporter, (H)TML(D): (Hydroxy) Trimethyllysine (Dioxygenase), CPT1: Carnitine Palmitoyltransferase 1, LCFA: Long-Chain Fatty Acids; LACS: Long-Chain Acyl-CoA Synthetase, TNFR: Tumor Necrosis Factor Receptor, NF-*κ*B: Nuclear Factor Kappa-light-chain-enhancer of activated B cells, NIK: NF-*κ*B-Inducing Kinase, NEMO: NF-*κ*B Essential Modulator, IKK*α *and β: I*κ*B*α *and β kinases, PI3K: Phosphoinositide 3-Kinase, Nrf2: Nuclear Factor Erythroid 2-Related Factor 2, HO-1: Heme Oxygenase 1, HMGB1 - High Mobility Group Box 1.*

IKKβ kinases (**Figure 5**) [81]. Further modulation is provided by the VitC induced decrease in the late pro-inflammatory cytokine HMGB1 (high mobility group box 1) secretion and through the lowering of histamine levels [82, 83].

#### *3.4.1 Clinical trials: vitamin C and sepsis or other critically ill conditions*

**Table 1** shows studies that were performed to evaluate VitC efficacy in many pathological conditions. In critically ill patients, several clinical trials have already been completed or are still ongoing. Until December 2020, 39 studies involving ascorbate and some critically ill conditions were registered at the United States National Library of Medicine (NLM) databank clinicaltrials.gov. The list with all referred studies and links to the clinicaltrials.gov forms are available at the end of this chapter.

To the present date, 25 of the cited trials are already finished, 12 are ongoing, and two will begin in 2021. Twelve of these studies tested VitC alone, with no other experimental therapeutics except the usually applied in sepsis cases (i.e., antimicrobial and fluid therapy, vasopressors, and inotropic agents). Seventeen trials used a combination of hydrocortisone, ascorbate, and thiamine (HAT).

Ten studies experimented a combination of VitC with a corticosteroid only (2 trials) or VitC with VitB1 (5 trials) or VitC in combination with some other therapeutic agent (3 trials). Even if there is no consensus about intravenous doses to be used in critically ill patients, 23 of the 39 trials employed 6 g/day doses, mostly in a 6 h-interval regimen (1.5 g each). Five studies used doses below 6 g/day, and nine studies used doses above 6 g/day, mostly in a protocol of 200 mg/kg/day in a 6 h interval regimen (about 14 g/day to a 70 kg patient).

Sadly, from the 25 already finished trials, only 5 reported their results to clinicaltrials.gov or published them in a peer-reviewed journal. One of those was a pharmacokinetic study [84], so no outcomes were evaluated. The other four studies that reported results were called REDOXS [85], ORANGES [86], VITAMINS [87], and CITRIS-ALI [88].

REDOXS used ascorbate in 1.5 g/day dose administered enterally associated with glutamine and other antioxidants. The study was planned to evaluate glutamine associated with a pool of antioxidants effect on critically ill patients. Results reported no difference when compared to placebo for the primary endpoint (28-day mortality rate).

ORANGES was a study intended to evaluate the HAT protocol in septic patients. They evaluated almost 70 patients (in each group) in a protocol that involved 6 g/day ascorbate (1.5 g per dose) for a maximum of 4 days after ICU admission. The study concluded that HAT could decrease the duration of shock, but not the 28-day mortality rate in patients with sepsis, probably due to ascorbate administration (they had an arm of the study that received only corticosteroids).

VITAMINS used the same HAT and ascorbate dosage as described above. They evaluated about 100 patients (in each group). The difference between the ORANGES trial is the control group. While ORANGES intervention in control was essentially placebo, the VITAMINS used a corticoid and thiamine (when clinicians evaluated its need). VITAMINS results indicate that treatment with intravenous ascorbate, hydrocortisone, and thiamine, did not significantly improve the duration of mortality rate and discontinuation of vasopressor administration over seven days.

The CITRIS-ALI trial evaluated the administration of VitC alone in sepsis, associated with acute respiratory distress syndrome (ARDS) patients, in a dose of 200 mg/kg/day (about 14 g/day to a 70 kg patient). The primary outcome evaluated

**291**

therapy [94].

*Vitamin C and Sepsis*

findings [92].

death [61, 93].

*DOI: http://dx.doi.org/10.5772/intechopen.95623*

the change in the Sequential Sepsis Related Organ Failure Score (SOFA) and two plasma biomarkers (C-reactive protein and thrombomodulin). The study assessed groups of about 80 patients. Circa 65% of patients (from both control and treatment groups) received corticosteroids during the study, and the mortality rate was significantly lower in the VitC group. However, since this outcome was not a primary outcome, the authors did not consider it in this study. Authors concluded that patients with sepsis and ARDS did not have an improvement in organ dysfunction scores, nor did they have altered markers of inflammation and vascular injury

Outside the clinicaltrial.gov, several studies have investigated the use of IV ascorbate in critically ill patients. Cases of trauma, severe burn, and septic shock were evaluated, in various dosage schemes, from 7 g until 110 g/day. No severe adverse effects related to the vitamin C infusion were reported in any of the studies. A decrease in the incidence of multiple system organ failure, trends to reduced

One of the most commented studies about the effects of the HAT approach, and maybe the reference for several of the clinical trials, was published by Dr. Marik from Eastern Virginia Medical School in 2017 [92]. This study proposed the early HAT protocol using ascorbate IV (1.5 g every 6 h for 4 days or until ICU discharge), hydrocortisone (50 mg every 6 h for 7 days or until ICU discharge), as well as IV thiamine (200 mg every 12 h for 4 days or until ICU discharge). VitC is administered as an infusion over 30 to 60 min and mixed in a 100 mL solution of either dextrose 5% in water or normal saline. Dr. Marik's results showed that early use of intravenous VitC, with hydrocortisone and thiamine, would be used effectively to prevent progressive organ impairment, including acute kidney damage, and reduce patients' mortality with severe sepsis and septic shock. However, the published work evaluated a small sample, and as the authors say at the end of the manuscript, additional studies are required to confirm their preliminary

High doses of IV ascorbate, thiamine, and glucocorticoids can reduce proinflammatory mediators, ROS, and decrease immunosuppression. Thiamine is useful to energy production as a precursor of thiamine pyrophosphate and acts as an antioxidant. Thiamine is essential because ascorbate may cause oxalate accumulation in the kidneys, and the concomitant use can prevent it since thiamine pyrophosphate is a cofactor required for the oxidation of glyoxylate to carbon dioxide by the enzyme glyoxylate aminotransferase. Thiamine deficiency increases the conversion of glyoxylate to oxalate. At the same time, thiamine deficiency is common in septic patients and is associated with an increased risk of

The VitC in critically ill patients is still a dilemma to be solved. There is a rationale behind its use that seems to be optimal. HAT therapy's premise is the use of a combination of drugs that aim at multiple sectors of the patient's response to an infectious agent, synergistically restoring the impaired immune system, avoid damage due to oxidants, and restore mitochondrial activity. However, to evaluate the clinical features and impact of this scheme, most of the studies performed were small, doses used between trials were highly different, and the risk of bias was usually uncertain or high. Secondary outcomes need bigger sample sizes, and so were yet harder to evaluate. The studies' duration was not uniform, so the follow-up and comparison analysis were possible only to the longest available time in each trial. Finally, the heterogeneity between treatment schemes made comparisons hard. Isolated analysis of VitC ignores any synergistic effects that could be seen with HAT

after a 96-hour infusion of vitamin C compared with placebo.

mortality, and ICU stay length was the usual results achieved [89–91].

#### *Vitamin C and Sepsis DOI: http://dx.doi.org/10.5772/intechopen.95623*

*Antioxidants - Benefits, Sources, Mechanisms of Action*

this chapter.

and CITRIS-ALI [88].

mortality rate).

IKKβ kinases (**Figure 5**) [81]. Further modulation is provided by the VitC induced decrease in the late pro-inflammatory cytokine HMGB1 (high mobility group box 1)

**Table 1** shows studies that were performed to evaluate VitC efficacy in many pathological conditions. In critically ill patients, several clinical trials have already been completed or are still ongoing. Until December 2020, 39 studies involving ascorbate and some critically ill conditions were registered at the United States National Library of Medicine (NLM) databank clinicaltrials.gov. The list with all referred studies and links to the clinicaltrials.gov forms are available at the end of

To the present date, 25 of the cited trials are already finished, 12 are ongoing, and two will begin in 2021. Twelve of these studies tested VitC alone, with no other experimental therapeutics except the usually applied in sepsis cases (i.e., antimicrobial and fluid therapy, vasopressors, and inotropic agents). Seventeen trials used a

Ten studies experimented a combination of VitC with a corticosteroid only (2 trials) or VitC with VitB1 (5 trials) or VitC in combination with some other therapeutic agent (3 trials). Even if there is no consensus about intravenous doses to be used in critically ill patients, 23 of the 39 trials employed 6 g/day doses, mostly in a 6 h-interval regimen (1.5 g each). Five studies used doses below 6 g/day, and nine studies used doses above 6 g/day, mostly in a protocol of 200 mg/kg/day in a 6 h

Sadly, from the 25 already finished trials, only 5 reported their results to clinicaltrials.gov or published them in a peer-reviewed journal. One of those was a pharmacokinetic study [84], so no outcomes were evaluated. The other four studies that reported results were called REDOXS [85], ORANGES [86], VITAMINS [87],

REDOXS used ascorbate in 1.5 g/day dose administered enterally associated with glutamine and other antioxidants. The study was planned to evaluate glutamine associated with a pool of antioxidants effect on critically ill patients. Results reported no difference when compared to placebo for the primary endpoint (28-day

ORANGES was a study intended to evaluate the HAT protocol in septic patients. They evaluated almost 70 patients (in each group) in a protocol that involved 6 g/day ascorbate (1.5 g per dose) for a maximum of 4 days after ICU admission. The study concluded that HAT could decrease the duration of shock, but not the 28-day mortality rate in patients with sepsis, probably due to ascorbate administration (they had an

VITAMINS used the same HAT and ascorbate dosage as described above. They evaluated about 100 patients (in each group). The difference between the ORANGES trial is the control group. While ORANGES intervention in control was essentially placebo, the VITAMINS used a corticoid and thiamine (when clinicians evaluated its need). VITAMINS results indicate that treatment with intravenous ascorbate, hydrocortisone, and thiamine, did not significantly improve the duration of mortality rate and discontinuation of vasopressor administration over

The CITRIS-ALI trial evaluated the administration of VitC alone in sepsis, associated with acute respiratory distress syndrome (ARDS) patients, in a dose of 200 mg/kg/day (about 14 g/day to a 70 kg patient). The primary outcome evaluated

secretion and through the lowering of histamine levels [82, 83].

combination of hydrocortisone, ascorbate, and thiamine (HAT).

interval regimen (about 14 g/day to a 70 kg patient).

arm of the study that received only corticosteroids).

*3.4.1 Clinical trials: vitamin C and sepsis or other critically ill conditions*

**290**

seven days.

the change in the Sequential Sepsis Related Organ Failure Score (SOFA) and two plasma biomarkers (C-reactive protein and thrombomodulin). The study assessed groups of about 80 patients. Circa 65% of patients (from both control and treatment groups) received corticosteroids during the study, and the mortality rate was significantly lower in the VitC group. However, since this outcome was not a primary outcome, the authors did not consider it in this study. Authors concluded that patients with sepsis and ARDS did not have an improvement in organ dysfunction scores, nor did they have altered markers of inflammation and vascular injury after a 96-hour infusion of vitamin C compared with placebo.

Outside the clinicaltrial.gov, several studies have investigated the use of IV ascorbate in critically ill patients. Cases of trauma, severe burn, and septic shock were evaluated, in various dosage schemes, from 7 g until 110 g/day. No severe adverse effects related to the vitamin C infusion were reported in any of the studies. A decrease in the incidence of multiple system organ failure, trends to reduced mortality, and ICU stay length was the usual results achieved [89–91].

One of the most commented studies about the effects of the HAT approach, and maybe the reference for several of the clinical trials, was published by Dr. Marik from Eastern Virginia Medical School in 2017 [92]. This study proposed the early HAT protocol using ascorbate IV (1.5 g every 6 h for 4 days or until ICU discharge), hydrocortisone (50 mg every 6 h for 7 days or until ICU discharge), as well as IV thiamine (200 mg every 12 h for 4 days or until ICU discharge). VitC is administered as an infusion over 30 to 60 min and mixed in a 100 mL solution of either dextrose 5% in water or normal saline. Dr. Marik's results showed that early use of intravenous VitC, with hydrocortisone and thiamine, would be used effectively to prevent progressive organ impairment, including acute kidney damage, and reduce patients' mortality with severe sepsis and septic shock. However, the published work evaluated a small sample, and as the authors say at the end of the manuscript, additional studies are required to confirm their preliminary findings [92].

High doses of IV ascorbate, thiamine, and glucocorticoids can reduce proinflammatory mediators, ROS, and decrease immunosuppression. Thiamine is useful to energy production as a precursor of thiamine pyrophosphate and acts as an antioxidant. Thiamine is essential because ascorbate may cause oxalate accumulation in the kidneys, and the concomitant use can prevent it since thiamine pyrophosphate is a cofactor required for the oxidation of glyoxylate to carbon dioxide by the enzyme glyoxylate aminotransferase. Thiamine deficiency increases the conversion of glyoxylate to oxalate. At the same time, thiamine deficiency is common in septic patients and is associated with an increased risk of death [61, 93].

The VitC in critically ill patients is still a dilemma to be solved. There is a rationale behind its use that seems to be optimal. HAT therapy's premise is the use of a combination of drugs that aim at multiple sectors of the patient's response to an infectious agent, synergistically restoring the impaired immune system, avoid damage due to oxidants, and restore mitochondrial activity. However, to evaluate the clinical features and impact of this scheme, most of the studies performed were small, doses used between trials were highly different, and the risk of bias was usually uncertain or high. Secondary outcomes need bigger sample sizes, and so were yet harder to evaluate. The studies' duration was not uniform, so the follow-up and comparison analysis were possible only to the longest available time in each trial. Finally, the heterogeneity between treatment schemes made comparisons hard. Isolated analysis of VitC ignores any synergistic effects that could be seen with HAT therapy [94].

## **4. Conclusions**

Vitamin C is a powerful antioxidant that takes part in many vital biological processes. Due to its properties, it has been proposed that VitC could improve sepsis and septic shock symptoms. Because of its pharmacokinetics, it is imperative that ascorbic acid is administered IV in high dosage to explore its full potential in sepsis. Furthermore, the inclusion of hydrocortisone and thiamine to compose the HAT protocol has shown to improve patients outcomes in some clinical trials. Nevertheless, there is still much debate on whether the HAT protocol can actually exert this improvement. To further investigate this proposal, trials should increase sample sizes and come to an agreement on treatment schemes so they can be accurately compared, in addition to sharing the results of the research on Clinical Trials.

## **Acknowledgements**

JChem for Word was used for **Figure 3**, Product version 20.21.0.768, ChemAxon (https://www.chemaxon.com). All other images were created with BioRender.com

## **Conflict of interest**

The authors declare no conflict of interest.

## **Appendix**


**293**

*Vitamin C and Sepsis*

Unit

Human Sepsis

Septic Shock

Shock

Recovery)

Vitamin C)

Dose in Septic Shock)

*DOI: http://dx.doi.org/10.5772/intechopen.95623*

High Dose of Vitamin C on Mechanically Ventilated Septic Patients in Intensive Care

Ascorbic Acid (Vitamin C) Infusion in

The Effect of Vitamin C, Thiamine and Hydrocortisone on Clinical Course and Outcome in Patients With Severe Sepsis and

Effect of Anti-inflammatory and Antimicrobial Cosupplementations in Traumatic ICU Patients at High Risk of Sepsis

Ascorbic Acid and Thiamine Effect in Septic

ASTER (Acetaminophen and Ascorbate in Sepsis: Targeted Therapy to Enhance

ViCiS (Vitamin C to Reduce Vasopressor

Comparative, Between Triple Therapy Regimen to Hydrocortisone Monotherapy in Reducing the MR in Septic Shock Patients

Ascorbic Acid and Thiamine

the Treatment of Septic Shock

Outcomes of Septic Shock Patients Treated With a Metabolic Resuscitation Bundle Consisting of Intravenous Hydrocortisone,

LOVIT (Lessening Organ Dysfunction With

Vitamin C, Thiamine and Hydrocortisone for

CORVICTES (Vitamin C, Hydrocortisone and Thiamine for Septic Shock)

CORVICTES-ΥΜ (Vitamin C, Steroids, and Thiamine, and Cerebral Autoregulation and Functional Outcome in Septic Shock)

Thiamine, Vitamin C and Hydrocortisone in

Effect of IV Vitamin C, Thiamine, and Steroids on Mortality of Septic Shock

Effects of Glucocorticoid Combined With Vitamin C and Vitamin B1 on Microcirculation in Severe Septic Shock

Clinical Trial of Antioxidant Therapy in

STASIS (Steroids, Thiamine and Ascorbic

HYVITS (Evaluation of Hydrocortisone, Vitamin C and Thiamine for the Treatment

AVoCaDO (Administration of Intravenous Vitamin C in Novel Coronavirus Infection (COVID-19) and Decreased Oxygenation)

the Treatment of Septic Shock

Patients With Septic Shock

Acid in Septic Shock)

of Septic Shock)

**Trial name Internet link to the trial**

https://clinicaltrials.gov/ct2/show/NCT04029675

https://clinicaltrials.gov/ct2/show/NCT01434121

https://clinicaltrials.gov/ct2/show/NCT03335124

https://clinicaltrials.gov/ct2/show/NCT04216459

https://clinicaltrials.gov/ct2/show/NCT03756220

https://clinicaltrials.gov/ct2/show/NCT03835286

https://clinicaltrials.gov/ct2/show/NCT03913468

https://clinicaltrials.gov/ct2/show/NCT03680274

https://clinicaltrials.gov/ct2/show/NCT03872011

https://clinicaltrials.gov/ct2/show/NCT03592693

https://clinicaltrials.gov/ct2/show/NCT03649633

https://clinicaltrials.gov/ct2/show/NCT03828929

https://clinicaltrials.gov/ct2/show/NCT03540628

https://clinicaltrials.gov/ct2/show/NCT03821714

https://clinicaltrials.gov/ct2/show/NCT03557229

https://clinicaltrials.gov/ct2/show/NCT04134403

https://clinicaltrials.gov/ct2/show/NCT0338050

https://clinicaltrials.gov/ct2/show/NCT04357782

Vitamin C and Septic Shock https://clinicaltrials.gov/ct2/show/NCT03338569

https://clinicaltrials.gov/ct2/show/study/NCT04291508

https://clinicaltrials.gov/ct2/show/study/NCT04508946

*Antioxidants - Benefits, Sources, Mechanisms of Action*

Vitamin C is a powerful antioxidant that takes part in many vital biological processes. Due to its properties, it has been proposed that VitC could improve sepsis and septic shock symptoms. Because of its pharmacokinetics, it is imperative that ascorbic acid is administered IV in high dosage to explore its full potential in sepsis. Furthermore, the inclusion of hydrocortisone and thiamine to compose the HAT protocol has shown to improve patients outcomes in some clinical trials. Nevertheless, there is still much debate on whether the HAT protocol can actually exert this improvement. To further investigate this proposal, trials should increase sample sizes and come to an agreement on treatment schemes so they can be accurately compared, in addition to sharing the results of the research on Clinical Trials.

JChem for Word was used for **Figure 3**, Product version 20.21.0.768, ChemAxon (https://www.chemaxon.com). All other images were created with BioRender.com

**4. Conclusions**

**Acknowledgements**

**Conflict of interest**

**Trial name Internet link to the trial**

The authors declare no conflict of interest.

Vitamin C & Thiamine in Sepsis https://clinicaltrials.gov/ct2/show/NCT03592277

Vitamin C, Vitamin B1 and Steroid in Sepsis https://clinicaltrials.gov/ct2/show/NCT04039815

https://clinicaltrials.gov/ct2/show/NCT04088591

https://clinicaltrials.gov/ct2/show/NCT01590303

https://clinicaltrials.gov/ct2/show/NCT03258684

https://clinicaltrials.gov/ct2/show/NCT04160676

https://clinicaltrials.gov/ct2/show/NCT03829683

https://clinicaltrials.gov/ct2/show/NCT04137276

https://clinicaltrials.gov/ct2/show/NCT03389555

https://clinicaltrials.gov/ct2/show/NCT04111822

https://clinicaltrials.gov/ct2/show/NCT04197115

https://clinicaltrials.gov/ct2/show/study/NCT03509350

High-dose Intravenous Vitamin C as an Adjunctive Treatment for Sepsis in Rwanda

VICTAS Vitamin C, Thiamine, and Steroids

Hydrocortisone, Vitamin C, and Thiamine for the Treatment of Sepsis and Septic Shock

Therapy With Hydrocortisone, Ascorbic Acid, Thamine in Patients With Sepsis

Vitamin C Infusion for Treatment in Sepsis

Effect of Intravenous Vitamin Con SOFA

Pilot Study on the Use of Hydrocortisone, Vitamin c and Thiamine in Patient With

Vitamin C, Thiamine, Cyanocobalamine, Pyridoxine and Hydrocortisone in Sepsis

Ascorbic Acid, Corticosteroids, and Thiamine in Sepsis (ACTS) Trial

and Alcoholic Hepatitis

Score Among Septic Patients

Sepsis and Septic Shock

Outcome Following Vitamin C Administration in Sepsis

in Sepsis

**Appendix**

**292**



## **Author details**

Adriana Françozo de Melo, Giulia Oliveira Timo and Mauricio Homem-de-Mello\* inSiliTox, University of Brasilia, Brasilia, Brazil

\*Address all correspondence to: mauriciohmello@unb.br

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**295**

*Vitamin C and Sepsis*

**References**

*DOI: http://dx.doi.org/10.5772/intechopen.95623*

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[4] Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med. 2003;348(2): 138-150. DOI: 10.1056/NEJMra021333

s00134-008-1337-8

archinte.167.15.1655

10.1159/000358835

10.1038/383787a0

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## **References**

*Antioxidants - Benefits, Sources, Mechanisms of Action*

REDOXS (Trial of Glutamine and Antioxidant Supplementation in Critically

Pharmacokinetics of Two Different Highdose Regimens of Intravenous Vitamin C in

High Dose Intravenous Ascorbic Acid in

ORANGES - Metabolic Resuscitation Using Ascorbic Acid, Thiamine, and

CITRIS-ALI Vitamin C Infusion for Treatment in Sepsis Induced Acute Lung

VITAMINS The Vitamin C, Hydrocortisone and Thiamine in Patients With Septic Shock

Ill Patients)

Severe Sepsis

Trial

Injury

Critically Ill Patients

Glucocorticoids in Sepsis.

**Trial name Internet link to the trial**

Adriana Françozo de Melo, Giulia Oliveira Timo and Mauricio Homem-de-Mello\*

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

https://clinicaltrials.gov/ct2/show/study/NCT00133978

https://clinicaltrials.gov/ct2/show/study/NCT02455180

https://clinicaltrials.gov/ct2/show/results/NCT02734147

https://clinicaltrials.gov/ct2/show/NCT03422159

https://clinicaltrials.gov/ct2/show/NCT03333278

https://clinicaltrials.gov/ct2/show/study/NCT02106975

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inSiliTox, University of Brasilia, Brasilia, Brazil

provided the original work is properly cited.

\*Address all correspondence to: mauriciohmello@unb.br

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DOI: 10.1007/s00134-016-4225-7

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CCM.0b013e31828e9b03

2013;41(9):2069-2079. DOI: 10.1097/

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10.1016/j.amjmed.2019.07.054

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[88] Fowler AA 3rd, Truwit JD, Hite RD, Morris PE, DeWilde C, Priday A, et al. Effect of Vitamin C Infusion on Organ Failure and Biomarkers of Inflammation and Vascular Injury in Patients With Sepsis and Severe Acute Respiratory Failure: The CITRIS-ALI Randomized Clinical Trial. JAMA. 2019;322(13):1261- 1270. DOI: 10.1001/jama.2019.11825

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[90] Tanaka H, Matsuda T,

archsurg.135.3.326

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10.4103/2279-042X.179569

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Vitamin C, and Thiamine for the Treatment of Severe Sepsis and Septic

10.1097/00000658-200212000-00014

Miyagantani Y, Yukioka T, Matsuda H, Shimazaki S. Reduction of resuscitation fluid volumes in severely burned patients using ascorbic acid administration: a randomized, prospective study. Arch Surg. 2000;135(3):326-331. DOI: 10.1001/

2020;158(1):164-173. DOI: 10.1016/j.

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2020;158(1):164-173. DOI: 10.1016/j. chest.2020.02.049

*Antioxidants - Benefits, Sources, Mechanisms of Action*

Parenter Enteral Nutr. 2014;38(7):825- 839. DOI: 10.1177/0148607113497760

Bórquez-Ojeda O, Golde DW. Vitamin C suppresses TNF alpha-induced NF kappa B activation by inhibiting I kappa B alpha phosphorylation. Biochemistry. 2002;41(43):12995-13002. DOI: 10.1021/

[82] Kim SR, Ha YM, Kim YM, Park EJ, Kim JW, Park SW, et al. Ascorbic acid

[83] Hattori M, Yamazaki M, Ohashi W, Tanaka S, Hattori K, Todoroki K, et al. Critical role of endogenous histamine in promoting end-organ tissue injury in sepsis. Intensive care Med Exp. 2016;4(1):36. DOI: 10.1186/

[84] de Grooth H-J, Manubulu-Choo W-P, Zandvliet AS, Spoelstra-de Man AME, Girbes AR, Swart EL, et al. Vitamin C Pharmacokinetics in Critically Ill Patients: A Randomized Trial of Four IV Regimens. Chest. 2018;153(6):1368-1377. DOI: 10.1016/j.

[81] Cárcamo JM, Pedraza A,

reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and improves survival rate in septic mice by activation of Nrf2/ HO-1 signals. Biochem Pharmacol. 2015;95(4):279-289. DOI: 10.1016/j.

bi0263210

bcp.2015.04.007

s40635-016-0109-y

chest.2018.02.025

NEJMoa1212722

[85] Heyland D, Muscedere J, Wischmeyer PE, Cook D, Jones G, Albert M, et al. A randomized trial of glutamine and antioxidants in critically ill patients. N Engl J Med. 2013;368(16):1489-1497. DOI: 10.1056/

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Integrative "omic" analysis of experimental bacteremia identifies

a metabolic signature that distinguishes human sepsis from systemic inflammatory response syndromes. Am J Respir Crit Care Med. 2014;190(4):445-455. DOI: 10.1164/

rccm.201404-0624OC

[75] Rumsey SC, Kwon O, Xu GW, Burant CF, Simpson I, Levine M. Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid. J Biol Chem. 1997;272(30):18982- 18989. DOI: 10.1074/jbc.272.30.18982

[76] Lowes DA, Webster NR, Galley HF. Dehydroascorbic acid as pre-conditioner: protection from lipopolysaccharide induced mitochondrial damage. Free Radic Res. 2010;44(3):283-292. DOI: 10.3109/10715760903468766

[77] Langley RJ, Tsalik EL, van Velkinburgh JC, Glickman SW, Rice BJ, Wang C, et al. An integrated clinico-metabolomic model improves prediction of death in sepsis. Sci Transl Med. 2013;5(195):195ra95. DOI: 10.1126/

scitranslmed.3005893

s40635-017-0134-5

[78] Haileselassie B, Su E,

Pozios I, Niño DF, Liu H, Lu D-Y, et al. Myocardial oxidative stress correlates with left ventricular dysfunction on strain echocardiography in a rodent model of sepsis. Intensive care Med Exp. 2017;5(1):21. DOI: 10.1186/

[79] Hao J, Li W-W, Du H, Zhao Z-F, Liu F, Lu J-C, et al. Role of Vitamin C in Cardioprotection of Ischemia/ Reperfusion Injury by Activation of Mitochondrial KATP Channel. Chem Pharm Bull (Tokyo). 2016;64(6):548- 557. DOI: 10.1248/cpb.c15-00693

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**300**

[87] Fujii T, Luethi N, Young PJ, Frei DR, Eastwood GM, French CJ, et al. Effect of Vitamin C, Hydrocortisone, and Thiamine vs Hydrocortisone Alone on Time Alive and Free of Vasopressor Support Among Patients With Septic Shock: The VITAMINS Randomized Clinical Trial. JAMA. 2020;323(5):423- 431. DOI: 10.1001/jama.2019.22176

[88] Fowler AA 3rd, Truwit JD, Hite RD, Morris PE, DeWilde C, Priday A, et al. Effect of Vitamin C Infusion on Organ Failure and Biomarkers of Inflammation and Vascular Injury in Patients With Sepsis and Severe Acute Respiratory Failure: The CITRIS-ALI Randomized Clinical Trial. JAMA. 2019;322(13):1261- 1270. DOI: 10.1001/jama.2019.11825

[89] Nathens AB, Neff MJ, Jurkovich GJ, Klotz P, Farver K, Ruzinski JT, et al. Randomized, prospective trial of antioxidant supplementation in critically ill surgical patients. Ann Surg. 2002;236(6):814-822. DOI: 10.1097/00000658-200212000-00014

[90] Tanaka H, Matsuda T, Miyagantani Y, Yukioka T, Matsuda H, Shimazaki S. Reduction of resuscitation fluid volumes in severely burned patients using ascorbic acid administration: a randomized, prospective study. Arch Surg. 2000;135(3):326-331. DOI: 10.1001/ archsurg.135.3.326

[91] Zabet MH, Mohammadi M, Ramezani M, Khalili H. Effect of highdose Ascorbic acid on vasopressor's requirement in septic shock. J Res Pharm Pract. 2016;5(2):94-100. DOI: 10.4103/2279-042X.179569

[92] Marik PE, Khangoora V, Rivera R, Hooper MH, Catravas J. Hydrocortisone, Vitamin C, and Thiamine for the Treatment of Severe Sepsis and Septic

Shock: A Retrospective Before-After Study. Chest. 2017;151(6):1229-1238. DOI: 10.1016/j.chest.2016.11.036

[93] Mitchell AB, Ryan TE, Gillion AR, Wells LD, Muthiah MP. Vitamin C and Thiamine for Sepsis and Septic Shock. Am J Med. 2020;133(5):635-638. DOI: 10.1016/j.amjmed.2019.07.054

[94] Zhang M, Jativa DF. Vitamin C supplementation in the critically ill: A systematic review and meta-analysis. SAGE open Med. 2018;6:2050312118807615. DOI: 10.1177/2050312118807615

**303**

Section 3

Phytochemical Antioxidants

Section 3
