DPPH Radical Scavenging Assay

Abstract

Today, there is an increasing interest in antioxidants, especially to prevent the known harmful effects of free radicals in human metabolism and their deterioration during processing and storage of fatty foods. In both cases, natural-source antioxidants are preferred over synthetic antioxidants. So, there has been a parallel increase in the use of assays to estimate antioxidant efficacy in human metabolism and food systems. Today, there are many bioanalytical methods that measure the antioxidant effect. Of these, the 1,1-diphenyl-2-picrylhydrazil (DPPH) removing assay is the most putative, popular, and commonly used method to determine antioxidant ability. In this review, a general approach to the DPPH radical scavenging assay has been taken. In this context, many studies, including attempts to adapt the DPPH radical scavenging method to different analytes, search for the highest antioxidant activity values, and optimize the method of measurement, have previously been performed. Therefore, it is highly important to introduce measures aimed at standardizing the conditions of the DPPH radical scavenging activity, including the various reaction media suitable for this assay. For this aim, the chemical and basic principles of DPPH free radical scavenging are defined and discussed in an outline. In addition, this study describes and defines the basic sections of DPPH free radical scavenging in food and biological systems. Additionally, some chemical, critical, and technical details of the DPPH free radical removal method are given. This is a simple assay in which the prospective compounds or herbal extracts are mixed with the DPPH solution and their absorbance is measured after a certain period. However, despite rapid advances in instrumental techniques and analysis, this method has not undergone extreme modification. This study presents detailed information about the DPPH method and an in-depth review of different developments.

Full text

Citation: Gulcin, ˙
I.; Alwasel, S.H.
DPPH Radical Scavenging Assay.
Processes 2023,11, 2248. https://
doi.org/10.3390/pr11082248
Academic Editor: Hoon Kim
Received: 26 June 2023
Revised: 22 July 2023
Accepted: 24 July 2023
Published: 26 July 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
processes
Review
DPPH Radical Scavenging Assay
˙
Ilhami Gulcin 1, * and Saleh H. Alwasel 2
1Department of Chemistry, Faculty of Sciences, Atatürk University, 25240 Erzurum, Turkey
2Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia;
sawasel10@hotmail.com
*Correspondence: igulcin@atauni.edu.tr; Tel.: +90-4422314375
Abstract:
Today, there is an increasing interest in antioxidants, especially to prevent the known
harmful effects of free radicals in human metabolism and their deterioration during processing
and storage of fatty foods. In both cases, natural-source antioxidants are preferred over synthetic
antioxidants. So, there has been a parallel increase in the use of assays to estimate antioxidant efficacy
in human metabolism and food systems. Today, there are many bioanalytical methods that measure
the antioxidant effect. Of these, the 1,1-diphenyl-2-picrylhydrazil (DPPH) removing assay is the most
putative, popular, and commonly used method to determine antioxidant ability. In this review, a
general approach to the DPPH radical scavenging assay has been taken. In this context, many studies,
including attempts to adapt the DPPH radical scavenging method to different analytes, search for
the highest antioxidant activity values, and optimize the method of measurement, have previously
been performed. Therefore, it is highly important to introduce measures aimed at standardizing the
conditions of the DPPH radical scavenging activity, including the various reaction media suitable
for this assay. For this aim, the chemical and basic principles of DPPH free radical scavenging are
defined and discussed in an outline. In addition, this study describes and defines the basic sections of
DPPH free radical scavenging in food and biological systems. Additionally, some chemical, critical,
and technical details of the DPPH free radical removal method are given. This is a simple assay in
which the prospective compounds or herbal extracts are mixed with the DPPH solution and their
absorbance is measured after a certain period. However, despite rapid advances in instrumental
techniques and analysis, this method has not undergone extreme modification. This study presents
detailed information about the DPPH method and an in-depth review of different developments.
Keywords: antioxidants; 1,1-diphenyl-2-picrylhydrazil; DPPH; antioxidant assay
1. Introduction
1.1. Reactive Species (RS) and Oxidative Stress (OS)
Oxidation processes are essential for the survival of cells. Aerobic cellular respiration
organisms provide energy from organic molecules such as glucose but also cause the for-
mation of free radicals that cause cellular damage in metabolism [
1
]. A free radical contains
an unpaired (free) electron with a quantum-mechanical property called spin. Such an entity
typically has high reactivity because of its open shell structure [
2
]. However, today there
are many free radicals that are stable under laboratory conditions, that is, in the air and
at room temperature [
3
]. Free radicals are known to be mostly associated with oxidative
stress [
4
,
5
]. Oxidative stress is a comparatively new concept that has been commonly used
in the medical sciences recently [
6
,
7
]. It occurs when there is an excess of reactive oxygen
species (ROS) produced by a cellular mitochondrion. It is inevitable that free radicals,
which are known to cause many degenerative diseases such as carcinogenesis, acute inflam-
mation, high blood pressure, diabetes, preeclampsia, acute renal failure, atherosclerosis,
Alzheimer’s disease and Parkinson’s disorders, mutagenesis, aging, and cardiovascular
disorders, are produced in biological systems [
8
,
9
]. There are many factors, including UV
radiation and pollutants, that contribute to oxidative stress, which has a daily influence
Processes 2023,11, 2248. https://doi.org/10.3390/pr11082248 https://www.mdpi.com/journal/processes

Processes 2023,11, 2248 2 of 20
on human health [
10
]. Cells metabolize oxygen, creating potentially harmful ROS. Under
normal conditions, the rate and amplitude of oxidant formation are balanced by the rate at
which they are removed [
11
]. However, disruption of the balance between antioxidants
and pro-oxidants causes oxidative stress [12].
Recent extensive scientific research has classified reactive species (RS) and free radi-
cals into three main categories: reactive nitrogen species (RNS), reactive oxygen species
(ROS), and reactive sulfur species (RSS), composed of nitrogen, oxygen, and sulfur atoms,
respectively [
13
–
15
]. Hydroxyl (HO
·
), superoxide anion (O
2−
), alkoxyl (RO
·
), nitric ox-
ide (NO
·
), and peroxyl (ROO
·
) radicals are radicals. Nitrogen monoxide (NO), singlet
oxygen (
1
O
2
), hydrogen peroxide (H
2
O
2
), ozone (O
3
), nitrous acid (HNO
2
), nitrous oxide
(N
2
O), lipid hydroperoxide (LOOH), and hypochlorous acid (HOCl) are non-radical re-
active species [
16
–
18
]. RS also occur in living organisms as part of their defense systems.
Phagocytes such as monocytes, macrophages, or neutrophils defend themselves against
foreign organisms by synthesizing large amounts of O
2·−
or NO
·
as part of their killing or
defense mechanisms [
19
–
24
]. Antioxidant molecules inhibit oxidative processes and reduce
the hazardous effects of RS. In this way, they are important in terms of health [25–29].
When free radicals occur excessively in the human body, they cause very serious
negative effects in different tissues [
30
]. One of the most important complications related
to this is the formation of lipid peroxidation in the plasma membrane. This event promotes
RNS and ROS formation. Meanwhile, metals such as iron and copper enable Fenton and
Haber–Weiss reactions and the formation of reactive species such as OH
·
[
31
–
33
]. In the
presence of metal ions and oxygen, H2O2can easily form OH·by the Fenton reaction [34].
In addition, the Haber–Weiss reaction produces OH
·
from O
2•−
and H
2
O
2
catalyzed by
iron ions. This impact was first suggested by Fritz Haber [
35
]. In later studies, it was known
that both reactions constitute the main source of radicals and are the most important ones
responsible for cellular damage [36–38].
Fe2++H2O2→Fe3++OH−+OH•(Fenton reaction)
O•−
2+H2O2→O2+H2O+OH•(Haber–Weiss reaction)
1.2. Antioxidants
Antioxidant molecules can be classified in different ways depending on their environ-
ment and the functions they perform [
39
–
42
]. An antioxidant is defined as a substance that
can significantly delay or completely prevent the oxidation of substrate molecules, even
at low concentrations [
43
]. They donate electrons to free radicals, rendering them harm-
less, and neutralize them by minimizing oxidative damage in biological processes [
44
–
46
].
Antioxidants prevent free radical formation by interfering with the free radical-mediated
oxidative process at any of its three main stages: initiation, propagation, and termina-
tion [
8
,
47
,
48
]. The effectiveness of an antioxidant compound depends on different parame-
ters and factors. The most important are the physical system state, temperature, structural
properties, properties of the oxidation-sensitive substrate, concentration, synergistic effect,
and presence of pro-oxidant compounds [
49
]. The chemical structure of an antioxidant
molecule determines its intrinsic reactivity and antioxidant ability towards free radicals
and other ROS [
50
]. In addition, the effectiveness of the antioxidant also depends on its
concentration in the system and localization, such as interface distribution [
51
,
52
]. The
reaction kinetics are another factor that plays an important role in the protective effect of
the antioxidant in the long or short term. This includes the thermodynamics of the reaction
between an antioxidant and a different oxidant, the reaction rate, and the antioxidant’s
ability to react. All of these parameters must be considered when testing the effectiveness
of a particular antioxidant substance [
53
]. In this way, they maintain the balance between
oxidants and antioxidants in metabolism [
54
]. In addition, antioxidants delay lipid per-
oxidation formation during storage and processing of foods, prevent the deterioration of
drugs and food products, and extend the shelf life of products [
55
]. For this purpose, a wide
variety of synthetic or natural antioxidants are often used to prevent food spoilage [
56
]. To

Processes 2023,11, 2248 3 of 20
address this, the pharmaceutical industry has mainly used synthetic antioxidants to block
or reduce the intracellular amounts of reactive oxygen or nitrogen species [
57
]. Of these,
synthetic antioxidants are widely used because they can be found in high purity, have low
costs, and are highly reactive even at low concentrations. However, some harmful effects
have been reported [58].
Therefore, antioxidants of natural origin rather than synthetic antioxidants are pre-
ferred. There has been a parallel increase in methods used to estimate the efficacy of
antioxidants [
59
]. The use of a free 1,1-diphenyl-2-picrylhydrazil radical (DPPH) is the
most common method. Butylated hydroxytoluene (BHT), propyl gallate (PG), butylated
hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ) are the synthetic antioxidants
that are most preferred by manufacturers, and therefore consumers have to use them
despite their known negative effects. The chemical structures of the synthetic antioxidant
molecules are given in Figure 1. These chemicals have been widely used as food additives
for the prevention of oxidative deterioration in food and pharmaceutical products [
60
].
However, new studies have raised concerns regarding the safety of these synthetic com-
pounds owing to unexpected consequences, particularly their inhibitory ability against
numerous enzymes [
61
]. Due to the toxic effects of these synthetic additives, researchers
are working hard to find new and alternative antioxidant substances with fewer side
effects [
62
]. In this context, there is a considerably increasing trend to replace synthetic
antioxidants with natural antioxidants, which have lower toxicity, high biodegradability,
and safer methods of action [63].
Processes 2023, 11, x FOR PEER REVIEW 3 of 21
delay lipid peroxidation formation during storage and processing of foods, prevent the
deterioration of drugs and food products, and extend the shelf life of products [55]. For
this purpose, a wide variety of synthetic or natural antioxidants are often used to prevent
food spoilage [56]. To address this, the pharmaceutical industry has mainly used synthetic
antioxidants to block or reduce the intracellular amounts of reactive oxygen or nitrogen
species [57]. Of these, synthetic antioxidants are widely used because they can be found
in high purity, have low costs, and are highly reactive even at low concentrations. How-
ever, some harmful effects have been reported [58].
Therefore, antioxidants of natural origin rather than synthetic antioxidants are pre-
ferred. There has been a parallel increase in methods used to estimate the efficacy of anti-
oxidants [59]. The use of a free 1,1-diphenyl-2-picrylhydrazil radical (DPPH) is the most
common method. Butylated hydroxytoluene (BHT), propyl gallate (PG), butylated hy-
droxyanisole (BHA), and tert-butylhydroquinone (TBHQ) are the synthetic antioxidants
that are most preferred by manufacturers, and therefore consumers have to use them de-
spite their known negative effects. The chemical structures of the synthetic antioxidant
molecules are given in Figure 1. These chemicals have been widely used as food additives
for the prevention of oxidative deterioration in food and pharmaceutical products [60].
However, new studies have raised concerns regarding the safety of these synthetic com-
pounds owing to unexpected consequences, particularly their inhibitory ability against
numerous enzymes [61]. Due to the toxic effects of these synthetic additives, researchers
are working hard to find new and alternative antioxidant substances with fewer side ef-
fects [62]. In this context, there is a considerably increasing trend to replace synthetic an-
tioxidants with natural antioxidants, which have lower toxicity, high biodegradability,
and safer methods of action [63].
OH
OCH3
BHA
OH
CH3
BHT
OH
OH
TBHQ PG
OH
O
O
H3COH
OH
Figure 1. The chemical structures of the most putative and commonly used synthetic antioxidants.
In the case of long-term use of these synthetic antioxidants, it has been stated that
they cause some health problems, including carcinogenesis, skin allergies, fatty liver, and
gastrointestinal distress [64]. Therefore, conscious consumers are concerned about the
negative effects of synthetic antioxidants and prefer natural antioxidants. The main and
most accessible sources of these natural and safer antioxidants are fruits, vegetables,
herbs, and spices [65]. For this purpose, plants such as tea, linden, cinnamon, cloves, fen-
nel, anise, and rosemary are used as sources of natural antioxidants due to their rich tan-
nin, catechin, theine, phenolic, and flavonoid contents [66]. Consumption of herbal prod-
ucts rich in phenolic content, which has an antioxidant effect, both reduces the risk of
catching diseases and prevents the development of degenerative disorders [67]. However,
the antioxidant capacity and quality of natural antioxidants and extracts depend not only
on the natural source but also on the applied isolation and extraction processes [68].
2. Antioxidant Methods
Several studies have been performed recently on the oxidation process of free radi-
cals and the general mechanism of action of antioxidants. This is because free radicals,
although neutral, have a significant effect on the biological system [1,14]. In fact, some
Figure 1. The chemical structures of the most putative and commonly used synthetic antioxidants.
In the case of long-term use of these synthetic antioxidants, it has been stated that
they cause some health problems, including carcinogenesis, skin allergies, fatty liver, and
gastrointestinal distress [
64
]. Therefore, conscious consumers are concerned about the
negative effects of synthetic antioxidants and prefer natural antioxidants. The main and
most accessible sources of these natural and safer antioxidants are fruits, vegetables, herbs,
and spices [
65
]. For this purpose, plants such as tea, linden, cinnamon, cloves, fennel, anise,
and rosemary are used as sources of natural antioxidants due to their rich tannin, catechin,
theine, phenolic, and flavonoid contents [
66
]. Consumption of herbal products rich in
phenolic content, which has an antioxidant effect, both reduces the risk of catching diseases
and prevents the development of degenerative disorders [
67
]. However, the antioxidant
capacity and quality of natural antioxidants and extracts depend not only on the natural
source but also on the applied isolation and extraction processes [68].
2. Antioxidant Methods
Several studies have been performed recently on the oxidation process of free radicals
and the general mechanism of action of antioxidants. This is because free radicals, although
neutral, have a significant effect on the biological system [
1
,
14
]. In fact, some lipid derivative
components, such as aldehyde, which can occur naturally during food processing and
have adverse effects on human health, can easily occur as a result of the heat treatment of
foods [
51
]. However, there are many antioxidant tests that directly measure the transfer of
H atoms or electrons from antioxidants to free radicals [
69
]. The methods for measuring the

Processes 2023,11, 2248 4 of 20
activities of antioxidants have recently made remarkable progress. Early methods measure
the effectiveness of antioxidants on the formation of certain types of oxidation products
and therefore rely on measuring lipid peroxidation. So far, different chemical methods have
been used for the evaluation of antioxidant activity by specific methods, combining highly
automated and sensitive detection technologies, such as removal activity against several
types of ROS or free radicals, reducing potency and metal chelation, and others [
70
]. The
concept of antioxidant capacity first emerged as a chemical concept, and later it was adapted
to fields such as medicine, biology, food, and epidemiology [
71
]. It is very important to
know the antioxidant profiles of these products in order to avoid loss of commercial and
nutritional value during processing and preservation of foods and pharmaceutical products.
Therefore, determining the potential antioxidant capacity of foods and pharmaceutical
products requires the development of a fast and simple method [
72
]. Today, many different
antioxidant procedures have been developed and used effectively [
73
]. In this context, the
most commonly used methods are inhibition of autoxidation of emulsions of linoleic acid,
the
β
-carotene bleaching method, total radical-trapping antioxidant parameter (TRAP) and
oxygen radical absorbance capacity (ORAC) analyses [
1
], ferric (Fe
3+
) and cupric (Cu
2+
)
ions reduction assays [
74
], DPPH
·
, N,N-dimethyl-p-phenylenediamine radicals (DMPD
·+
),
2,2-azinobis 3-ethylbenzthiazoline-6-sulfonic acid radicals (ABTS
·+
), superoxide anion
radicals (O
2·−
) removal experiments, and metal chelation tests [
75
]. As is known, most
of these methods use similar principles and techniques. Measuring the ability of these
antioxidant techniques is based on a suitable standard spectrophotometer measurement [
76
].
Antioxidant ability should not be tested with a single method; at least three different
in vitro
antioxidant methods must be performed together to determine antioxidant activity.
A pure-only method does not reflect antioxidant activity. Given these, it is quite difficult
to compare one method with another. Therefore, the methods to be used in analysis
for research purposes should be carefully selected and applied [
6
]. Additionally, one of
the most important objectives of this review is to detail the chemistry, mechanism, and
application of the DPPH radical scavenging assay after giving some basic information
about the antioxidant methods used to evaluate antioxidant properties. In recent years,
researchers have focused on the DPPH radical scavenging method.
3. Radical Scavenging Methods
Despite the antioxidant defense mechanisms found in living things, especially humans,
cell damage accelerates the aging process and plays an important role in the development
of diseases. Tissue damage may occur as a result of the oxidative modification of biological
macromolecules such as lipids, proteins, and DNA [
77
]. In order to understand and prevent
these events, radical chain reactions in metabolism should be well understood.
Radical chain reactions are common mechanisms of lipid autoxidation and peroxi-
dation. Radical scavenging agents can scavenge peroxide radicals to terminate radical
chain reactions and improve the stability and quality of food products [
14
]. The radical-
scavenging properties of antioxidants are the most important lipid oxidation inhibition
mechanism. This method is an indispensable and standard test in antioxidant activity de-
termination studies. Radical scavenging-based methods such as DPPH
·
, DMPD
+
, ABTS
+
,
and O
2−
are the most popular and putative spectrophotometric assays used for the de-
termination of antioxidant activities of beverages, foods, and vegetable and fruit extracts.
These chromogen radicals can react directly with antioxidant compounds. These assays are
also commonly used because they are sensitive, simple, fast, and reproducible [1].
4. What Are DPPH Radicals?
The 1,1-diphenyl-2-picrylhydrazil (DPPH) radical was discovered 100 years ago by
Goldschmidt and Renn in 1922 [
78
]. This method was developed by Blois [
79
] using a stable
free radical, DPPH, to similarly determine antioxidant activity. The chemical structures
of the 1,1-diphenyl-2-picrylhydrazil radical (DPPH
·
) are given in Figure 2. This assay is
based on spectrophotometric measurements of the capacity of antioxidants to scavenge

Processes 2023,11, 2248 5 of 20
DPPH radicals. Later, this test was developed in 1995 by Brand-Williams and his team
and adopted by the vast majority of researchers. This antioxidant application was used
very effectively by Gulcin’s research group with a slight modification [
80
]. The single
electron of the nitrogen atom in DPPH is reduced to the corresponding hydrazine by
taking a hydrogen atom from the antioxidants. The DPPH
·
radical has a remarkably stable
and intense color. Due to these two properties of the radical, its solution has been used
intensively. This radical has been frequently used in polymer chemistry, especially in EPR
spectroscopy, and in the evaluation of the antioxidant capacities of chemicals [
80
,
81
]. The
use of this last feature in the evaluation of antioxidant capacities was first discovered by
Blois in 1958 [
79
]. The stability of the radicals is due to the steric crowding on the first-order
divalent N atom and the “push-pull” effect exerted by the second-order diphenylamino
group, an electron donor, and picryl, an electron acceptor. This effect stabilizes the canonical
structure considerably. EPR measures the spin densities at the two hydrazil N atoms, which
are large and essentially equal. It has been reported that the two distinct bands seen
in the UV-vis spectrum of DPPH
·
are produced by
π−π
* transitions, with the unpaired
electron making a major contribution to the band in the visible region [
82
,
83
]. When a
DPPH solution is mixed with a solution of a substance capable of donating a hydrogen
atom, this violet color disappears, resulting in the reduced form of the DPPH radical
(
DPPH-H
) [
84
]. The wider band is responsible for the deep violet color of the DPPH
·
solution. The formation of hydrazine (DPPH-H) induces the disappearance of the visible
band as the color of the solution changes from violet to pale yellow as a result of radical
reduction by hydrogen atom transfer from antioxidants, which are H donors. The color
intensity of this reaction, known as the “DPPH test” in the literature, can be easily recorded
by UV-vis spectroscopy. This method is widely used to evaluate the antioxidant capacity of
pure antioxidant molecules, especially herbal extracts or phenolic compounds [85].
Processes 2023, 11, x FOR PEER REVIEW 5 of 21
4. What Are DPPH Radicals?
The 1,1-diphenyl-2-picrylhydrazil (DPPH) radical was discovered 100 years ago by
Goldschmidt and Renn in 1922 [78]. This method was developed by Blois [79] using a
stable free radical, DPPH, to similarly determine antioxidant activity. The chemical struc-
tures of the 1,1-diphenyl-2-picrylhydrazil radical (DPPH·) are given in Figure 2. This assay
is based on spectrophotometric measurements of the capacity of antioxidants to scavenge
DPPH radicals. Later, this test was developed in 1995 by Brand-Williams and his team
and adopted by the vast majority of researchers. This antioxidant application was used
very effectively by Gulcin’s research group with a slight modification [80]. The single elec-
tron of the nitrogen atom in DPPH is reduced to the corresponding hydrazine by taking a
hydrogen atom from the antioxidants. The DPPH· radical has a remarkably stable and
intense color. Due to these two properties of the radical, its solution has been used inten-
sively. This radical has been frequently used in polymer chemistry, especially in EPR spec-
troscopy, and in the evaluation of the antioxidant capacities of chemicals [80,81]. The use
of this last feature in the evaluation of antioxidant capacities was first discovered by Blois
in 1958 [79]. The stability of the radicals is due to the steric crowding on the first-order
divalent N atom and the “push-pull” effect exerted by the second-order diphenylamino
group, an electron donor, and picryl, an electron acceptor. This effect stabilizes the canon-
ical structure considerably. EPR measures the spin densities at the two hydrazil N atoms,
which are large and essentially equal. It has been reported that the two distinct bands seen
in the UV-vis spectrum of DPPH· are produced by π−π* transitions, with the unpaired
electron making a major contribution to the band in the visible region [82,83]. When a
DPPH solution is mixed with a solution of a substance capable of donating a hydrogen
atom, this violet color disappears, resulting in the reduced form of the DPPH radical
(DPPH-H) [84]. The wider band is responsible for the deep violet color of the DPPH· so-
lution. The formation of hydrazine (DPPH-H) induces the disappearance of the visible
band as the color of the solution changes from violet to pale yellow as a result of radical
reduction by hydrogen atom transfer from antioxidants, which are H donors. The color
intensity of this reaction, known as the “DPPH test” in the literature, can be easily rec-
orded by UV-vis spectroscopy. This method is widely used to evaluate the antioxidant
capacity of pure antioxidant molecules, especially herbal extracts or phenolic compounds
[85].
Figure 2. The chemical structures of a 1,1-diphenyl-2-picrylhydrazil radical (DPPH·).
DPPH remains a stable free radical thanks to the delocalization of the spare electron
in the whole molecule (Figure 2). In this way, the DPPH radical does not dimerize like
many other free radicals. Additionally, this electron delocalization causes a dark purple
color to appear in the molecule and a maximum absorption of the ethanol solution at 517
nm [86]. Absorption is lost as the electron pairs are removed from the DPPH radical. The
resulting color flare is dependent on the stoichiometry of the electron number. A concen-
trated solution of 0.5 mM colored alcohol also obeys Lambert-Beer’s law [79].
While DPPH· is slightly soluble in nonpolar solvents, it dissolves quite well in differ-
ent polar organic solvents. It is almost insoluble in water at room temperature. DPPH·
Figure 2. The chemical structures of a 1,1-diphenyl-2-picrylhydrazil radical (DPPH·).
DPPH remains a stable free radical thanks to the delocalization of the spare electron in
the whole molecule (Figure 2). In this way, the DPPH radical does not dimerize like many
other free radicals. Additionally, this electron delocalization causes a dark purple color to
appear in the molecule and a maximum absorption of the ethanol solution at 517 nm [
86
].
Absorption is lost as the electron pairs are removed from the DPPH radical. The resulting
color flare is dependent on the stoichiometry of the electron number. A concentrated
solution of 0.5 mM colored alcohol also obeys Lambert-Beer’s law [79].
While DPPH
·
is slightly soluble in nonpolar solvents, it dissolves quite well in different
polar organic solvents. It is almost insoluble in water at room temperature. DPPH
·
selec-
tively reacts with radicals and hydrogen atom donors at different reaction sites. Radicals
usually attack the phenyl ring, while hydrogen donors react with the divalent nitrogen
atom. The limited space around the nitrogen atom sterically inhibits the addition of bulky
radicals to this region. Hydrogen atom donors can approach the nitrogen atom and release
the hydrogens there with the formation of hydrazine (DPPH-H) [
81
]. The researchers
purposefully used a methanolic DPPH radical solution (simple to use and available as
ready-made radicals) to study the antioxidant ability of food and pharmaceutical ingredi-
ents by measuring the absorbance of radicals remaining in the reaction environment [
80
].

Processes 2023,11, 2248 6 of 20
They measured residual DPPH radicals until they reached an equilibrium plateau. The
simplicity of the procedure and the short reaction time made working with these radicals
popular [
79
]. Molecular oxygen (O
2
) does not react with DPPH
·
. However, in the presence
of light, molecular oxygen reacts slightly with DPPH
·
. In addition, DPPH
·
solutions kept
in the dark can remain stable for a long time. DPPH radicals do not dimer and exist in free
monomeric form in alcohol solutions [87].
5. The Synthesis of DPPH Radicals
DPPH radicals are easily obtained by the oxidation of hydrazines with lead dioxide,
lead tetraacetate, potassium permanganate, or silver oxide (Figure 3). These reactions
are carried out in non-polar solvents such as benzene or dichloromethane. With simple
filtration, the desired radical is obtained in quantitative yield. In this way, many hydrazyl
permanent or stable free radicals containing carboxyl or sulfono groups are obtained from
these derivatives, generally in a single step with high yield [3].
Processes 2023, 11, x FOR PEER REVIEW 6 of 21
selectively reacts with radicals and hydrogen atom donors at different reaction sites. Rad-
icals usually attack the phenyl ring, while hydrogen donors react with the divalent nitro-
gen atom. The limited space around the nitrogen atom sterically inhibits the addition of
bulky radicals to this region. Hydrogen atom donors can approach the nitrogen atom and
release the hydrogens there with the formation of hydrazine (DPPH-H) [81]. The research-
ers purposefully used a methanolic DPPH radical solution (simple to use and available as
ready-made radicals) to study the antioxidant ability of food and pharmaceutical ingredi-
ents by measuring the absorbance of radicals remaining in the reaction environment [80].
They measured residual DPPH radicals until they reached an equilibrium plateau. The
simplicity of the procedure and the short reaction time made working with these radicals
popular [79]. Molecular oxygen (O2) does not react with DPPH·. However, in the presence
of light, molecular oxygen reacts slightly with DPPH·. In addition, DPPH· solutions kept
in the dark can remain stable for a long time. DPPH radicals do not dimer and exist in free
monomeric form in alcohol solutions [87].
5. The Synthesis of DPPH Radicals
DPPH radicals are easily obtained by the oxidation of hydrazines with lead dioxide,
lead tetraacetate, potassium permanganate, or silver oxide (Figure 3). These reactions are
carried out in non-polar solvents such as benzene or dichloromethane. With simple filtra-
tion, the desired radical is obtained in quantitative yield. In this way, many hydrazyl per-
manent or stable free radicals containing carboxyl or sulfono groups are obtained from
these derivatives, generally in a single step with high yield [3].
Figure 3. The synthesis route of 1,1-diphenyl-2-picrylhydrazil radicals (DPPH·).
6. Interactions of DPPH· with Phenols as H-Atom Donors
Some chemicals easily react with DPPH radicals by electron transfer or by donating
H atoms. Especially phenolic compounds are the most reactive and important ones that
react easily with DPPH·. Hydrogen atom abstraction contains reactions that form through
electron transfer followed by or preceded by proton transfer and can be formally classified
as Hydrogen atom transfer reactions. The DPPH· test therefore gives an estimate of the
total content of reductants present in the solution in plant extracts. The antioxidant capa-
bilities of phenolic compounds (ArOH) are quantified by the following reaction [1,14]:
ArOH + ROO•→ArO
•+ ROOH
This radical scavenging reaction of phenolic compounds had great industrial and bi-
ological importance because it was used to reduce the oxidation rate of organic matter
exposed to molecular oxygen in the air [88,89]. However, since peroxyl reacts very quickly
with DPPH radicals, these reactions are difficult to monitor, and sophisticated devices are
required. In contrast, the colored DPPH· radical is available and has much less reactivity
than ROO· [90]. As seen in Figure 4, the best example of this situation is the electron-
transfer reaction of cinnamic acids with DPPH radicals in alcoholic solutions [91].
Figure 3. The synthesis route of 1,1-diphenyl-2-picrylhydrazil radicals (DPPH·).
6. Interactions of DPPH·with Phenols as H-Atom Donors
Some chemicals easily react with DPPH radicals by electron transfer or by donating
H atoms. Especially phenolic compounds are the most reactive and important ones that
react easily with DPPH
·
. Hydrogen atom abstraction contains reactions that form through
electron transfer followed by or preceded by proton transfer and can be formally classified
as Hydrogen atom transfer reactions. The DPPH
·
test therefore gives an estimate of the total
content of reductants present in the solution in plant extracts. The antioxidant capabilities
of phenolic compounds (ArOH) are quantified by the following reaction [1,14]:
ArOH +ROO•→ArO•+ROOH
This radical scavenging reaction of phenolic compounds had great industrial and
biological importance because it was used to reduce the oxidation rate of organic matter
exposed to molecular oxygen in the air [
88
,
89
]. However, since peroxyl reacts very quickly
with DPPH radicals, these reactions are difficult to monitor, and sophisticated devices are
required. In contrast, the colored DPPH
·
radical is available and has much less reactivity
than ROO
·
[
90
]. As seen in Figure 4, the best example of this situation is the electron-transfer
reaction of cinnamic acids with DPPH radicals in alcoholic solutions [91].
The DPPH radical interaction of quercetin as an H-donor phenolic compound is a
good example of this situation. Quercetin is a flavonoid widely found in plants and is a very
important component of a regular diet [
92
]. In particular, the total flavonoid amount of plant
extracts is given as the equivalent of this polyphenolic compound. Two-electron oxidation
of quercetin yields quinomethide/quinone products, an intensely colored compound
(Figure 4). Several tautomeric forms of quercetin can be found in solution, but the second
tautomeric form shown in Figure 5has been reported to be the most stable and abundant
in solution. The interesting thing about these compounds is that their UV-vis spectrum
and colors are coincidentally very similar to the DPPH radical. This was interpreted as a
change in absorbance of 519 nm and a relatively different loss of DPPH radicals [93].

Processes 2023,11, 2248 7 of 20
Processes 2023, 11, x FOR PEER REVIEW 7 of 21
Figure 4. The mechanism between cinnamic acids and 1,1-diphenyl-2-picrylhydrazil (DPPH·) radi-
cals.
The DPPH radical interaction of quercetin as an H-donor phenolic compound is a
good example of this situation. Quercetin is a flavonoid widely found in plants and is a
very important component of a regular diet [92]. In particular, the total flavonoid amount
of plant extracts is given as the equivalent of this polyphenolic compound. Two-electron
oxidation of quercetin yields quinomethide/quinone products, an intensely colored com-
pound (Figure 4). Several tautomeric forms of quercetin can be found in solution, but the
second tautomeric form shown in Figure 5 has been reported to be the most stable and
abundant in solution. The interesting thing about these compounds is that their UV-vis
spectrum and colors are coincidentally very similar to the DPPH radical. This was inter-
preted as a change in absorbance of 519 nm and a relatively different loss of DPPH radicals
[93].
Figure 5. Enol and keto tautomeric forms of a quercetin molecule.
In radical scavenging studies, the antioxidant effects of phenolic compounds (Ar–
OH) generally occur by two mechanisms, including hydrogen atom transfer (HAT) or sin-
gle-electron transfer followed by proton transfer (SET-PT). However, in some cases, it may
not be possible to separate these two mechanisms with clear boundaries [94]. In a HAT-
based assay, an antioxidant molecule can quench free radicals through H-donation, while
in a SET-based method, a potential antioxidant agent exhibits antioxidant ability by trans-
ferring an electron (e-) to reduce any compound, including radicals, metals, and carbonyls
[95]. Recently, in addition to these two mechanisms, a third mechanism called the sequen-
tial proton loss electron transfer (SPLET) mechanism has been developed [14].
Figure 4.
The mechanism between cinnamic acids and 1,1-diphenyl-2-picrylhydrazil (DPPH
·
) radicals.
Processes 2023, 11, x FOR PEER REVIEW 7 of 21
Figure 4. The mechanism between cinnamic acids and 1,1-diphenyl-2-picrylhydrazil (DPPH·) radi-
cals.
The DPPH radical interaction of quercetin as an H-donor phenolic compound is a
good example of this situation. Quercetin is a flavonoid widely found in plants and is a
very important component of a regular diet [92]. In particular, the total flavonoid amount
of plant extracts is given as the equivalent of this polyphenolic compound. Two-electron
oxidation of quercetin yields quinomethide/quinone products, an intensely colored com-
pound (Figure 4). Several tautomeric forms of quercetin can be found in solution, but the
second tautomeric form shown in Figure 5 has been reported to be the most stable and
abundant in solution. The interesting thing about these compounds is that their UV-vis
spectrum and colors are coincidentally very similar to the DPPH radical. This was inter-
preted as a change in absorbance of 519 nm and a relatively different loss of DPPH radicals
[93].
Figure 5. Enol and keto tautomeric forms of a quercetin molecule.
In radical scavenging studies, the antioxidant effects of phenolic compounds (Ar–
OH) generally occur by two mechanisms, including hydrogen atom transfer (HAT) or sin-
gle-electron transfer followed by proton transfer (SET-PT). However, in some cases, it may
not be possible to separate these two mechanisms with clear boundaries [94]. In a HAT-
based assay, an antioxidant molecule can quench free radicals through H-donation, while
in a SET-based method, a potential antioxidant agent exhibits antioxidant ability by trans-
ferring an electron (e-) to reduce any compound, including radicals, metals, and carbonyls
[95]. Recently, in addition to these two mechanisms, a third mechanism called the sequen-
tial proton loss electron transfer (SPLET) mechanism has been developed [14].
Figure 5. Enol and keto tautomeric forms of a quercetin molecule.
In radical scavenging studies, the antioxidant effects of phenolic compounds (Ar–OH)
generally occur by two mechanisms, including hydrogen atom transfer (HAT) or single-
electron transfer followed by proton transfer (SET-PT). However, in some cases, it may not
be possible to separate these two mechanisms with clear boundaries [
94
]. In a HAT-based
assay, an antioxidant molecule can quench free radicals through H-donation, while in a
SET-based method, a potential antioxidant agent exhibits antioxidant ability by transferring
an electron (e-) to reduce any compound, including radicals, metals, and carbonyls [
95
].
Recently, in addition to these two mechanisms, a third mechanism called the sequential
proton loss electron transfer (SPLET) mechanism has been developed [14].
ArOH →ArO−+H+(HAT)
ArOH →ArO•+H•(SET −PT)
ArOH →ArO•++e−
ArO•+→ArO•+H+
ArO−+ROO•→ArO•+e−
ArOH +ROO•→ArO•+ROO−(SPLET)
The -OH group in the 7th position of flavonoids had great importance as the site of
ionization and electron transfer, according to SPLET. This mechanism has been discovered
recently [
96
,
97
]. In the first step, the reaction enthalpy corresponds to the proton affinity of
the phenoxide anion (ArO
−
), while in the second step, the phenoxy radical is formed by
electron transfer from the phenoxide anion to ROO
·
. In terms of antioxidant effect, SPLET
is similar to free radicals in the HAT mechanism. For example, the possible mechanism for

Processes 2023,11, 2248 8 of 20
the reactions of quercetin and taxifolin (dihydroquercetin) with DPPH radicals is shown in
Figures 6and 7. Quercetin and taxifolin, as stable antioxidant flavonoids, can easily convert
purple-colored DPPH radicals to yellow-colored DPPH-H. In addition, it has been reported
that taxifolin, which is naturally bioactive, significantly inhibits some metabolic enzymes
associated with some diseases [1,14].
Processes 2023, 11, x FOR PEER REVIEW 8 of 21
ArOH → ArO+H (HAT)
ArOH → ArO•+H• (SET − PT)
ArOH → ArO• +e

ArO• → ArO•+H
ArO+ ROO•→ArO
•+e

ArOH + ROO•→ArO
•+ROO
 (SPLET)
The -OH group in the 7th position of flavonoids had great importance as the site of
ionization and electron transfer, according to SPLET. This mechanism has been discov-
ered recently [96,97]. In the first step, the reaction enthalpy corresponds to the proton af-
finity of the phenoxide anion (ArO−), while in the second step, the phenoxy radical is
formed by electron transfer from the phenoxide anion to ROO·. In terms of antioxidant
effect, SPLET is similar to free radicals in the HAT mechanism. For example, the possible
mechanism for the reactions of quercetin and taxifolin (dihydroquercetin) with DPPH rad-
icals is shown in Figures 6 and 7. Quercetin and taxifolin, as stable antioxidant flavonoids,
can easily convert purple-colored DPPH radicals to yellow-colored DPPH-H. In addition,
it has been reported that taxifolin, which is naturally bioactive, significantly inhibits some
metabolic enzymes associated with some diseases [1,14].
HO
OH
O
O
OH
OH
OH
-O
OH
O
O
OH
OH
OH
O
OH
O
O
OH
OH
OH
ET
(Very fast)
O
OH
O
O
OH
O-
OH
O
OH
O
O
OH
O
OH
HO
OH
O
O
OH
O
OH
DPPH DPPH-H
DPPH
DPPH-H
Quercetin
HAT HAT
+H+
(Non-polar solvent)
Figure 6. The reactions between DPPH free radicals and a quercetin molecule.
These reactions are strongly and effectively accelerated due to the increased electron
density in the A and C rings found in quercetin. Moreover, a fast electron transfer from
the phenolate anion to the DPPH radicals is also an alternative route. The A ring in the
quercetin molecule strongly attracts electrons, creating a positive effect on conjugation.
On the other hand, the catechol moiety within the B ring constitutes the most likely site of
deprotonation. Due to the existence of -OH groups in flavonoid molecules, many flavo-
noid compounds are present in the water phase of biological systems. The interactions of
flavonoids with electron-deficient radicals can be accelerated by the SPLET mechanism to
effectively minimize the generation and consequent accumulation of reactive oxygen spe-
cies in the cells [98]. In addition, since the SET-PT and SPLET mechanisms in the solvent
environment are important, the effect of water on the three mechanisms also has signifi-
cance. Additionally, Litwinienko and Ingold (2005) proposed a different mechanism for
SPLET. Among organic solvents, methanol is the leading solvent that supports ionization.
This mechanism is preferred in that phenols with low pKa react with electron-deficient
radicals with relatively lower HAT activities and yield product molecules with low pKa
Figure 6. The reactions between DPPH free radicals and a quercetin molecule.
Processes 2023, 11, x FOR PEER REVIEW 9 of 21
[96]. In addition, SPLET formation in methanol and ethanol solutions was also reported
by Foti et al. [91]. Many studies have shown that DPPH· reacts with phenolic acids. As a
result of the suppression of the ionization of the phenolic hydroxyl group by the free car-
boxylic acid, the rate constants of the reactions for methyl esters of these acids are several
times higher than for free acids. These experiments nicely confirm the effective role of
ionization of phenolic compounds in the reaction of phenols with DPPH· in solvents that
can promote ionization [97,98].
Figure 7. The reactions between taxifolin and DPPH free radicals.
α-Tocopherol is a natural phenolic compound commonly added to food products as
a preservative. Tocopherols show antioxidant activity by donating the hydrogen from the
-OH group to DPPH·. The formation of an α-tocopherol radical is stabilized by the delo-
calization of the solitary electron on the structure of the aromatic ring (Figure 8). These
compounds are highly lipophilic and more active in lipoproteins and membranes. The
most crucial antioxidant effect is the inhibition of lipid peroxidation, which scavenges li-
pid peroxyl radicals, resulting in lipid hydroperoxides and the formation of a tocopher-
oxyl radical [14].
Figure 7. The reactions between taxifolin and DPPH free radicals.
These reactions are strongly and effectively accelerated due to the increased elec-
tron density in the A and C rings found in quercetin. Moreover, a fast electron transfer
from the phenolate anion to the DPPH radicals is also an alternative route. The A ring
in the quercetin molecule strongly attracts electrons, creating a positive effect on conju-
gation. On the other hand, the catechol moiety within the B ring constitutes the most
likely site of deprotonation. Due to the existence of -OH groups in flavonoid molecules,
many flavonoid compounds are present in the water phase of biological systems. The
interactions of flavonoids with electron-deficient radicals can be accelerated by the SPLET
mechanism to effectively minimize the generation and consequent accumulation of reactive
oxygen species in the cells [
98
]. In addition, since the SET-PT and SPLET mechanisms

Processes 2023,11, 2248 9 of 20
in the solvent environment are important, the effect of water on the three mechanisms
also has significance. Additionally, Litwinienko and Ingold (2005) proposed a different
mechanism for SPLET. Among organic solvents, methanol is the leading solvent that sup-
ports ionization. This mechanism is preferred in that phenols with low pKa react with
electron-deficient radicals with relatively lower HAT activities and yield product molecules
with low pKa [
96
]. In addition, SPLET formation in methanol and ethanol solutions was
also reported by
Foti et al. [91]
. Many studies have shown that DPPH
·
reacts with phenolic
acids. As a result of the suppression of the ionization of the phenolic hydroxyl group by
the free carboxylic acid, the rate constants of the reactions for methyl esters of these acids
are several times higher than for free acids. These experiments nicely confirm the effective
role of ionization of phenolic compounds in the reaction of phenols with DPPH
·
in solvents
that can promote ionization [97,98].
α
-Tocopherol is a natural phenolic compound commonly added to food products as
a preservative. Tocopherols show antioxidant activity by donating the hydrogen from
the -OH group to DPPH
·
. The formation of an
α
-tocopherol radical is stabilized by the
delocalization of the solitary electron on the structure of the aromatic ring (Figure 8). These
compounds are highly lipophilic and more active in lipoproteins and membranes. The
most crucial antioxidant effect is the inhibition of lipid peroxidation, which scavenges lipid
peroxyl radicals, resulting in lipid hydroperoxides and the formation of a tocopheroxyl
radical [14].
Processes 2023, 11, x FOR PEER REVIEW 10 of 21
Figure 8. The reaction mechanism between DPPH free radicals and α-tocopherol as a commonly
used food additive.
Similarly, the phenolic structure of usnic acid, a lichen metabolite, is highly suitable
for interaction with DPPH·. After the mutual effect of usnic acid and DPPH radicals,
DPPH radicals readily convert to DPPH-H by accepting an electron or hydrogen radical
from usnic acid. This possible interaction pattern between usnic acid and DPPH· is shown
in Figure 9. The phenolic group in usnic acid has two -OH units. The withdrawal of H
atoms from phenolic -OH groups by a reactive radical can occur quite easily. It can remove
three DPPH radicals due to the resonance structures that occur with the delocalization of
electrons in the phenolic ring of usnic acid [99]. In this study, the interaction between usnic
acid and DPPH· is documented and summarized in Figure 9. According to our best
knowledge, a phenol group easily stabilizes radicals formed on the phenolic carbon with
its resonance structures. The phenolic group in the usnic acid molecule has two hydroxyl
groups. It is quite easy to withdraw H atoms from phenolic -OH groups. It can acquire a
triradical one by inactivating three DPPH molecules using resonance structures, as shown
in Figure 8.
Figure 8.
The reaction mechanism between DPPH free radicals and
α
-tocopherol as a commonly
used food additive.
Similarly, the phenolic structure of usnic acid, a lichen metabolite, is highly suitable
for interaction with DPPH
·
. After the mutual effect of usnic acid and DPPH radicals, DPPH
radicals readily convert to DPPH-H by accepting an electron or hydrogen radical from usnic
acid. This possible interaction pattern between usnic acid and DPPH
·
is shown in Figure 9.
The phenolic group in usnic acid has two -OH units. The withdrawal of H atoms from
phenolic -OH groups by a reactive radical can occur quite easily. It can remove three DPPH
radicals due to the resonance structures that occur with the delocalization of electrons in
the phenolic ring of usnic acid [
99
]. In this study, the interaction between usnic acid and
DPPH
·
is documented and summarized in Figure 9. According to our best knowledge, a
phenol group easily stabilizes radicals formed on the phenolic carbon with its resonance
structures. The phenolic group in the usnic acid molecule has two hydroxyl groups. It is
quite easy to withdraw H atoms from phenolic -OH groups. It can acquire a triradical one
by inactivating three DPPH molecules using resonance structures, as shown in Figure 8.

Processes 2023,11, 2248 10 of 20
Processes 2023, 11, x FOR PEER REVIEW 11 of 21
Figure 9. DPPH radical scavenging mechanism by usnic acid as a lichen metabolite.
Phenolic compounds with very electron-rich substituents are excellent H donors.
Therefore, phenolic compounds have the ability to quickly extinguish all types of radicals,
especially ROO radicals. In this context, the possible scavenging mechanisms of DPPH
radicals in some phenolic compounds, which have a very rich biological activity spectrum
and are recorded in the literature, have been predicted and clarified [14].
7. DPPH Radical Scavenging Assays
Radical chain reactions serve as a common mechanism for lipid peroxidation. Radical
scavengers increase the stability and quality of food products by ending peroxidation
chain reactions. For this purpose, radical scavenger molecules interact directly with per-
oxide radicals and scavenge them quickly [100]. Free radical scavenging has a known
mechanism where antioxidants directly inhibit lipid peroxidation. This method is a stand-
ard, most widely used, and very fast and practical technique in antioxidant activity stud-
ies. Radical removal activity has great importance due to the hazardous effects of free
radicals in foods and pharmaceutical systems. Many assays are used for the evaluation of
the antioxidant activity of herbal extracts or phenolics. Different radicals and methods are
used for antioxidant analyses and the determination of the final product of oxidation.
ABTS+, DPPH·, DMPD+, or O2− radical removal methods are the most commonly used
spectrophotometric methods for this purpose. When antioxidants are added to these rad-
icals, color removal occurs with a mechanism that reverses the formation of DPPH·,
ABTS+, and DMPD+ cations [1,14].
DPPH•+ AH → DPPH+A
•
Figure 9. DPPH radical scavenging mechanism by usnic acid as a lichen metabolite.
Phenolic compounds with very electron-rich substituents are excellent H donors.
Therefore, phenolic compounds have the ability to quickly extinguish all types of radicals,
especially ROO radicals. In this context, the possible scavenging mechanisms of DPPH
radicals in some phenolic compounds, which have a very rich biological activity spectrum
and are recorded in the literature, have been predicted and clarified [14].
7. DPPH Radical Scavenging Assays
Radical chain reactions serve as a common mechanism for lipid peroxidation. Radical
scavengers increase the stability and quality of food products by ending peroxidation chain
reactions. For this purpose, radical scavenger molecules interact directly with peroxide
radicals and scavenge them quickly [
100
]. Free radical scavenging has a known mechanism
where antioxidants directly inhibit lipid peroxidation. This method is a standard, most
widely used, and very fast and practical technique in antioxidant activity studies. Radical
removal activity has great importance due to the hazardous effects of free radicals in foods
and pharmaceutical systems. Many assays are used for the evaluation of the antioxidant
activity of herbal extracts or phenolics. Different radicals and methods are used for antiox-
idant analyses and the determination of the final product of oxidation. ABTS
+
, DPPH
·
,
DMPD
+
, or O
2−
radical removal methods are the most commonly used spectrophotometric
methods for this purpose. When antioxidants are added to these radicals, color removal
occurs with a mechanism that reverses the formation of DPPH
·
, ABTS
+
, and DMPD
+
cations [1,14].

Processes 2023,11, 2248 11 of 20
DPPH•+AH →DPPH2+A•
ABTS•++AH →ABTS++A•
DMPD•++AH →DMPD++A•
These three radical scavenging methods are extremely fast, requiring no expensive
reagents or sophisticated instruments. Preparing and analyzing a sample takes half an hour
and requires very little labor. These methods, which have high sensitivity, are very easy to
use. The analysis of antioxidant activity in many samples can be performed quickly and
spontaneously [14].
7.1. Evaluation of DPPH Radical Scavenging
DPPH radical scavenging ability is determined mostly in organic solvents such as
methanol or ethanol by measuring the absorbance drop at 517 nm [
101
]. The use of
methanol is not preferred because of its toxic properties. Analyses were performed with
a UV-vis spectrophotometer in 1 mL or 3 mL cuvettes. For this purpose, a stock solution
of 10
−3
M DPPH radicals in ethanol or methanol was freshly prepared before analysis. To
prepare the DPPH solution, 3 mL of the stock solution was diluted to 50 mL with methanol
in a volumetric flask and protected from light with aluminum foil. Absorbance values
were set to 1.00
±
0.200. Then, 3 mL of DPPH working solution was transferred to the
0.5 mL extract, mixed, and left in the dark for 30 min. The purple color disappears when
an antioxidant agent is present in the reaction medium. A reference sample containing
0.5 mL of solvent was similarly prepared. A newly prepared DPPH radical solution
shows maximal absorption at 517 nm. All analyses were carried out in 3 replicates, and
absorbance was recorded at 517 nm. The blank is the reaction mixture that does not contain
test compounds [1,102].
7.2. Evaluation of DPPH Radical Scavenging as TEAC
Another way to evaluate the results of DPPH’s radical-removing ability is to express
them as Trolox equivalent antioxidant capacity (TEAC). For this purpose, the radical-
removing activity (RSA) of Trolox standard solution at different concentrations is deter-
mined. In this assay, Trolox, as a standard radical scavenger compound, is interpolated into
a dose–response curve. Then, using these values (%RSA)—(Trolox;
µ
M/L), a calibration
curve is prepared. For herbal extracts or chemicals prepared at different concentrations, the
TEAC (
µ
M/L) was calculated, and Trolox equivalent antioxidant capacities were calculated
using the linear regression equation obtained in the linearity range (0.01–0.05 µM/L) [14].
7.3. The Importance of the IC50 Value in DPPH Radical Scavenging Activity
Different antioxidant concentrations are used to determine the antioxidant concentra-
tion that scavenges 50% of the initial DPPH radicals in a specific but arbitrary time interval.
This concentration was also referred to as “EC
50
”, short for “efficient concentration” or
sometimes as “IC
50
”, short for “inhibitory concentration”. Indeed, this EC
50
designation
has found appropriate scientific use in drug testing under a different name “LD
50
”. These
terms became widely accepted as “IC
50
” to indicate the practicality of antioxidant testing
using DPPH radicals. The lower the IC
50
values, the higher the DPPH radical-removing
ability of the antioxidants. In this context, the IC
50
value is widely used in biochemistry to
compare the radical scavenging capacities of different antioxidants [
103
]. The IC
50
values
quantitatively describe the radical scavenging affinity. For all these reasons, the IC
50
value
is one of the most practical ways to evaluate DPPH radical scavenging affinities. The radical
scavenging activity (RSA) of maca extracts was calculated using the following equation:
RSA(%)=(Ac −As)
Ac ×100 or RSA(%)=1−Ac
As×100

Processes 2023,11, 2248 12 of 20
where A
c
is the absorbance at 517 nm of the control sample, and A
s
is the absorbance at
517 nm that contains the test sample, including plant extracts or pure compounds. The
IC
50
was calculated from the graph plotting scavenging percentage against test sample
concentration (
µ
g/mL). DPPH radicals decrease significantly upon exposure to radical
remover [104].
7.4. Scope of DPPH Radical Scavenging Applications
DPPH radical scavenging is a popular spectrophotometric method that has a wide
application area and is used for determining the antioxidant capacity of beverages, pure
substances, foods, and herbal extracts. This method is simple, sensitive, fast, and repro-
ducible, making it the most convenient and common radical removal method for evaluating
the antioxidant capacity of compounds and herbal extracts. For this purpose, the IC
50
values of herbal extracts and pure compounds in recent studies on DPPH radical removal
are given in Tables 1and 2.
Table 1. Half maximal inhibition concentration (IC50,µg/mL) of different antioxidant molecules.
Antioxidants DPPH•Scavenging (IC50,µg/mL) References
Curcumin 34.86 [105]
Resveratrol 17.80 [106]
Eugenol 16.06 [107]
Coumestrol 25.95 [108]
Magnofluorine 10.58 [109]
Hederin 69.40 [110]
Hederasaponin 82.40 [110]
Hederacolchiside 73.50 [110]
Hederagenin 28.50 [111]
Morphine 56.82 [112]
Uric acid 17.80 [113]
Caffeic acid 10.64 [114]
Usnic acid 49.50 [109]
Tannic acid 23.65 [115]
Rosmarinic acid 3.07 [116]
L-Carnitine 58.90 [117]
L-Dopa 12.41 [18]
L-Tyrosine 43.86 [18]
L-Adrenaline 30.60 [118]
Propofol 16.23 [26]
Dipropofol 31.29 [24]
Silymarin 20.80 [119]
Cepharanthine 22.20 [43]
Fangchinoline 6.40 [43]
CAPE 3.30 [47]
Taxifoline 77.00 [120]
Cynarine 3.98 [32]
Olivetol 17.77 [121]
Nordihydroguaiaretic acid 6.60 [122]
(-)-Secoisolariciresinol 14.14 [122]
Secoisolariciresinol diglycoside 16.97 [122]
α-(-)-Conidendrin 23.29 [122]
Phillyrin 11.75 [111]
Pinoresinol-β-D-glycoside 19.60 [111]
Pinoresinol di-β-D-glycoside 26.52 [111]
Ligustroside 12.00 [123]
Oleuropein 57.50 [123]
Pelargonin 67.73 [63]
Silychristin 86.16 [63]
Callistephin 20.64 [63]
Oenin 16.72 [63]
Malvin 21.36 [63]
Arachidonoyl dopamine 84.10 [63]

Processes 2023,11, 2248 13 of 20
Table 2.
Half maximal DPPH radical scavenging concentration (IC
50
,
µ
g/mL) of water and ethanol
extracts of different antioxidant plants.
Antioxidant Plants
DPPH•Scavenging (IC50,µg/mL)
Water Extract Ethanol Extract References
Anise (Pimpinella anisum) 11.74 18.12 [124]
Fennel (Foeniculum vulgare) 263.21 343.41 [125]
Sage (Salvia pilifera) 30.95 28.92 [126]
Giant fennel (Ferula orientalis) 88.60 346.50 [127]
Thyme (Thymus vulgaris) 13.40 12.10 [59]
Clove (Eugenia caryophylata) 48.39 40.54 [128]
Lavender (Lavandula stoechas) 66.67 60.00 [128]
Black pepper (Piper nigrum) 68.18 78.13 [129]
Juniper (Juniperus communis) 22.27 23.81 [130]
Bay (Laurus nobilis) 38.46 32.60 [131]
Basil (Ocimum basilicum) 44.64 38.46 [132]
Lemon balm (Melissa officinalis) 31.40 202.70 [10]
Cauliflower (Brassica oleracea) 29.30 23.21 [133]
Liquorice (Glycyrrhiza glabra) 52.2 54.4 [134]
Cherry stem (Cerasus avium) 23.38 17.36 [135]
Galanga (Alpinia officinarum) 14.75 31.51 [136]
Ginger (Zingiber offcinale) 16.20 43.80 [137]
Flaxseed (Linum usitatissimum) 53.30 49.50 [138]
Cinnamon (Cinnamomum verum) 21.25 15.71 [139]
Pennyroyal (Mentha pulegium) 18.52 16.92 [140]
Avocado (Folium perseae) 601.00 240.40 [141]
Kınkor (Ferulago stellata) 57.80 34.70 [142]
Bindweed (Convulvulus betonicifolia) 346.50 77.00 [143]
Mint (Cyclotrichium leucotrichum) 28.85 23.74 [144]
Pomegranate (Punica granatum) 31.50 16.100 [145]
Achillea pseudoaleppica 25.57 23.24 [146]
Acantholimon caryophyllaceum 69.30 19.80 [147]
Salvia eriophora 9.94 9.21 [148]
Lecokia cretica 78.13 77.32 [149]
Stachys annua 8.90 7.80 [150]
Astragalus alopecurus 115.53 99.02 [151]
Verbascum speciousum 173.25 24.75 [152]
Cyclotrichium niveum 9.17 14.45 [153]
Nettle (Urtica dioica) 81.08 - [154]
Cornelian cherry (Cornus mas) 91.77 - [155]
Kiwifruit (Actinidia deliciosa) 83.40 - [156]
Cranberries (Vaccinium macrocarpon) 86.63 - [157]
Spearmint (Mentha spicata) - 97.82 [158]
Yarrow (Achillea cucullata) 132.55 [70]
Sahlep (Dactylorhiza osmanica) - 86.63 [159]
8. Limitations of the DPPH Assay
In DPPH removal activity, it is very important to convert the moles of DPPH lost
by using the change in absorbance, Beer’s law, and damping
ε
values of 10,900–12,500.
Another important point is that this method does not detect reaction rates and ignores some
crucial information in the reaction curves [
160
]. The reactions of DPPH and antioxidants
appear to be complex when changes in absorbance are continuously monitored. Although
the reaction curves were similar to ABTS
•+
removal, there were some differences when
comparing the studied phenolic compounds [
85
]. In DPPH radical scavenging, antioxidants
can react with DPPH radicals by very fast electron transfer and slow hydrogen atom
transfer. Although electron transfer is quite fast, it is slower than ABTS
•+
’s reactions with
antioxidants due to the difficult accessibility of phenolic compounds to the radical site of
the DPPH molecule. This barrier of access inhibits all reactions, particularly the hydrogen
transfer, which is necessary for the formation of a hydrogen-bonded complex between the

Processes 2023,11, 2248 14 of 20
α
-C—H radical and the N lone pair required [
161
,
162
]. Complex molecules get in the way
of each other more easily and block access to DPPH radicals at low concentrations and
strongly block the reaction at high concentrations. Additionally, methyl alcohol, which is
generally used as a solvent for the DPPH method, strongly binds H atoms and inhibits HAT
processes
[163–165]
. However, when water is added to the reaction, it disrupts the bonding
and facilitates hydrogen atom transfer. Any test compound with H-atom transfer capability
will increase the reaction rate. In the same manner, electron transfer is pH-dependent,
with speed increasing with pH and ionization degree, while HAT is pH-independent. The
dominant mechanism of a test compound can be evaluated by reacting it with DPPH
radicals in methanol and 50% methanol, where the water phase is buffered to a pH range
from acidic to alkaline [85,166,167].
9. Conclusions
Antioxidant compounds play a vital role in reducing oxidative damage caused by
ROS. The DPPH radical scavenging ability of the compounds used for this purpose can
be extremely valuable for antioxidant profiles. DPPH radical removal is one of the most
widely applied and used methods in food and pharmaceutical applications. As a result of
the review, it has been reported that the DPPH method gives a better response for mostly
phenolic compounds and then for compounds with limited polarity. In the case of polar
and phenolic compounds, adding water to the reaction medium, that is, aqueous methanol,
gives better results. When testing low-polarity compounds, ethyl acetate with a radical is
suitable. All results show that DPPH reaction rates depend on the steric accessibility of the
radical site rather than the chemical properties of the tested antioxidant compounds. The
rate at which DPPH reacts with antioxidants depends on the varying ratios of mixed SET
and HAT mechanisms. The reaction mechanisms of DPPH
·
scavenging and responses are
modified by many environmental and experimental factors.
The interaction of antioxidants with DPPH radicals was less than the total activity
of the individual compounds. It clearly shows that radical scavenging by extracts from
mixtures is actually suppressed in the DPPH assay. Therefore, the assay does not fully
demonstrate potential radical scavenging in cells or food, and steric interferences do not
account for all synergisms or antagonisms in the test.
Author Contributions: Investigation, writing—original draft preparation and writing—review and
editing, ˙
I.G. and S.H.A. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: Data are available in a publicly accessible repository.
Acknowledgments: I. Gulcin would like to extend his sincere appreciation to the Turkish Academy
of Sciences (TÜBA). S. H. Alwasel would like to extend his sincere appreciation to the Researchers
Supporting Project (RSP-2023/59), King Saud University, Saudi Arabia.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Gulcin, I. Antioxidants and antioxidant methods-An updated overview. Arch. Toxicol. 2020,94, 651–715. [PubMed]
2.
Buyukokuroglu, M.E.; Gulcin, I.; Oktay, M.; Küfrevioglu, O.I.
In vitro
antioxidant properties of dantrolene sodium. Pharmacol.
Res. 2001,44, 491–494. [CrossRef] [PubMed]
3. Ionita, P. The chemistry of DPPH·free radical and congeners. Int. J. Mol. Sci. 2021,22, 1545. [CrossRef] [PubMed]
4.
Gulcin, I.; Mshvildadze, V.; Gepdiremen, A.; Elias, R. Screening of antioxidant and antiradical activity of monodesmosides and
crude extract from Leontice smirnowii Tuber. Phytomedicine 2006,13, 343–351. [CrossRef]
5.
Altay, A.; Tohma, H.; Durmaz, L.; Taslimi, P.; Korkmaz, M.; Gulcin, I.; Koksal, E. Preliminary phytochemical analysis and
evaluation of
in vitro
antioxidant, antiproliferative, antidiabetic and anticholinergics effects of endemic Gypsophila taxa from
Turkey. J. Food Biochem. 2019,43, e12908. [CrossRef]
6.
Munteanu, I.G.; Apetrei, C. Analytical methods used in determining antioxidant activity: A review. Int. J. Mol. Sci.
2021
,22, 3380.
[CrossRef]

Processes 2023,11, 2248 15 of 20
7.
Balaydın, H.T.; Gulcin, I.; Menzek, A.; Goksu, S.; Sahin, E. Synthesis and antioxidant properties of diphenylmethane derivative
bromophenols including a natural product. J. Enzym. Inhib. Med. Chem. 2010,25, 685–695. [CrossRef]
8.
Kedare, S.B.; Sing, R.P. Genesis and development of DPPH method of antioxidant assay. J. Food Sci. Technol.
2011
,48, 412–422.
[CrossRef]
9.
Cetinkaya, Y.; Gocer, H.; Menzek, A.; Gulcin, I. Synthesis and antioxidant properties of (3,4-dihydroxyphenyl) (2,3,4-
trihydroxyphenyl)methanone and its derivatives. Arch. Pharm. 2012,345, 323–334. [CrossRef]
10.
Koksal, E.; Bursal, E.; Dikici, E.; Tozoglu, F.; Gulcin, I. Antioxidant activity of Melissa officinalis leaves. J. Med. Plants Res.
2011
,5,
217–222.
11.
Tohma, H.; Altay, A.; Koksal, E.; Gören, A.C.; Gulcin, I. Measurement of anticancer, antidiabetic and anticholinergic properties of
sumac (Rhus coriaria)-Analysis of its phenolic compounds by LC-MS/MS. J. Food Meas. Charac. 2019,13, 1607–1619. [CrossRef]
12. Rodrigo, R. Oxidative Stress and Antioxidants: Their Role in Human Diseases; Nova: New York, NY, USA, 2009; pp. 9–10.
13.
Christodoulou, M.C.; Orellana Palacios, J.C.; Hesami, G.; Jafarzadeh, S.; Lorenzo, J.M.; Domínguez, R.; Moreno, A.; Hadidi, M.
Spectrophotometric methods for measurement of antioxidant activity in food and pharmaceuticals. Antioxidants
2022
,11, 2213.
[CrossRef] [PubMed]
14. Gulcin, I. Antioxidant activity of food constituents: An overview. Arch. Toxicol. 2012,86, 345–391. [CrossRef] [PubMed]
15.
Sehitoglu, M.H.; Han, H.; Kalin, P.; Gucin, I.; Ozkan, A.; Aboul-Enein, H.Y. Pistachio (Pistacia vera L.) gum: A potent inhibitor of
reactive oxygen species. J. Enzyme Inhib. Med. Chem. 2015,30, 264–269. [CrossRef] [PubMed]
16.
Gulcin, I.; Bursal, E.; Sehitoglu, H.M.; Bilsel, M.; Goren, A.C. Polyphenol contents and antioxidant activity of lyophilized aqueous
extract of propolis from Erzurum, Turkey. Food Chem. Toxicol. 2010,48, 2227–2238. [CrossRef]
17.
Karagecili, H.; Yılmaz, M.A.; Erturk, A.; Kızıltas, H.; Güven, L.; Alwasel, S.H.; Gulcin, I. Comprehensive metabolite profiling
of Berdav propolis using LC-MS/MS: Determination of antioxidant, anticholinergic, antiglaucoma, and antidiabetic effects.
Molecules 2023,28, 1739. [CrossRef]
18.
Gulcin, I. Comparison of
in vitro
antioxidant and antiradical activities of L-Tyrosine and L-Dopa. Amino Acids
2007
,32, 431–843.
[CrossRef]
19.
Aytac, S.; Gundogdu, O.; Bingol, Z.; Gulcin, I. Synthesis of Schiff bases containing phenol ring and investigation of their
antioxidant capacity, anticholinesterase, butyrylcholinesterase and carbonic anhydrase inhibition properties. Pharmaceutics
2023
,
15, 779.
20.
Elmastas, M.; Turkekul, I.; Ozturk, L.; Gulcin, I.; Isıldak, O.; Aboul-Enein, H.Y. The antioxidant activity of two wild edible
mushrooms (Morchella vulgaris and Morchella esculanta). Comb. Chem. High Throughput Screen. 2006,9, 443–448. [CrossRef]
21.
Bae, J.H.; Park, Y.J.; Namiesnik, J.; Gulcin, I.; Kim, T.C.; Kim, H.C.; Heo, B.G.; Gorinstein, S.; Ku, Y.G. Effects of artificial lighting
on bioactivity of sweet red pepper (Capsicum annuum L.). Int. J. Food Sci. Technol. 2016,51, 1378–1385. [CrossRef]
22.
Rezai, M.; Bayrak, C.; Taslimi, P.; Gulcin, I.; Menzek, A. The first synthesis, antioxidant and anticholinergic activities of
1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols including natural products. Turk. J. Chem. 2018,42, 808–825.
23.
Talaz, O.; Gulcin, I.; Goksu, S.; Saracoglu, N. Antioxidant activity of 5,10-dihydroindeno[1,2-b]indoles containing substituents on
dihydroindeno part. Bioorg. Med. Chem. 2009,17, 6583–6589. [CrossRef]
24.
Gulcin, I.; Dastan, A. Synthesis of dimeric phenol derivatives and determination of
in vitro
antioxidant and radical scavenging
activities. J. Enzym. Inhib. Med. Chem. 2007,22, 685–695. [CrossRef]
25.
Oztaskin, N.; Kaya, R.; Maras, A.; Sahin, E.; Gulcin, I.; Goksu, S. Synthesis and characterization of novel bromophenols:
Determination of their anticholinergic, antidiabetic and antioxidant activities. Bioorg. Chem.
2019
,87, 91–102. [CrossRef]
[PubMed]
26.
Gulcin, I.; Alici, H.A.; Cesur, M. Determination of
in vitro
antioxidant and radical scavenging activities of propofol. Chem. Pharm.
Bull. 2005,53, 281–285. [CrossRef] [PubMed]
27.
Gulcin, I.; Buyukokuroglu, M.E.; Oktay, M.; Kufrevioglu, O.I. On the
in vitro
antioxidant properties of melatonin. J. Pineal Res.
2002,33, 167–171. [CrossRef]
28.
Lourenco, S.C.; Moldao-Martins, M.; Alves, V.D. Antioxidants of natural plant origins: From sources to food industry applications.
Molecules 2019,24, 4132. [CrossRef]
29.
Gulcin, I.; Sat, I.G.; Beydemir, S.; Kufrevioglu, O.I. Evaluation of the
in vitro
antioxidant properties of extracts of broccoli
(Brassica oleracea L.). Ital. J. Food Sci. 2004,16, 17–30.
30.
Hamad, H.O.; Alma, M.H.; Gulcin, I.; Yılmaz, M.A.; Karaogul, E. Evaluation of phenolic contents and bioactivity of root and
nutgall extracts from Iraqian Quercus infectoria Olivier. Rec. Nat. Prod. 2017,11, 205–210.
31.
Gulcin, I. Measurement of antioxidant ability of melatonin and serotonin by the DMPD and CUPRAC methods as trolox
equivalent. J. Enzym. Inhib. Med. Chem. 2008,23, 871–876. [CrossRef]
32.
Topal, M.; Gocer, H.; Topal, F.; Kalin, P.; Polat Kose, P.; Gulcin, I.; Cakmak, K.C.; Kucuk, M.; Durmaz, L.; Goren, A.C.; et al.
Antioxidant, antiradical and anticholinergic properties of cynarin purified from the illyrian thistle (Onopordum illyricum L.). J.
Enzym. Inhib. Med. Chem. 2016,31, 266–275. [CrossRef]
33.
Cakmakcı, S.; Topdas, E.F.; Kalın, P.; Han, H.; Sekerci, P.; Polat Kose, L.; Gulcin, I. Antioxidant capacity and functionality of
oleaster (Elaeagnus angustifolia L.) flour and crust in a new kind of fruity ice cream. Int. J. Food Sci. Technol.
2015
,50, 472–481.
[CrossRef]
34. Fenton, H.J.H. Oxidation of tartaric acid in the presence of iron. J. Chem. Soc. Trans. 1984,65, 899–910.

Processes 2023,11, 2248 16 of 20
35.
Haber, F.; Weiss, J. The catalytic decomposition of hydrogen peroxide by iron salts. Proc. Roy. Soc. Lond. Ser. A
1934
,147, 332–351.
36.
Gulcin, I.; Elias, R.; Gepdiremen, A.; Boyer, L.; Koksal, E. A comparative study on the antioxidant activity of fringe tree
(Chionanthus virginicus L.) extracts. Afr. J. Biotechnol. 2007,6, 410–418.
37.
Gulcin, I.; Berashvili, D.; Gepdiremen, A. Antiradical and antioxidant activity of total anthocyanins from Perilla pankinensis decne.
J. Ethnopharmacol. 2005,101, 287–293. [CrossRef] [PubMed]
38.
Gulcin, I.; Buyukokuroglu, M.E.; Kufrevioglu, O.I. Metal chelating and hydrogen peroxide scavenging effects of melatonin. J.
Pineal Res. 2003,34, 278–281. [CrossRef]
39.
Aksu, K.; Ozgeri¸s, B.; Taslimi, P.; Naderi, A.; Gulcin, I.; Goksu, S. Antioxidant activity, acetylcholinesterase and carbonic anhydrase
inhibitory properties of novel ureas derived from phenethylamines. Arch. Pharm. 2016,349, 944–954.
40.
Gulcin, I.; Oktay, M.; Küfrevioglu, O.I.; Aslan, A. Determinations of antioxidant activity of lichen Cetraria islandica (L.) Ach. J.
Ethnopharmacol. 2002,79, 325–329. [PubMed]
41.
Buyukokuroglu, M.E.; Gulcin, I.
In vitro
antioxidant and antiradical properties of Hippophae rhamnoides L. Pharmacog. Mag.
2009
,
4, 189–195.
42.
Gulcin, I.; Beydemir, S.; Hisar, O.; Koksal, E.; Reiter, R.J. Melatonin administration increases antioxidant enzymes activities and
reduce lipid peroxidation in the rainbow trout (Oncorhynchus mykiss, Walbaum) erythrocytes. Turk. J. Vet. Anim. Sci.
2009
,33,
241–245.
43.
Gulcin, I.; Elias, R.; Gepdiremen, A.; Chea, A.; Topal, F. Antioxidant activity of bisbenzylisoquinoline alkaloids from Stephania
rotunda: Cepharanthine and fangchinoline. J. Enzym. Inhib. Med. Chem. 2010,25, 44–53. [CrossRef] [PubMed]
44.
Gulcin, I.; Topal, F.; Ozturk Sarikaya, S.B.; Bursal, E.; Gören, A.C.; Bilsel, M. Polyphenol contents and antioxidant properties of
medlar (Mespilus germanica L.). Rec. Nat. Prod. 2011,5, 158–175.
45.
Gulcin, I.; Topal, F.; Cakmakcı, R.; Goren, A.C.; Bilsel, M.; Erdogan, U. Pomological features, nutritional quality, polyphenol
content analysis and antioxidant properties of domesticated and three wild ecotype forms of raspberries (Rubus idaeus L.). J. Food
Sci. 2011,76, C585–C593. [CrossRef]
46.
Shantabi, L.; Jagetia, G.C.; Ali, M.A.; Singh, T.T.; Devi, S.V. Antioxidant potential of Croton caudatus leaf extract
in vitro
.Transl.
Med. Biotechnol. 2014,2, 1–15.
47.
Gocer, H.; Gulcin, I. Caffeic acid phenethyl ester (CAPE): Correlation of structure and antioxidant properties. Int. J. Food Sci. Nutr.
2011,62, 821–825. [CrossRef]
48.
Gülçin, ˙
I.; Beydemir, S.; Topal, F.; Gagua, N.; Bakuridze, A.; Bayram, R.; Gepdiremen, A. Apoptotic, antioxidant and antiradical
effects of majdine and isomajdine from Vinca herbacea Waldst. and kit. J. Enzym. Inhib. Med. Chem. 2012,27, 587–594. [CrossRef]
49.
Gulcin, I.; Elmastas, M.; Aboul-Enein, H.Y. Antioxidant activity of clove oil-A powerful antioxidant source. Arab. J. Chem.
2012
,5,
489–499. [CrossRef]
50.
Gulcin, I.; Beydemir, S. Phenolic compounds as antioxidants: Carbonic anhydrase isoenzymes inhibitors. Mini Rev. Med. Chem.
2013,13, 408–430. [PubMed]
51.
Shahidi, F.; Zhong, Y. Revisiting the polar paradox theory: A critical overview. J. Agric. Food Chem.
2011
,59, 3499–3504. [CrossRef]
52.
Gocer, H.; Akıncıoglu, A.; Oztaskin, N.; Goksu, S.; Gulcin, I. Synthesis, antioxidant and antiacetylcholinesterase activities of
sulfonamide derivatives of dopamine related compounds. Arch. Pharm. 2013,346, 783–792. [CrossRef]
53. Aksu, K.; Topal, F.; Gulcin, I.; Tumer, F.; Goksu, S. Acetylcholinesterase inhibitory and antioxidant activities of novel symmetric
sulfamides derived from phenethylamines. Arch. Pharm. 2015,348, 446–455. [CrossRef] [PubMed]
54.
Halliwell, B. Oxidative stress, nutrition and health. Experimental strategies for optimization of nutritional antioxidant intake in
humans. Free Radic. Res. 1996,25, 57–74. [CrossRef] [PubMed]
55.
Isık, M.; Korkmaz, M.; Bursal, E.; Gulcin, I.; Koksal, E.; Tohma, H. Determination of antioxidant properties of Gypsophila bitlisensis.
Int. J. Pharmacol. 2015,11, 366–371. [CrossRef]
56.
Boztas, M.; Taslimi, P.; Yavari, M.A.; Gulcin, I.; Sahin, E.; Menzek, A. Synthesis and biological evaluation of bromophenol
derivatives with cyclopropyl moiety: Ring opening of cyclopropane with monoester. Bioorg. Chem. 2019,89, 103017. [CrossRef]
57.
Garibov, E.; Taslimi, P.; Sujayev, A.; Bingol, Z.; Cetinkaya, S.; Gulcin, ˙
I.; Beydemir, S.; Farzaliyev, V.; Alwasel, S.H.; Supuran,
C.T. Synthesis of 4,5-disubstituted-2-thioxo-1,2,3,4-tetrahydropyrimidines and investigation of their acetylcholinesterase, bu-
tyrylcholinesterase, carbonic anhydrase I/II inhibitory and antioxidant activities. J. Enzym. Inhib. Med. Chem.
2016
,31, 1–9.
[CrossRef]
58.
Ekinci Akdemir, F.N.; Gulcin, I.; Alwasel, S. A comparative study on the antioxidant effects of hesperidin and ellagic acid against
skeletal muscle ischemia/reperfusion injury. J. Enzym. Inhib. Med. Chem. 2016,31, 114–118. [CrossRef] [PubMed]
59.
Koksal, E.; Bursal, E.; Gulcin, I.; Korkmaz, M.; Caglayan, C.; Goren, A.C.; Alwasel, S.H. Antioxidant activity and polyphenol
content of Turkish thyme (Thymus vulgaris) monitored by LC-MS/MS. Int. J. Food Prop. 2017,20, 514–525. [CrossRef]
60.
Koksal, E.; Tohma, S.H.; Kılıc, O.; Alan, Y.; Aras, A.; Gulcin, I.; Bursal, E. Assessment of antimicrobial and antioxidant activities of
Nepeta trachonitica-Analysis of its phenolic compounds using HPLC-MS/MS. Sci. Pharmaceut. 2017,15, 85. [CrossRef]
61. Shahidi, F.; Zhong, Y. Measurement of antioxidant activity. J. Funct. Foods 2015,18, 757–781. [CrossRef]
62.
Oztaskın, N.; Taslimi, P.; Maras, A.; Goksu, S.; Gulcin, I. Novel antioxidant bromophenols with acetylcholinesterase, butyryl-
cholinesterase and carbonic anhydrase inhibitory actions. Bioorg. Chem. 2017,74, 104–114. [CrossRef] [PubMed]
63.
Huyut, Z.; Beydemir, S.; Gulcin, I. Antioxidant and antiradical properties of some flavonoids and phenolic compounds. Biochem.
Res. Int. 2017,2017, 7616791. [CrossRef] [PubMed]

Processes 2023,11, 2248 17 of 20
64.
Bulut, N.; Koçyi˘git, U.M.; Gecibesler, I.H.; Dastan, T.; Karci, H.; Taslimi, P.; Durna Dastan, S.; Gulcin, I.; Cetin, A. Synthesis of
some novel pyridine compounds containing bis-1,2,4-triazole moiety and investigation of their antioxidant properties, carbonic
anhydrase and acetylcholinesterase enzymes inhibition profiles. J. Biochem. Mol. Toxicol. 2018,32, e22006.
65.
Taslimi, P.; Sujayev, E.; Turkan, F.; Garibov, E.; Huyut, Z.; Farzaliyev, F.; Mamedova, S.; Gulcin, I. Synthesis and investigation of
the conversion reactions of pyrimidine-thiones with nucleophilic reagent and evaluation of their acetylcholinesterase, carbonic
anhydrase inhibition and antioxidant activities. J. Biochem. Mol. Toxicol. 2018,32, e22019.
66.
Elmastas, M.; Celik, S.M.; Genc, N.; Aksit, H.; Erenler, R.; Gulcin, I. Antioxidant activity of an Anatolian herbal tea-Origanum
minutiflorum: Isolation and characterization of its secondary metabolites. Int. J. Food Prop. 2018,21, 374–384. [CrossRef]
67.
Gulcin, I.; Taslimi, P.; Aygun, A.; Sadeghian, N.; Bastem, E.; Kufrevioglu, O.I.; Turkan, F.; Sen, F. Antidiabetic and antiparasitic
potentials: Inhibition effects of some natural antioxidant compounds on
α
-glycosidase,
α
-amylase and human glutathione
S-transferase enzymes. Int. J. Biol. Macromol. 2018,119, 741–746. [CrossRef]
68.
Askın, H.; Yılmaz, B.; Gulcin, I.; Taslimi, P.; Bakırcı, S.; Yıldız, M.; Kandemir, N. Antioxidant activities of aqueous extract from Iris
taochia and identification of its natural chemical compounds pharmacognosy and phytochemistry. Ind. J. Pharm. Sci.
2018
,80,
802–812. [CrossRef]
69.
Maharramova, G.; Taslimi, P.; Sujayev, A.; Farzaliyev, F.; Durmaz, L.; Gulcin, I. Synthesis, characterization, antioxidant, antidia-
betic, anticholinergic, and antiepileptic properties of novel N-substituted tetrahydropyrimidines based on phenylthiourea. J.
Biochem. Mol. Toxicol. 2018,32, e22221. [CrossRef]
70.
Eruygur, N.; Atas, M.; Tekin, M.; Taslimi, P.; Koçyigit, U.M.; Gulcin, I.
In vitro
antioxidant, antimicrobial, anticholinesterase and
antidiabetic activities of Turkish endemic Achillea cucullata (Asteraceae) from ethanol extract. S. Afr. J. Bot.
2019
,120, 141–145.
[CrossRef]
71.
Floegel, A.; Kim, D.O.; Chung, S.J.; Koo, S.I.; Chun, O.K. Comparison of ABTS/DPPH assays to measure antioxidant capacity in
popular antioxidant-rich US foods. J. Food Comp. Anal. 2011,24, 1043–1048.
72. Shahidi, F.; Wanasundara, P.D. Phenolic antioxidants. Crit. Rev. Food Sci. Nutr. 1992,32, 67–103. [CrossRef]
73.
Çakmakcı, S.; Oz, E.; Cakıroglu, K.; Polat, A.; Gulcin, I.; Ilgaz, S.; Cheraghi, K.S.; Ozhamamcı, I. Probiotic shelf life, antioxidant,
sensory, physical and chemical properties of yogurts produced with Lactobacillus acidophilus and green tea powder. Kafkas Univ.
Vet. Fak. Der. 2019,25, 673–682.
74.
Buldurun, K.; Turan, N.; Bursal, E.; Mantarcı, A.; Turkan, F.; Taslimi, P.; Gulcin, I. ; Synthesis, spectroscopic properties, crystal
structures, antioxidant activities and enzyme inhibition determination of Co(II) and Fe(II) complexes of Schiff base. Res. Chem.
Intermed. 2020,46, 283–297. [CrossRef]
75.
Turkan, F.; Atalar, M.N.; Aras, A.; Gulcin, I.; Bursal, E. ICP-MS and HPLC analyses, enzyme inhibition and antioxidant potential
of Achillea schischkinii Sosn. Bioorg. Chem. 2020,94, 103333. [CrossRef] [PubMed]
76.
Taslimi, P.; Koksal, E.; Goren, A.C.; Bursal, E.; Aras, A.; Kılıc, O.; Alwasel, S.; Gulcin, I. Anti-Alzheimer, antidiabetic and
antioxidant potential of Satureja cuneifolia and analysis of its phenolic contents by LC-MS/MS. Arab. J. Chem.
2020
,13, 4528–4537.
[CrossRef]
77.
Artunc, T.; Menzek, A.; Taslimi, P.; Gulcin, I.; Kazaz, C.; Sahin, E. Synthesis and antioxidant activities of phenol derivatives from
1,6-bis(dimethoxyphenyl)hexane-1,6-dione. Bioorg. Chem. 2020,100, 103884. [CrossRef]
78.
Goldschmidt, S.; Renn, K. Zweiwertiger sticlstoff: Über das
α
,
α
-diphenyl-
β
-trinitrophenyl hydrazyl. Ber. Dtsch. Chem. Ges. B
2022,B55, 628–643.
79. Blois, M.S. Antioxidant determinations by the use of a stable free radical. Nature 1958,181, 1199–1200. [CrossRef]
80.
Bondet, V.; Brand-Williams, W.; Berset, C. Kinetics and mechanism of antioxidant activity using the DPPH free radical method.
Lebensm. Wiss. Technol. 1997,30, 609–615. [CrossRef]
81. Foti, M.C. Use and abuse of the DPPH·radical. J. Agric. Food Chem. 2015,63, 8765–8776. [CrossRef]
82.
Kawai, A.; Shibuya, K. Energy separation between quartet and doublet spin states of radical-triplet encounter pairs; unusual
ferromagnetic interaction in a 1,1-diphenyl-2-picrylhydrazyl and triplet coronene pair. J. Phys. Chem. A
2002
,106, 12305–12314.
[CrossRef]
83.
Chen, O.; Zhuang, J.; Guzzetta, F.; Lynch, J.; Angerhofer, A.; Cao, Y.C. Synthesis of water-soluble 2,2
0
-diphenyl-1-picrylhydrazyl
nanoparticles: A new standard for electron paramagnetic resonance spectroscopy. J. Am. Chem. Soc.
2009
,131, 12542–12543.
[CrossRef]
84.
Yapıcı, I.; Altay, A.; Ozturk Sarikaya, S.B.; Korkmaz, M.; Atila, A.; Gulcin, I.; Koksal, E.
In vitro
antioxidant and cytotoxic
activities of extracts of endemic Tanacetum erzincanense together with phenolic content by LC-ESI-QTOF-MS. Chem. Biodivers.
2021,18, e2000812. [CrossRef]
85.
Xie, J.; Schaich, K.M. Re-evaluation of the 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) assay for antioxidant activity. J.
Agric. Food Chem. 2014,62, 4251–4260. [CrossRef] [PubMed]
86.
Erdogan, M.; Polat Kose, L.; Essiz, S.; Gulcin, I. Synthesis and biological evaluation of some L-naphthol derivatives as antioxidants,
acetylcholinesterase, carbonic anhydrase inhibitors. Arch. Pharm. 2021,354, e2100113. [CrossRef]
87.
Ozcelik, B.; Lee, J.H.; Min, D.B. Effects of light, oxygen, and pH on the absorbance of 2,2-diphenyl-1-picrylhydrazyl. J. Food Sci.
2003,68, 487–490. [CrossRef]
88.
Ingold, K.U.; Pratt, D.A. Advances in radical-trapping antioxidant chemistry in the 21st century: A kinetics and mechanisms
perspective. Chem. Rev. 2014,114, 9022–9046.

Processes 2023,11, 2248 18 of 20
89. Foti, M.C. Antioxidant properties of phenols. J. Pharm. Pharmacol. 2007,59, 1673–1685. [CrossRef] [PubMed]
90. Alfassi, Z. Peroxyl Radicals; Wiley: Chichester, UK, 1997.
91.
Foti, M.C.; Daquino, C.; Geraci, C. Electron-transfer reaction of cinnamic acids and their methyl esters with the DPPH radical in
alcoholic solutions. J. Org. Chem. 2004,69, 2309–2314. [CrossRef] [PubMed]
92.
Conquer, J.A.; Maiani, G.; Azzini, E.; Raguzzini, A.; Holub, B.J. Supplementation with quercetin markedly increases plasma
quercetin concentration without effect on selected risk factors for heart disease in healthy subjects. J. Nutr.
1998
,128, 593–597.
[CrossRef]
93.
Foti, M.C.; Daquino, C.; DiLabio, G.A.; Ingold, K.U. Kinetics of the oxidation of quercetin by 2,2-diphenyl-1-picrylhydrazyl
(DPPH·). Org. Lett. 2011,13, 4826–4829. [CrossRef] [PubMed]
94.
Apak, R.; Calokerinos, A.; Gorinstein, S.; Segundo, M.A.; Hibbert, D.B.; Gulcin, I.; Demirci Cekic, S.; Guclu, K.; Ozyurek, M.;
Esin Celik, S.
; et al. Methods to evaluate the scavenging activity of antioxidants toward reactive oxygen and nitrogen species.
Pure Appl. Chem. 2022,94, 87–144. [CrossRef]
95.
Shahidi, F.; Ambigaipalan, P. Phenolics and polyphenolics in foods, beverages and spices: Antioxidant activity and health
effects-A review. J. Funct. Foods 2015,18, 820–897.
96.
Litwinienko, G.; Ingold, K.U. Abnormal solvent effects on hydrogen atom abstraction. 3. Novel kinetics in sequential proton loss
electron transfer chemistry. J. Org. Chem. 2005,70, 8982–8990. [CrossRef] [PubMed]
97.
Musialik, M.; Kuzmicz, R.; Pawlowski, T.S.; Litwinienko, G. Acidity of hydroxyl groups: An overlooked influence on antiradical
properties of flavonoids. J. Org. Chem. 2009,74, 2699–2709. [CrossRef] [PubMed]
98.
Diplock, A.T.; Charleux, J.L.; Crozier-Willi, G.; Kok, F.J.; Rice-Evans, C.; Roberfroid, M.; Stahl, W.; Vina-Ribes, J. Functional food
science and defence against reactive oxidative species. Br. J. Nutr. 1998,80, S77–S112. [CrossRef]
99.
Cetin Cakmak, K.; Gulcin, I. Anticholinergic and antioxidant activities of usnic acid-An activity-structure insight. Toxicol. Rep.
2019,6, 1273–1280. [CrossRef]
100.
Soares, J.R.; Dinis, T.C.; Cunha, A.P.; Almeida, L.M. Antioxidant activities of some extracts of Thymus zygis.Free Radic. Res.
1997
,
26, 469–478. [CrossRef]
101.
Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. Lebensm. Wissen.
Technol. 1995,28, 25–30. [CrossRef]
102.
Ozaslan, M.S.; Saglamtas, R.; Demir, Y.; Genc, Y.; Saracoglu, I.; Gulcin, I. Isolation of some phenolic compounds from Plantago
subulata L. and determination of its antidiabetic, anticholinesterase, antiepileptic and antioxidant activity. Chem. Biodivers. 2022,
19, e202200280. [CrossRef]
103.
Koca, M.; Gulcin, I.; Uç, E.M.; Bilginer, S.; Aydın, A.S. Evaluation of antioxidant potentials and acetylcholinesterase inhibitory
efects of some new salicylic acid-salicylamide hybrids. J. Iran. Chem. Soc. 2023,20, 1535–1543. [CrossRef]
104.
Gulcin, I.; Alwasel, S.H. Metal ions, metal chelators and metal chelating assay as antioxidant method. Processes
2022
,10, 132.
[CrossRef]
105.
Ak, T.; Gulcin, I. Antioxidant and radical scavenging properties of curcumin. Chem. Biol. Interact.
2008
,174, 27–37. [CrossRef]
[PubMed]
106.
Gulcin, I. Antioxidant properties of resveratrol: A structure-activity insight. Innov. Food Sci. Emerg.
2010
,11, 210–218. [CrossRef]
107.
Gulcin, I. Antioxidant activity of eugenol-a structure and activity relationship study. J. Med. Food
2011
,14, 975–985. [CrossRef]
[PubMed]
108.
Durmaz, L.; Erturk, A.; Akyuz, M.; Polat Kose, L.; Uc, E.M.; Bingol, Z.; Sa˘glamtas, R.; Alwasel, S.; Gulcin, I. Screening of carbonic
anhydrase, acetylcholinesterase, butyrylcholinesterase and
α
-glycosidase enzymes inhibition effects and antioxidant activity of
coumestrol. Molecules 2022,27, 3091. [CrossRef] [PubMed]
109.
Durmaz, L.; Kiziltas, H.; Guven, L.; Karagecili, H.; Alwasel, S.; Gulcin, I. Antioxidant, antidiabetic, anticholinergic, and
antiglaucoma effects of magnofluorine. Molecules 2022,27, 5902. [CrossRef] [PubMed]
110.
Gulcin, I.; Mshvildadze, V.; Gepdiremen, A.; Elias, R. Antioxidant activity of saponins isolated from ivy: (-Hederin, hederasaponin-
C, hederacolchiside-E and hederacolchiside F. Planta Med. 2004,70, 561–563. [CrossRef]
111.
Gulcin, I.; Mshvildadze, V.; Gepdiremen, A.; Elias, R. Antioxidant activity of a triterpenoid glycoside isolated from the berries of
Hedera colchica: 3-O-(β-D-glucopyranosyl)-hederagenin. Phytother. Res. 2004,20, 130–134. [CrossRef] [PubMed]
112.
Gulcin, I.; Beydemir, S.; Alici, H.A.; Elmastas, M.; Büyükokuroglu, M.E.
In vitro
antioxidant properties of morphine. Pharmacol.
Res. 2004,49, 59–66. [CrossRef]
113.
Gulcin, I.; Oktay, M.; Koksal, E.; Serbetci, H.; Beydemir, S.; Kufrevioglu, O.I. Antioxidant and radical scavenging activities of uric
acid. Asian J. Chem. 2008,20, 2079–2090.
114. Gulcin, I. Antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid). Toxicology 2006,217, 213–220. [CrossRef] [PubMed]
115.
Gulcin, I.; Huyut, Z.; Elmastas, M.; Aboul-Enein, H.Y. Radical scavenging and antioxidant activity of tannic acid. Arab. J. Chem.
2010,3, 43–53. [CrossRef]
116.
Topal, M.; Gulcin, I. Evaluation of the
in vitro
antioxidant, antidiabetic and anticholinergic properties of rosmarinic acid from
rosemary (Rosmarinus officinalis L.). Biocat. Agric. Biotechnol. 2022,43, 102417. [CrossRef]
117. Gulcin, I. Antioxidant and antiradical activities of L-carnitine. Life Sci. 2006,78, 803–811. [CrossRef] [PubMed]
118. Gulcin, I. Antioxidant activity of L-Adrenaline: An activity-structure insight. Chem. Biol. Interact. 2009,179, 71–80. [CrossRef]

Processes 2023,11, 2248 19 of 20
119.
Koksal, E.; Gulcin, I.; Ozturk Sarikaya, S.B.; Bursal, E. On the
in vitro
antioxidant activity of silymarin. J. Enzym. Inhib. Med. Chem.
2009,24, 395–405. [CrossRef]
120.
Topal, F.; Topal, M.; Gocer, H.; Kalın, P.; Kocyigit, U.M.; Gulcin, I.; Alwasel, S.H. Antioxidant activity of taxifolin: An activity-
structure relationship. J. Enzym. Inhib. Med. Chem. 2016,31, 674–683. [CrossRef]
121. Taslimi, P.; Gulcin, I. Antioxidant and anticholinergic properties of olivetol. J. Food Biochem. 2018,42, e12516. [CrossRef]
122.
Polat Kose, L.; Gulcin, I. Evaluation of the antioxidant and antiradical properties of some phyto and mammalian lignans. Molecules
2021,26, 7099. [CrossRef]
123.
Gulcin, I.; Elias, R.; Gepdiremen, A.; Taoubi, K.; Koksal, E. Antioxidant secoiridoids from fringe tree (Chionanthus virginicus L.).
Wood Sci. Technol. 2009,43, 195–212. [CrossRef]
124.
Gulcin, I.; Oktay, M.; Kirecci, E.; Kufrevioglu, O.I. Screening of antioxidant and antimicrobial activities of anise (
Pimpinella anisum L.
)
seed extracts. Food Chem. 2003,83, 371–382. [CrossRef]
125.
Oktay, M.; Gulcin, I.; Kufrevioglu, O.I. Determination of
in vitro
antioxidant activity of fennel (Foeniculum vulgare) seed extracts.
Lebens. Wissen. Technol. 2003,36, 263–271. [CrossRef]
126.
Gulcin, I.; Tel, A.Z.; Goren, A.C.; Taslimi, P.; Alwasel, S. Sage (Salvia pilifera): Determination its polyphenol contents, anticholiner-
gic, antidiabetic and antioxidant activities. J. Food Meas. Charac. 2019,13, 2062–2074. [CrossRef]
127.
Kiziltas, H.; Bingol, Z.; Goren, A.C.; Alwasel, S.H.; Gulcin, I. Anticholinergic, antidiabetic and antioxidant activities of
Ferula orientalis L.-Analysis of its polyphenol contents by LC-HRMS. Rec. Nat. Prod. 2021,15, 513–528. [CrossRef]
128.
Gulcin, I.; Sat, I.G.; Beydemir, S.; Elmastas, M.; Kufrevioglu, O.I. Comparison of antioxidant activity of clove (Eugenia caryophylata
Thunb) buds and lavender (Lavandula stoechas L.). Food Chem. 2004,87, 393–400. [CrossRef]
129.
Gulcin, I. The antioxidant and radical scavenging activities of black pepper (Piper nigrum) seeds. Int. J. Food Sci. Nutr.
2005
,56,
491–499. [CrossRef]
130.
Elmastas, M.; Gulcin, I.; Beydemir, ¸S.; Kufrevioglu, O.I.; Aboul-Enein, H.Y. A study on the
in vitro
antioxidant activity of juniper
(Juniperus communis L.) seeds extracts. Anal. Lett. 2006,39, 47–65. [CrossRef]
131.
Elmastas, M.; Gulcin, I.; Isıldak, O.; Kufrevioglu, O.I.; Ibaoglu, K.; Aboul-Enein, H.Y. Antioxidant capacity of bay (
Laurus nobilis L.
)
leaves extracts. J. Iran. Chem. Soc. 2006,3, 258–266. [CrossRef]
132.
Gulcin, I.; Elmastas, M.; Aboul-Enein, H.Y. Determination of antioxidant and radical scavenging activity of basil
(Ocimum basilicum) assayed by different methodologies. Phytother. Res. 2007,21, 354–361. [CrossRef]
133.
Koksal, E.; Gulcin, I. Purification and characterization of peroxidase from cauliflower (Brassica oleracea L.) buds. Protein Peptide
Lett. 2008,15, 320–326. [CrossRef]
134.
Serbetci Tohma, H.; Gulcin, I. Antioxidant and radical scavenging activity of aerial parts and roots of Turkish liquorice
(Glycyrrhiza glabra L.). Int. J. Food Prop. 2010,13, 657–671. [CrossRef]
135.
Bursal, E.; Koksal, E.; Gulcin, I.; Bilsel, G.; Goren, A.C. Antioxidant activity and polyphenol content of cherry stem
(Cerasus avium L.) determined by LC-MS/MS. Food Res. Int. 2013,51, 66–74. [CrossRef]
136.
Polat Kose, L.; Gulcin, I.; Goren, A.C.; Namiesnik, J.; Martinez-Ayala, A.L.; Gorinstein, S. LC-MS/MS analysis, antioxidant and
anticholinergic properties of galanga (Alpinia officinarum Hance) rhizomes. Ind. Crops Prod. 2015,74, 712–721. [CrossRef]
137.
Tohma, H.; Gulcin, I.; Bursal, E.; Goren, A.C.; Alwasel, S.H.; Koksal, E. Antioxidant activity and phenolic compounds of ginger
(Zingiber officinale Rosc.) determined by HPLC-MS/MS. J. Food Meas. Charac. 2017,11, 556–566. [CrossRef]
138.
Han, H.; Yılmaz, H.; Gulcin, I. Antioxidant activity of flaxseed (Linum usitatissimum L.) and analysis of its polyphenol contents by
LC-MS/MS. Rec. Nat. Prod. 2018,12, 397–402. [CrossRef]
139.
Gulcin, I.; Kaya, R.; Goren, A.C.; Akıncıoglu, H.; Topal, M.; Bingol, Z.; Cetin Cakmak, K.; Ozturk Sarikaya, S.B.; Durmaz, L.;
Alwasel, S. Anticholinergic, antidiabetic and antioxidant activities of cinnamon (Cinnamomum verum) bark extracts: Polyphenol
contents analysis by LC-MS/MS. Int. J. Food Prop. 2019,22, 1511–1526. [CrossRef]
140.
Gulcin, I.; Goren, A.C.; Taslimi, P.; Akyuz, B.; Tuzun, B. Anticholinergic, antidiabetic and antioxidant activities of Anatolian
pennyroyal (Mentha pulegium)-Analysis of its polyphenol contents by LC-MS/MS. Biocat. Agric. Biotechnol.
2020
,23, 101441.
[CrossRef]
141.
Polat Kose, L.; Bingol, Z.; Kaya, R.; Goren, A.C.; Akincioglu, H.; Durmaz, L.; Koksal, E.; Alwasel, S.; Gulcin, I. Anticholinergic
and antioxidant activities of avocado (Folium perseae) leaves—Phytochemical content by LC-MS/MS analysis. Int. J. Food Prop.
2020,23, 878–893. [CrossRef]
142.
Kızıltas, H.; Bingol, Z.; Goren, A.C.; Polat Kose, L.; Durmaz, L.; Topal, F.; Alwasel, S.H.; Gulcin, I. LC-HRMS profiling, antidiabetic,
anticholinergic and antioxidant activities of aerial parts of kınkor (Ferulago stelleta). Molecules 2021,26, 2469. [CrossRef]
143. Bingol, Z.; Kızıltas, H.; Goren, A.C.; Polat Kose, L.; Topal, M.; Durmaz, L.; Alwasel, S.H.; Gulcin, I. Antidiabetic, anticholinergic
and antioxidant activities of aerial parts of shaggy bindweed (Convulvulus betonicifolia Miller subsp.)-profiling of phenolic
compounds by LC-HRMS. Heliyon 2021,7, e06986. [CrossRef]
144.
Gulcin, I.; Bingol, Z.; Taslimi, P.; Goren, A.C.; Alwasel, S.H.; Tel, A.Z. Polyphenol contents, potential antioxidant, anticholinergic
and antidiabetic properties of mountain mint (Cyclotrichium leucotrichum). Chem. Biodivers. 2022,19, e202100775. [CrossRef]
145.
Karagecili, H.; Izol, E.; Kireçci, E.; Gulcin, I. Determination of antioxidant, anti-Alzheimer, antidiabetic, antiglaucoma and
antimicrobial effects of zivzik pomegranate (Punica granatum)-A chemical profiling by LC-MS/MS. Life
2023
,13, 735. [CrossRef]
[PubMed]

Processes 2023,11, 2248 20 of 20
146.
Yılmaz, M.A.; Taslimi, P.; Kilic, O.; Gulcin, I.; Dey, A.; Bursal, E. Unravelling the phenolic compound reserves, antioxidant
and enzyme inhibitory activities of an endemic plant species, Achillea pseudoaleppica.J. Biomol. Struct. Dyn.
2023
,41, 445–456.
[CrossRef] [PubMed]
147.
Kiziltas, H.; Goren, A.C.; Alwasel, S.; Gulcin, I. Comprehensive metabolic profiling of Acantholimon caryophyllaceum using
LC-HRMS and evaluation of antioxidant activities, enzyme inhibition properties and molecular docking studies. S. Afr. J. Bot.
2022,151, 743–751. [CrossRef]
148.
Bursal, E.; Aras, A.; Kilic, O.; Taslimi, P.; Goren, A.C.; Gulcin, I. Phytochemical content, antioxidant activity and enzyme inhibition
effect of Salvia eriophora Boiss. & Kotschy against acetylcholinesterase,
α
-amylase, butyrylcholinesterase and
α
-glycosidase
enzymes. J. Food Biochem. 2019,43, e12776.
149.
Aras, A.; Bursal, E.; Turkan, F.; Tohma, H.; Kılıc, O.; Gulcin, I.; Koksal, E. Phytochemical content, antidiabetic, anticholinergic, and
antioxidant activities of endemic Lecokia cretica extracts. Chem. Biodivers. 2019,16, e1900341. [CrossRef]
150.
Bursal, E.; Taslimi, P.; Goren, A.; Gulcin, I. Assessments of anticholinergic, antidiabetic, antioxidant activities and phenolic content
of Stachys annua.Biocat. Agric. Biotechnol. 2020,28, 101711. [CrossRef]
151.
Guven, L.; Erturk, A.; Demirkaya Miloglu, F.; Alwasel, S.; Gulcin, I. Screening of antiglaucoma, antidiabetic, anti-Alzheimer, and
antioxidant activities of Astragalus alopecurus Pall-Analysis of phenolics profiles by LC-MS/MS. Pharmaceuticals
2023
,16, 659.
[CrossRef]
152.
Kızıltas, H.; Bingol, Z.; Goren, A.C.; Alwasel, S.H.; Gulcin, I. Verbascum speciousum Schrad: Analysis of phenolic compounds by
LC-HRMS and determination of antioxidant and enzyme inhibitory properties. Rec. Nat. Prod. 2023,17, 485–500.
153.
Gulcin, I.; Tel, A.Z.; Kirecci, E. Antioxidant, antimicrobial, antifungal and antiradical activities of Cyclotrichium niveum (Boiss.)
Manden and Scheng. Int. J. Food Prop. 2008,11, 450–471. [CrossRef]
154.
Gulcin, I.; Kufrevioglu, O.I.; Oktay, M.; Buyukokuroglu, M.E. Antioxidant, antimicrobial, antiulcer and analgesic activities of
nettle (Urtica dioica L.). J. Ethnopharmacol. 2004,90, 205–215. [CrossRef] [PubMed]
155.
Gulcin, I.; Beydemir, S.; Sat, I.G.; Kufrevioglu, O.I. Evaluation of antioxidant activity of cornelian cherry (Cornus mas L.). Acta
Aliment. Hung. 2005,34, 193–202. [CrossRef]
156.
Bursal, E.; Gulcin, I. Polyphenol contents and
in vitro
antioxidant activities of lyophilized aqueous extract of kiwifruit
(Actinidia deliciosa). Food Res. Int. 2011,44, 1482–1489. [CrossRef]
157.
Kalin, P.; Gulcin, I.; Goren, A.C. Antioxidant activity and polyphenol content of cranberries (Vaccinium macrocarpon). Rec. Nat.
Prod. 2015,9, 496–502.
158.
Elmastas, M.; Gulcin, I.; Ozturk, L.; Gokce, I. Investigation of antioxidant properties of spearmint (Mentha spicata L.). Asian J.
Chem. 2005,17, 137–148.
159.
Kiziltas, H.; Goren, A.C.; Alwasel, S.; Gulcin, I. Sahlep (Dactylorhiza osmanica): Phytochemical analyses by LC-HRMS, molecular
docking, antioxidant activity and enzyme inhibition profiles. Molecules 2022,27, 6907. [CrossRef]
160.
Lebeau, J.; Furman, C.; Bernier, J.L.; Duriez, P.; Teissier, E.; Cotelle, N. Antioxidant properties of di-tert-butylhydroxylated
flavonoids. Free Radic. Biol. Med. 2020,29, 900–912. [CrossRef]
161.
Salamone, M.; DiLabio, G.A.; Bietti, M. Hydrogen atom abstraction reactions from tertiary amines by benzyloxyl and cumyloxyl
radicals: Influence of structure on the rate determining formation of a hydrogen-bonded prereaction complex. J. Org. Chem.
2011
,
76, 6264–6270. [CrossRef]
162.
Salamone, M.; Martella, R.; Bietti, M. Hydrogen abstraction from cyclic amines by the cumyloxyl and benzyloxyl radicals. The
role of stereoelectronic effects and of substrate/radical hydrogen bonding. J. Org. Chem. 2012,77, 8556–8561. [CrossRef]
163.
Barclay, L.R.C.; Edwards, C.E.; Vinqvist, M.R. Media effects on antioxidant activities of phenols and catechols. J. Am. Chem. Soc.
1999,121, 6226–6231. [CrossRef]
164.
Hogg, J.S.; Lohmann, D.H.; Russell, K.S. The kinetics of reaction of 2,2-diphenyl-1-picrylhydrazyl with phenols. Canad. J. Chem.
1961,39, 1588–1594. [CrossRef]
165.
Oztaskin, N.; Cetinkaya, Y.; Taslimi, P.; Goksu, S.; Gulcin, I. Antioxidant and acetylcholinesterase inhibition properties of novel
bromophenol derivatives. Bioorg. Chem. 2015,60, 49–57. [CrossRef]
166.
Tohma, H.; Koksal, E.; Kılıc, O.; Alan, Y.; Yılmaz, M.A.; Gulcin, I.; Bursal, E.; Alwasel, S.H. RP-HPLC/MS/MS analysis of the
phenolic compounds, antioxidant and antimicrobial activities of Salvia L. species. Antioxidants
2016
,5, 38. [CrossRef] [PubMed]
167.
Schaich, K.M.; Tian, X.; Xie, J. Hurdles and pitfalls in measuring antioxidant efficacy: A critical evaluation of ABTS, DPPH, and
ORAC assays. J. Func. Foods 2015,14, 111–125. [CrossRef]
Disclaimer/Publisher’s Note:
The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.