Chapter

Flavonoids and Phenolic Acids as

Potential Natural Antioxidants
Biljana Kaurinovic and Djendji Vastag

Abstract

For centuries, aromatic herbs and spices have been added to different foods to
improve the flavor and organoleptic properties. The use of aromatic plants and
spices in phytotherapy is mostly related to different activities of their essential oils,
such as antimicrobial, spasmolytic, carminative, hepatoprotective, antiviral, and
anticarcinogenic activities. Furthermore, many studies point to strong antioxidant
activities of aromatic plants and their essential oils. Knowing that phenolic com-
pounds are the most responsible for the antioxidant activity, the amount of total
phenolic contents and content of flavonoids have also been determined. In order to
examine the antioxidant properties of five different extracts of Laurus nobilis L.
leaves, various assays which measure free radical scavenging ability were carried
out: 1,1-diphenyl-2-picrylhydrazyl, hydroxyl, superoxide anion, nitric oxide and
hydroxyl radical scavenger capacity test, and lipid peroxidation assay. In all of the
tests, only the EtOAc extract showed a potent antioxidant effect.

Keywords: aromatic plants, flavonoids, phenolic acids, ROS, oxidative stress

1. Introduction

The history of medicinal herb usage dates back to the distant past, many centu-
ries and civilizations ago. Plants have played an important role in many cultures in
the treatment of various diseases, and floral fragrances have been used to refine the
spirit and body, to attract partners, and to establish a psychophysical balance. The
first written testimonials on the use of herbs for treatment are found in China.
Emperor Kin-Nong knew about 100 medicinal plants in 3000 years BC. One of the
oldest classical medical texts of ancient China is “Pent-Sao,” which was written
2500 years BC and is composed of 52 books; of which, two books are dedicated to
herbal remedies. In the nineteenth century, medicinal and exotic plants have
become lucrative, as more and more people began growing plants in their homes.
China, Japan, and South America were overwhelmed by collectors from plant com-
panies who looked for tropical plants to meet the needs of society. This instigated
scientific pharmacy and the start of chemical and physiological research on medic-
inal herbs. It can be said that the nineteenth century was the century of alkaloids,
because hundreds were isolated from plants from all over the world. The beginning
of the twentieth century threatened medicinal herbs to be completely thrown out of
use. Thus, “medicines” that have been successfully used for thousands of years have
become subject to mockery and disdain. The expulsion of medicinal herbs from
therapy can be compared to the darkness of the Middle Ages that had ruled Europe.

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Antioxidants

In the last four decades, especially in the developed countries of Europe and
America, scientists have shown increasing interest in plant research. It is estimated
that today about 60% of the total world population in treatment relies on herbs and
natural products that are thus recognized as an important source of drugs [1].
Phytochemistry studies a huge variety of organic substances that have been discov-
ered and which accumulate in plants. Furthermore, phytochemistry is also defining
the structure of these compounds, their biosynthesis, metabolism, natural distribu-
tion, and biological activities [2]. An important place among them is occupied by
aromatic plants, whose aroma is associated with the presence of essential oils and
complex mixtures of volatile compounds, dominated by mono- and sesquiterpenes.
In addition to essential oils, aromatic plants are characterized by the presence of
plant phenolic compounds, primarily coumarins and phenylpropanoids, that have
been shown to possess multiple pharmacological activities. Investigations of these
secondary biomolecules intensified when some commercial synthetic antioxidants
were found to exhibit toxic, mutagenic, and carcinogenic effects [3]. It was also
found that excessive production of oxygen radicals in the body initiates the oxida-
tion and degradation of polyunsaturated fatty acids. It is known that free radicals
attack the highly unsaturated fatty acid membrane systems and induce lipid perox-
idation, which is a key process in many pathological conditions and one of the
reactions that cause oxidative stress. Particularly, the biological membrane lipids in
the spinal cord and brain are vulnerable, because they contain high levels of poly-
unsaturated fatty acids. Moreover, the brain contains significant amounts of transi-
tional prooxidant metals and consumes a lot of oxygen. These features facilitate the
formation of oxygen radicals involved in the processes of aging, Alzheimer’s and
Parkinson’s disease, ischemic heart damage, arthritis, myocardial infarction, arte-
riosclerosis, and cancer. Phenolic antioxidants “stop” free oxygen radicals and free
radicals formed from the substrate by donating hydrogen atoms or electrons. Many
plant species and aromatic plants have been tested because of their antioxidant and
antiradical activities [4].
The aim of this chapter was to show the antioxidant role of phenolic acids and
flavonoids presented in aromatic plants and to assess their potential capacity as
scavengers of different free radicals.

2. Oxygen as a toxic molecule

Atmospheric oxygen (O2) is present as a biradical with two unpaired electrons,

which have the same spin quantum number and are located opposite the orbited
orbits. This electronic structure of molecular oxygen determines its chemical reac-
tivity and allows the absorption of individual electrons, with the formation of
numerous intermediate, partially reduced oxygen species that are commonly
referred to as reactive oxygen species (ROS) [5, 6]. These reactive oxygen species
are able to react with basic cellular structures and biomolecules [7] and are respon-
sible for the emergence of many diseases and degenerative damage [8].
The normal concentration of free radicals in the body is very low. However, the
effects are very disruptive, as the chain reaction allows one free radical to cause
changes in thousands of molecules and damage DNA, RNA, and enzymes in cell
membranes and leads to the formation of lipoxygenation products before being
inactivated. Which part of the cell (proteins, nucleic acids, membrane lipids, cyto-
solic molecules) or the extracellular component (hyaluronic acid, collagen) will
react with free radicals depends on the nature of the radical and the site of its
formation (e.g., cytosolic membranes, mitochondria, endoplasmic reticulum, per-
oxisome, cell membranes). Due to the presence of molecular oxygen in aerobic

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organisms and its ability to easily receive electrons, free radicals of oxygen origin
start more reactions in the cell. The reactions responsible for their formation are
respiration, processes of autoxidation of hydroquinone and catecholamine, reduced
transition metals, some herbicides and drugs, as well as irradiation that causes water
decomposition.

2.1 The role of ROS and RNOS in the onset of many diseases

Any disorder of oxygen species’ regulation resulting from a disturbance in the

balance between the formation of reactive oxygen metabolites and their elimination
by the antioxidant protection system is the state of oxidative stress. In oxidative
stress, the formation and accumulation of reactive metabolites are increased,
resulting in oxidative processes of destruction of cellular components and genetic
material.

2.1.1 Cardiovascular disease

ROS, RNOS, and LP are considered to be the major contributors to the etiology
of atherosclerosis and various chronic disorders such as coronary disease, stroke,
and ischemic dementia [9]. Antioxidants introduced through food can reduce the
occurrence of cardiovascular diseases by inhibiting the production of free radicals
and oxidative stress, protecting LDL from oxidation and aggregation, and inhibiting
the synthesis of proinflammatory cytokines [10].

2.1.2 Neurodegenerative diseases

Oxidative stress often occurs in the brain, because although it represents only
2% of the body weight, the brain uses up to 20% of oxygen added. Also, the brain
contains large amounts of polyunsaturated fatty acids subject to lipid peroxidation
under conditions of high oxygen concentration [11, 12].

2.1.3 Carcinogenesis

Although there are insufficient facts to confirm that the presence of free radicals
is necessary in the process of carcinogenesis, it is clear that they can lead to muta-
tions, transformations, and cancers [13]. Regarding the development of cancer, the
most important target for ROS is DNA. Carcinogenesis is the result of successive
mutations in DNA molecules leading to uncontrolled growth and cell phenotypic
modification. One of the first steps in this process is the direct interaction of
electrophiles or free radicals with cellular DNA in which promutagen lesions
develop. If no repair is performed, these lesions result in mutations in the next
generation of cells [14]. An increased intake of antioxidants through diet or dietary
supplements is associated with a reduction in the onset of cancer.

2.1.4 Aging

A reduced amount of free radicals or a reduction in the speed of their production

postpones the aging process and a whole series of diseases related to the aging
process [15]. A certain maximum life potential characterizes each animal species.
There is a reciprocal correlation between the speed of oxygen consumption (and
therefore the production of free radicals) and the maximum life potential. Some
studies have shown that the aging process can be slowed by increased food intake

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Antioxidants

that increases antioxidant capacity (e.g., fruit and vegetables) or by supplemental

intake of vitamins E, C, and β-carotene [16].

3. Antioxidant protection systems

The process of oxidative modification of proteins, carbohydrates, DNA, and

lipids is a universal mechanism of damage to the cell, especially at the membrane
level. On the other hand, the numerous roles of free radicals in physiological pro-
cesses make their creation a mandatory precondition of life, which is why a protec-
tive system has been established during evolutionary development. The basic role of
this protection system is to reduce the amount and uncontrolled creation of free
radicals and their precursors in the cell.
From a functional point of view, the antioxidant protection of the organism
includes three levels of action:

1. Antioxidant protection systems that prevent the endogenous formation of free

radicals. This level of protection is ensured by the spatial separation of
processes in which free radicals are formed.

2. Engagement of the system in conditions of normal and enhanced formation of

free radicals. According to the nature and method of action, antioxidants are
divided into two types:

a. Enzymatic (superoxide-dismutase, catalase, xanthine oxidase,

peroxidase, glutathione peroxidase, glutathione reductase, glutathione-
S-transferase). These enzymes make the so-called primary line of
antioxidant protection.

b.Nonenzymatic or the so-called secondary line of defense.

3. Enzymatic antioxidants involved in the reparation of oxidative damage of

lipids, proteins, carbohydrates, and nucleic acids.

3.1 Phenolic compounds

During the evolution, the plants developed effective defense mechanisms

against the harmful effects of visible, ultraviolet light and radiation and are a
natural source of various antioxidants. Several thousands of biologically active
secondary biomolecules of higher plants for phenolic compounds (vitamin E, fla-
vonoids, biflavonols, benzophenones, xanthones, stilbene, quinones, betacities,
phenolic acids, acetophenones, phenylpropanoids, coumarins, isocoumarins,
chromones, phenols, and diterpenic alcohols) and different nitrogen compounds
(alkaloids, amines, amino acids, and chlorophyll derivatives) have been shown to
exhibit strong antioxidant activity, but antioxidant activity of essential oils of many
spice plants is intense. Their significance is higher because it has been found that
many synthetic antioxidants exhibit undesired effects after a prolonged use (e.g.,
some of them are withdrawn from the market as a possible carcinogen). These
biomolecules exhibit their activity through various mechanisms: removing free
radicals, binding metal ions, inhibiting enzymatic systems that produce free radical
forms, increasing the concentration of biologically important endogenous antioxi-
dants, and inducing the expression of a variety of genes responsible for the synthe-
sis of enzymes that inhibit oxidative stress [14]. The term “herbal phenols”

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encompasses a wide range of plant substances that form one of the most numerous
classes of secondary biomolecules that have a common characteristic of an aromatic
ring carrying one or more hydroxyl groups as substituents, including functional
derivatives (esters, glycosides, etc.). However, this broad definition also includes
some non-phenolic substances. For this reason, it is recommended to combine a
definition that includes a chemical description and a biogenetic origin. In nature,
there are two general biosynthetic pathways for the synthesis of plant phenols:
(1) a polyacetate route and (2) a phenylpropanoid route with scrub acid as an
intermediate. Some phenols are formed by a combination of these two times [17].
The efficiency of phenolic compounds in protection against oxidative stress
depends on their reactivity in relation to toxic oxygen species and the reactivity of
phenoxy radicals relative to critical biomolecules. Chemical or enzymatic oxidation
of phenolic components of plant tissue results in a dark color which is of particular
importance in food technology. Their susceptibility to oxidation allows their use in
the protection of fats and oils.
Phenolic compounds also increase the activity of antioxidant enzymes, thus
indirectly affecting the concentration of harmful oxygen radicals in the living cell.
In high concentrations, radical reactions such as DNA damage, superoxide anion
production, etc. can also be act as a prooxidant [18].

3.1.1 Phenolic acids

The term “phenolic acid” includes hydroxy and other functional derivatives of
benzoic acid (C6▬C1) and cinnamic acid (C6▬C3) [19, 20]. Figures 1 and 2 give
the structures of the basic representatives of these acids.

Figure 1.
Chemical compounds of basic benzoic acid derivatives.

Figure 2.
Chemical formulas of basic derivatives of cinnamic acid.

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Antioxidants

Cinnamic acids, especially hydroxy-cinnamic acids, have the role of basic pre-
cursors in the biosynthesis of various plant phenols. The cinnamic acid and its
derivatives are produced by condensation of the acidic acids with phosphoenolpyr-
uvate to give the horizmic acid. Additional reactions of interconversion, decarbox-
ylation, transamination, and disinfection lead to the formation of cinnamon
(3-phenylpropenoic acid) and hydroxy-cinnamic acid. Subsequent reactions of
hydroxylation, methoxylation, etc. produce cimetic acid derivatives such as
p-coumaric acid (p-hydroxy cinnamic acid), β-acid (2,3-dihydroxy cinnamic acid),
ferulic acid (2-methoxy-3-hydroxy cinnamic acid), and synapartoic acid
(2,4-dimethoxy-3-hydroxy cinnamic acid).
The derivatives of cinnamic acid, in particular hydroxy-cinnamic acids, are the
basis of the overall phenylpropanoid metabolism consisting of complex
biochemical reactions which as a result supply the plant with important phenolic
components [21].

3.1.2 Flavonoids

The term “flavonoids” was proposed by Geisman and Heinseiner [21] to

describe all plant pigments having a C6▬C3▬C6 skeleton, in which two benzene
rings are linked via the C3 unit. These natural products, varying in color from white
to yellow, except anthocyanidins responsible for almost all pink and violet shades
[20], are widely distributed in the plant kingdom with the exception of algae and
fungi. So far, more than 4000 flavonoids have been found in plants, fruits, and
vegetables [22]. The most common are seeds, citrus fruits, olive oil, tea, and red
wine [23]. They are found in vacuoles, chloroplasts, and chromoplasts, in the form
of glycosides, and in the extinct cells free of glycosides. The presence of OH groups
directly linked to the carbon atoms of the benzene ring determines the antioxidant
role of flavonoids, phenolic acids, and their esters. The expressed activity is shown
by compounds with two hydroxyl groups, arranged as for catechol, and three
hydroxyl groups arranged as in pyrogallol.
The structure of all flavonoids is based on the C15 skeleton of the chromatic
structure for which the secondary ring (B) is attached (Figure 3) [24, 25].
Flavonoids are divided according to the substitution profile of the heterocyclic
ring. In the classification of flavonoids, the oxidation state of the heterocyclic ring as
well as the position of the secondary aromatic ring is taken into account. A total of
about 12 subgroups of flavonoids are distinguished. The secondary (B) ring may be
in position 2 (flavones, flavonols, dihydroflavonols, catechins, flavans, and

Figure 3.
Basic structure of flavonoids.

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anthocyanidins), position 3 (isoflavonoids), or position 4 (4-phenyl-coumarins,

neoflavonoids). In a few cases, the six-membered heterocyclic ring occurs in an
open isomeric form (chalcones and dihydrochalcones) or is replaced by a five-
membered ring.
The most widespread of all flavonoids are flavonols (3-hydroxyflavones) and
flavones. The most commonly used flavonoids are quercetin, kaempferol, and
myricetin. Quercetin is considered the most widespread component of all plant
phenols. More than 100 glycosides of quercetin are known. Among flavonols there
are about 200–300 known aglycons of these compounds [26].

3.1.3 The importance of phenolic compounds

Phenolic acids are important not only for ensuring the construction of lignin but
also for regulating plant growth and disease resistance. Hydroxy-cinnamic acids are
associated with the role of growth regulators and proteins in the development of
certain diseases. In addition, it is possible that they are important for chloroplasts
and the process of photosynthesis itself. Benzoic acid has been shown to inhibit
photosynthesis in chloroplasts of spinach [17]. p-Coumaric acid is the most wide-
spread compound among plant phenols. Furthermore, rosmarinic acid has antioxi-
dant, anti-inflammatory, and antimicrobial effects. Its antioxidant effect is stronger
than vitamin E. Rosemary acid prevents damage to cells caused by free radicals and
reduces the risk of cancer and atherosclerosis. Unlike antihistamines, rosemary acid
prevents the activation of immune system cells that cause swelling and fluid collec-
tion. It is used in the treatment of stomach ulcers, arthritis, cataracts, cancer, and
bronchial asthma [27, 28]. Caffeic acid far exceeds other antioxidants because it
reduces the production of α-toxin by more than 95%. It has been proven that high
doses of coffee acids have a detrimental effect on the rats because they cause gastric
papillomas. However, the combination of different antioxidants, including baconic
acid, had a pronounced effect on the reduction of colon tumors in the same rats. The
harmful effects of bicarbonate on human health are not known [29]. Calcium acid
and its derivative caffeic acid phenethyl ester (CAPE) show a reduction in tumors
and show anti-inflammatory and anticancer effects on ultraviolet-exposed skin,
especially UVC and UVB rays [30]. Anticancer activity was observed in mice whose
skin was treated with bee propolis and a papilloma-causing agent (TPA). CAPE
significantly reduced the number of papillomas [31].
Flavonoids have a high ecological significance. They function as pigments that
attract insect pollinators, not only as signal molecules for microorganisms that are
useful for the plant but also as antimicrobial agents [32]. In this sense, yellow
flavones and flavonols are particularly important. Because of the intense absorption
of UV radiation, flavonoids protect the plant tissue from UV radiation, thereby
influencing vital processes in chloroplasts.
In a pharmacological view, flavonoids show antiviral, antiallergic, antitumor,
antibacterial, antifungal, and antithrombotic activity [33]. They act on blood ves-
sels, namely, flavanones and catechins, that increase the resistance of the capillaries.
They show an anti-inflammatory activity that depends on the structure of flavo-
noids [34]. The flavonoid anti-inflammatory activity was also confirmed by in vitro
testing of the ability to inhibit lipoxygenase and cyclooxygenase [35]. Flavonoids
eliminate pathological changes on capillaries and are used against diabetes, hyper-
tension, and atherosclerosis. Flavonoids have been found to stimulate the secretion
of bile and inhibit enzymes and enzymatic systems. Many flavonoids have antimi-
crobial and antiviral activity. A certain number of flavonoids show some cytotoxic
activity. The common structural feature of cytotoxic flavonoids is trisubstituted
ring A, methylation at position C4 [21].

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For many flavonoids, high antioxidant activity has been demonstrated in var-
ious in vitro systems [36–38]. It has been shown that quercetin, rhamnetin, and
isorhamnetin can reduce the amount of serum and liver cholesterol in addition to
the in vivo antioxidant activity they show [39]. Flavonoids have been found to
inhibit the activity of XOD and have the ability to capture superoxide radicals.
Based on this, it is assumed that flavonoids can help in the treatment of gout and
ischemia by reducing the amount of uric acid and superoxide anion of radicals in
tissues [40]. Two flavonol glycoside-gallate esters showed inhibitory activity on
human immunodeficiency virus-1 (HIV-1) integrase [41]. The HIV-1 integrase
manages the process of incorporating viral DNA into the DNA of the host cell
molecule, which is necessary for the virus to reproduce and produce virions. In
this way, the inhibition of the given enzyme can be effective in anti-AIDS therapy.
For example, quercetin has a beneficial effect on human health: it improves heart
rate and reduces the risk of cancer. It has an anti-inflammatory and antiallergic
effect. All of these effects are caused by a strong antioxidant effect of quercetin.
Like many other flavonoids, quercetin inhibits the oxidation of LDL cholesterol,
and its anti-inflammatory activity derives from inhibition of lipoxygenase enzyme
and inhibition of inflammatory mediators. Quercetin also inhibits the release of
histamine. Studies have shown that quercetin lowers the risk of prostate, uterine,
breast, tissue, and colon cancer. It is presumed to reduce the production of uric
acid by inhibiting XOD. It also shows NO inhibitory activity. Rutin has a strong
antioxidant effect, as well as the ability to build chelates with metal ions (e.g.,
iron) and reduces Fenton’s reaction in which harmful oxygen radicals are pro-
duced. It is supposed to stabilize vitamin C. If rutin is taken along with vitamin C,
the activity of ascorbic acid increases. Rutin strengthens the capillaries, which
helps people who easily bleed or get bruises. It prevents the formation of various
edemas, which is an early symptom of a chronic vein disease. It has an anti-
inflammatory effect. There are indications that rutin can inhibit some carcino-
genic and precancerous conditions, prevent atherogenesis, and reduce the cyto-
toxicity of oxidized LDL cholesterol [22]. Furthermore, kaempferol prevents
arteriosclerosis by inhibiting the oxidation of low-density lipoproteins and the
formation of blood platelets. It has a role of a chemopreventive agent, which
means it prevents the formation of cancer cells. Quercetin has a synergistic effect
in reducing the proliferation of malignant cells, so treatment with quercetin and
kaempferol combinations is more effective than their individual use [42]. In
addition, tangeretin acts as an anticancer agent, and in in vitro studies, it has been
shown to act against some forms of malignant cells. It strengthens the cell wall and
protects it from attack. It also causes apoptosis of cells suffering from leukemia,
while normal cells remain undamaged [43]. Tangeretin prevents tumor suppres-
sion of intercellular bonds when transmitting the signal [44]. In the G1 phase of
the cell cycle, it “freezes” the cancer cells and prevents their replication. In short,
in vitro studies have shown that tangerine exhibits antimutagenic, noniinvasive,
and antiproliferative activity [45]. Animal studies have shown that tangeretin
reduces cholesterol levels [46] and has a potentially protective effect from
Parkinson’s disease [47].

4. Lauraceae family

The Lauraceae family comprises over 2500 species, which occur within the
subtropics and tropics of Eastern Asia and South and North America. Most species
possess aromatic roots, stems, and fruits. One of the most well-known and most
frequently used plants from this family is Laurus nobilis L., also called bay laurel.

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Figure 4.
Laurus nobilis L [21].

L. nobilis is a species held in high esteem since ancient times. It was dedicated to
Apollo, the ancient Greek god of light, and a symbol of peace and victory used to
make wreaths for emperors, generals, and poets (Figure 4) [48].

4.1 Laurus nobilis L.

Laurel is a tree or a large bush of pyramidal shape with aromatic, constantly

green leaves and shiny gray corn. It reaches a height of up to 5.5 m, but the
cultivated form is usually lower (1–3 m). The leaves are elliptical, fairly thick,
leathery, and shiny green. Clusters of tiny, yellow, single-polar flowers appear in
the spring. Berries (fruit) (Lauri Fructus), when dry, are black and wrinkled and
contain two oval fat seeds. Laurel is cultivated in several cultivated forms: spp.
aurea with yellowish young leaves, spp. angustifolia with narrow leaves (often called
Vrbolik laurel), and spp. undulata with corrugated leaf edges. Laurel is commer-
cially grown for aromatic leaves in Turkey, Algeria, Morocco, Portugal, Spain, Italy,
France, and Mexico [49, 50].
The distillation of laurel leaves produces green-yellow volatile oil that contains a
high percentage of oxidized components. Essential oil leaf (0.8–3%) contains
mainly 1,8-cineol (50%) and then eugenol, acetyleugenol, methyl eugenol, α- and
β-pinene, felsenren, linalool, geraniol, and terpineol. Dried berries can extract green
mass (melting point about 30°C) containing several percent essential oils
(0.6–10%), depending on the conditions of breeding and storage. Berries contain
both volatile and fixed oils. The others are known under the common name “laurel
oil” (Oleum Lauri expressum, Oleum laurinum, and Oleum Lauri unguinosum). As
essential ingredients, the oil contains laurosterin, glycerol ester with lauric acid, and
sesquiterpenoid (the costume and dehydrocostus lactone), while the rest is made up
of fats: triglycerides with lauric, myristic, and elastic acids. As with leaves, the
aroma is mainly due to terpenes (cineol, terpineol, α- and β-pinene, citral) but also
cinnamic acid and its methyl ester [51].
The main flavonoids in bay leaf are quercetin, kaempferol, rutin, and their
derivatives (Figure 5).
Kaempferol appears in the form of four nonpolar glycosides (Figure 6) [52, 53].
Laurus nobilis is characterized by the presence of the other important plant
phenolic substances such as phenolic acids (rosmarinic and caffeic acids)
(Figure 7).
As a medicinal plant, bay leaves and fruits have been employed against rheuma-
tism, skin rashes, and earaches. In addition, it has been used as a stomachic, astrin-
gent, carminative, diaphoretic, stimulant, emetic, emmenagogue, abortifacient,

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Figure 5.
Structures of the main flavonoids present in L. nobilis [21].

and insect repellent. The essential oil is used by the cosmetic industry in creams,
perfumes, and soaps.
Numerous investigations of qualitative composition of plant extracts have
revealed the presence of high concentration of phenols in the extracts obtained
using polar solvents [54]. The extracts that display the highest antioxidant activity
have the highest concentration of phenols. Because of that, our research on laurel
was recently extended to the comprehensive in vitro and in vivo studies of antiox-
idant activity of different extracts of leaves, to assess their potential capacity as
scavengers of free radicals. Results of determination of total phenolic contents and
total flavonoid contents in laurel leaf extracts are given in Table 1.
The amount of total phenolics in L. nobilis extracts ranged from 2.41 mg GAE/g
d.w. (Et2O extract) to 4.53 mg GAE/g d.w. (EtOAc extract). A significant amount of
these compounds has also been observed in the n-BuOH extract (3.96 mg GAE/g d.e.).
Furthermore, a considerable total flavonoid content was determined in the EtOAc and
n-BuOH extracts. A little less amount of total flavonoids was determined in the CHCl3
extract, while the smallest quantity of these compounds was found in the Et2O and
H2O extracts. HPLC-DAD analysis indicates a significant presence of flavonoids and
phenolic in the EtOAc and n-BuOH extracts. Quercetin glycosides and flavonoids

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Figure 6.
Structures of kaempferol and its glucosides present in L. Nobilis [21].

Figure 7.
Structures of two phenolic acids in L. nobilis.

Extracts Et2O CHCl3 EtOAc n-BuOH H2O

Total phenolic content 2.41 2.85 4.53 3.96 3.20

Total flavonoid content 0.76 1.02 1.56 1.07 0.68

Table 1.
The amount of total phenolic contents (mg GAE/g d.w.) and content of total flavonoids (mg QE/g d.w.) in
L. nobilis extracts.

(e.g., kaempferol-3-O-Glc) were detected in EtOAc extract. In addition, the presence

of phenolic acids (such as caffeic acid) and flavonoids (rutin and kaempferol) was
proven in the H2O extract. The amount of flavonoids in extracts plays a significant role
in their antioxidant capacity. Differences in flavonoid content between extracts and
between plant organs can be explained by different numbers of secretory structures in
various plant tissues [42, 55, 56].
It should be considered that the number of identified and quantified compounds
in MeOH extract of L. nobilis L. has been expanded in the present work (Table 2).
The results indicate that the major bioactive compounds in L. nobilis extracts
were kaempferol-3-O-glucoside, quercetin, and rutin. Phenolic acids were also

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Compounds Extract

Phenolic acid p-Hydroxybenzoic acid 38.46

Protocatechuic acid n.d.

p-Coumaric acid n.d.

Vanillic acid n.d.

Gallic acid n.d.

Caffeic acid 16.18

Quinic acid n.d.

Ferulic acid n.d.

Syringic acid n.d.

Chlorogenic acid 13.11

Cinnamic acid n.d.

Flavonoids Apigenin n.d.

Naringenin n.d.

Luteolin 5.19

Kaempferol 11.97

Apigenin-7-O-β-glucoside n.d.

Luteolin-7-O-β-glucoside n.d.

Kaempferol-3-O-glucoside 56.15

Quercetin-3-O-glucoside 31.18

Rutin 17.44

Quercetin 21.62

Quercitrin 7.14

Table 2.
LC-MS-MS quantification of bioactive compounds presented in L. nobilis L. crude MeOH extract (μg/g d.w.).

observed in the high level, where the antioxidant, caffeic, and chlorogenic acids
were found in the highest amount. Furthermore, p-hydroxybenzoic acid was also
found in very high amount. The rest of the phenolic acids were not detected [57–59].
Furthermore, antioxidant activity was observed in the study of laurel leaf
extracts in different solvents on the content of DPPH•, O2• , NO•, and OH• radicals
(Table 3).
The obtained results could point to strong quenching activities of flavonoids
present in the leaves of laurel against DPPH radicals, and a high degree of correla-
tion is observed between total phenol content and the ability of EtOAc extract to
neutralize DPPH radicals. This is indicated by the fact that phenolic compounds

Extract Et2O CHCl3 EtOAc n-BuOH H2O

DPPH radical 127.38 139.42 83.24 181.35 161.83

O2• radical 327.60 429.43 163.57 288.64 486.32

NO radical 168.77 322.84 158.63 386.80 618.42

OH radical 442.84 241.18 121.84 213.36 187.65

Table 3.
IC50 values (μg/mL) of L. nobilis for different antioxidant assays.

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Flavonoids and Phenolic Acids as Potential Natural Antioxidants
DOI: http://dx.doi.org/10.5772/intechopen.83731

play a key role in neutralizing free radical species which occurs by the mechanism of
electron transfer. But, it can be supposed that such antiradical activity is also
caused, besides flavonoids, by terpenoids, since nonpolar solvents also exhibited
high antiradical potential. When investigating neutralization of O2• and NO radi-
cals, ethyl acetate extract has also exhibited the greatest ability of their scavenging.
These results can be attributed to the presence of sesquiterpene lactones isolated
from the plant that possess certain biological and pharmacological activity [60, 61].
Matsuda et al. [62] have also established that the methanolic extract from the leaves
of L. nobilis was found to inhibit nitric oxide (NO) production in lipopolysaccharide
(LPS)-activated mouse peritoneal macrophages. It was concluded that seven ses-
quiterpene lactones (costunolide, dehydrocostus lactone, eremanthine, zaluzanin C,
magnolialide, santamarine, and spirafolide) potently inhibited LPS-induced NO
production. Inhibition of NO radicals with laurel extracts is very significant, having
in mind the ability to neutralize the superoxide anion radicals as well. The common
reaction between superoxide anion radical and nitrogen oxide radical yields a very
reactive peroxynitrite anion (ONOO ) which is very active in reaction of nitrifica-
tion of phenols—e.g., nitrification of thyroxine causes enzyme dysfunctions, and
increased amounts of 3-nitrothyrozine were found in various pathological states
[63]. If formation of nitroderivatives of thyroxine is prevented, the occurrence of
these diseases due to oxidative stress is reduced. Ethyl acetate extract of laurel
leaves is especially suited in this process since it neutralizes both superoxide anion
radical and NO radical. Obtained results can be related to the experiments in which
the total amount of phenols and flavonoids were determined (Table 1), which show
that ethyl acetate extract of laurel leaf contains the largest amounts of total phenolic
content and total flavonoid content. The cellular damage resulting from hydroxyl
radical is strongest among free radicals. Hydroxyl radical can be generated by
biochemical reaction. Superoxide radical is converted by superoxide dismutase
(SOD) to H2O2, which can subsequently produce extremely reactive OH• radicals in
the presence of transition metal ions such as iron and cooper [64, 65]. A good
antioxidant potential of neutralization OH radical was shown by the EtOAc
(IC50 = 121.84 μg/mL) and H2O (IC50 = 187.65 μg/mL) extracts. Such a good anti-
oxidant activity of H2O and EtOAc extracts is expected, because it is known that the
antioxidant activity of phenols is primarily a result of the ability of these com-
pounds to act as donors of hydrogen atoms removing free radicals with the forma-
tion of less reactive phenoxyl radicals [66]. The increased stability of the formed
phenoxyl radicals primarily attributed to electron delocalization and the existence
of multiple resonant forms. Researching dependence of activity on the structure
was found to have three structural features as important factors of radical removal
potential and/or antioxidant potential of flavonoids: (1) o-dihydroxy function of
ring B, which serves as the target of radicals; (2) 2,3-double bond in conjugation
with 4-oxo function, which is responsible for electron delocalization of the ring B;
and (3) the additional presence of 3- and 5-hydroxyl groups for the maximum
radical scavenging potential [67]. The positive relationship between increased
hydroxylation and increased antioxidant activity of flavonoids was found in differ-
ent lipid systems, such as oil and liposome systems. Also, for phenolic acids and
coumarins, it has been shown that vicinal diol groups are important for radical
scavenging capacity and that methoxylation or glycosylation of o-hydroxy group in
the coumarins and esterification of phenolic acids reduce the antioxidant activity of
these compounds [68]. For example, it was determined that rosmarinic acid has
stronger antioxidant effect than vitamin E. Rosmarinic acid prevents cell damage
caused by free radicals and reduces the risk of cancer and atherosclerosis. In con-
trast to the histamines, rosmarinic acid prevents activation of the immune system
cells that cause swelling and fluid collection [27, 69]. Furthermore, the action of

13
Antioxidants

some flavonoids is based on their ability to chelate transition metal ions, thereby
preventing the formation of radicals (initiators of LP), catching radical initiators of
LP (ROS), scavenging lipid alkoxyl and lipid peroxyl radicals, and regenerating
α-tocopherol by reduction of α-tocopherol radicals. Different metals have different
binding affinities of the flavonoids. Thus, for example, iron has the highest binding
affinity for 3-OH group of ring C, then catechol group ring B, and at the end of
5-OH group of ring A, while the copper ions bind to the first ring catechol group B
[70]. Also, in the previous investigation, on L. nobilis, different groups of chemicals
were isolated (luteolin, apigenin, alkaloids, monoterpene, and germacrane
alcohols) [71].

5. Conclusions

One of the paradoxes of life on Earth is that, on the one hand, oxygen is
necessary for the life of aerobic organisms. On the other hand, increased concen-
trations of oxygen and especially its reactive metabolites (reactive oxygen species)
may lead to the development of numerous diseases. A major source of free radicals
in biological systems is molecular oxygen (O2). The results of our in vitro assays
of examined five different extracts of Laurus nobilis leaves expressed significant
protective effects on ROS (DPPH, O2• , NO, and OH radicals), which was found to
be correlated to different compounds. HPLC-DAD analysis indicates a significant
presence of flavonoids and phenolic in the EtOAc and n-BuOH extracts. Quercetin
glycosides and flavonoids (e.g., kaempferol-3-O-Glc) were detected in EtOAc
extract. In addition, the presence of phenolic acids (such as caffeic acid) and
flavonoids (rutin and kaempferol) was proven in the H2O extract. The amount of
flavonoids in extracts plays a significant role in their antioxidant capacity, and it can
be concluded that ethyl acetate proved to be the best solvent for extraction of plant
material. Furthermore, it can be concluded that these extracts can be used in the
preparation of various herbal medicines.

Acknowledgements

This work was supported by the Ministry of Science and Environmental Protec-
tion of the Republic of Serbia (Project No. 172058).

Conflict of interest

The authors declare that there is no conflict of interest.

14
Flavonoids and Phenolic Acids as Potential Natural Antioxidants
DOI: http://dx.doi.org/10.5772/intechopen.83731

Author details

Biljana Kaurinovic* and Djendji Vastag

Department of Chemistry, Biochemistry and Environmental Protection,
University of Novi Sad, Republic of Serbia

*Address all correspondence to: [email protected]

© 2019 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.

15
Antioxidants

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