Description of an article over antioxidant. Introduction Definition Why antioxidant need? Classifications Schiff base synthesis example Suzuki Reaction Amine synthesis Reaction conditions Optimization Optimizations for Suzuki cross-coupling reaction Catalytic Activities catalytic mechanism (mercury test) DPPH principle Antioxidant activities DPPH scavenging activity half maximal inhibitory concentration (IC50) Ferrous ions (Fe2+) chelating activity

1. Presented by
Abdoul Salam
Issiaka Ibrahim

2. Introduction
With advent in knowledge prevalence regarding health issues, natural
antioxidants are getting popular. These are compounds responsible for
hindering autoxidation reactions in food system and reducing oxidative stress
in human body.
In this article, new Schiff base derivatives have been synthesized and all
structures have been identified by using NMR, FTIR, and GC–MS techniques.
In addition, in situ catalytic activities for Suzuki cross-coupling reactions and
the antioxidant activities via DPPH (2,2-diphenyl-1-picrylhydrazyl) and Ferrous
ions (Fe2+) chelating methods have been examined for all synthesized
molecules.
Before starting our assignment. We will present a quick summary of the main
words used in this paper.
In this following presentation we will envisage to expose a brief overview of
new Schiff bases synthesis and its optimization. Thus, we will explain the
antioxidant activities during the them synthesis consisting our work.

3. Definition
Antioxidant means "against oxidation”. Any substance at low concentrations
compared to that of an oxidizable substrate that significantly delays or prevents
oxidation of that substrate is called as antioxidant. Antioxidants play vital role in
preserving the quality of food and maintaining health of human being.

4. Why antioxidant need?
Oxidation reaction depending upon site of occurrences presents specific
repercussions. If the site of occurrence is food system, then food deteriorates.
When oxidation occurs in biological cell system, it causes damage or death to
the cell.

5. Classifications
Primary antioxidants or chain
breaking antioxidants are
those compounds, mainly
phenolic substances that
terminate the free radical
chains in lipid oxidation and
function as hydrogen and
electron donors
Chelating agents are
synergistic substances which
greatly enhance the action of
phenolic antioxidants. Most of
these synergists exhibit little
or no antioxidant activity, for
example citric acid, amino
acid etc.
Oxygen scavengers are those
substances which react with
oxygen and can thus remove
it in a closed system, e.g.,
ascorbic acid (vitamin C).
Secondary antioxidants are
those compounds which
function by decomposing the
lipid hydroperoxides into
stable end products.
Enzymatic antioxidants are
those enzymes which function
either by removing dissolved
or head space oxygen, e.g.,
glucose oxidase, or by
removing highly oxidative
species, e.g., super oxide
dismutase.

6. In-Vitro Estimation Antioxidant Activity
Estimation by Free Radical Scavenging Capacity: The primary mode of action
of an antioxidant is scavenging free radicals either by hydrogen atom or single
electron transfer. Based on this mechanism, antioxidant assays have been
designed and utilized to assess potentiality of compound as an antioxidant. In
these assays free radicals are added/generated and the potency is calculated
based on decrease in concentration of free radicals. The scavenging activity is
either conducted versus stable radical like 2,2-diphenyl-1- picrylhydrazyl (DPPH)
or done on comparison basis with other standard antioxidants like trolox, BHT,
tocopheroal, gallic acid etc.

7. In-Vitro Estimation Antioxidant Activity
Estimation by Reduction of Metal Ions: Transition metals act as catalyst in free radical chain
reaction or autoxidation. Antioxidants, particularly phenolic compounds are capable of chelating metal
ions and non-phenolic can reduce metal ions by single electron transfer mechanism. Based on action
mechanism against metal ions, two popular assays known as Ferric Reducing/Antioxidant Power
(FRAP) and Cupric Ion Reducing Antioxidant Capacity (CUPRAC) have been established and utilized
in assessment of antioxidant activity. In these the potency of the compound to reduce ferric [Fe(III)]
and cupric [Cu(II)] to their respective lower valency states. In FRAP, the potency is measured through
reaction between FRAP reagent [mixture of 2,4,6-TPTZ [2,4,6-tri (2-pyridyl)- 1,3,5-triazine] solution in
hydrochloric acid + ferric chloride + acetate buffer (pH 3.6)] and compound. The blue color developed
is measured at 593 nm in spectrophotometer. Ironically, Fe(II) and Cu(I) are more reactive ions than
Fe(III) and Cu(II) in decomposing hydrogen peroxide and hydroperoxides, resultant will be prooxidant
effect of antioxidants showing reducing action on ferric and cupric ions.

8. In-Vitro Estimation Antioxidant Activity
Estimation by Inhibition of Lipid Peroxidation in Plasma: This method, using
plasma as reaction medium, has biological relevance with respect to evaluation of
potency of hydrophilic and lipophilic antioxidants and their interaction in biological
fluid. The assessment is done either by adding antioxidant to separated blood
plasma of test animal or by first administering antioxidant to test animal and then
separating plasma for further analysis. The testing parameters include cholesteryl
linoleate hydroperoxide, a marker of lipid peroxidation in human plasma.

9. In-Vitro Estimation Antioxidant Activity
Estimation using Cultured Cell against Oxidative Stress: Cultured cells have
often been used as a substrate to elucidate the underlying mechanisms of
oxidative stress and also to evaluate the protective effects of antioxidants against
various oxidative stressors. The estimation provides the functional aspects of the
antioxidant in human body for the suppression of Reactive oxygen species (ROS)
formation, oxidation of lipids, proteins and DNA, and cell death. In this method,
the antioxidants are added to the culture medium simultaneously with the stressor
or pre-incubated to incorporate in the cells. The advantage of using cultured cells
is that various different stressors and cell types for some specific disease can be
used for evaluation of the antioxidant effects. It may also be noted that cultured
cells may overcome the difficulty involved in procuring experimental animals in the
future.

10. SCHIFF BASE
A Schiff base, named after Hugo Schiff, is a compound having a C=N
double bond with the nitrogen atom bonded to an aryl or alkyl group,
and not a hydrogen: thus, they are the secondary imines.
Schiff bases in the broad sense have a general formula of the type
R1R2C=NR3, where R is an organic chain. In this definition, Schiff's
base is synonymous with azomethine. Some restrict the definition to
secondary aldimines (azomethines where the carbon is bonded to
only one hydrogen), and therefore have the general formula
RCH=NR'.
The carbon chain on the nitrogen atom makes Schiff bases a stable
imine. Schiff bases derived from aniline, where R3 is therefore phenyl
or substituted phenyl are called aniles.
General structure of Schiff Base

11. SCHIFF BASE SYNTHESIS EXAMPLE
THEORY
Schiff bases can be synthesized from
aromatic amines and a carbonyl compound by
nucleophilic addition forming a hemiaminal,
followed by dehydration to form an imine. A
typical reaction is the reaction between 4,4'-
diaminodiphenyl ether and o-vanillin.
A mixture of 4,4'-diaminodiphenyl ether (1)
(1.00 g, 5.00 mmol) and o-vanillin (2) (1.52 g,
10.00 mmol) in methanol ( 40.00 ml) is stirred
at room temperature for one hour to give an
orange precipitate which is then filtered and
rinsed with methanol to give pure Schiff's
base (3) (2.27 g, 97.00%)

12. SUZUKI REACTION
The Suzuki reaction is an organic reaction, classified as a cross-coupling reaction,
where the coupling partners are a boronic acid and an organohalide and the catalyst is
a palladium complex. This reaction is also known as the Suzuki–Miyaura reaction or
simply as the Suzuki coupling. It is widely used to synthesize polyolefins, styrenes, and
substituted biphenyls. The general scheme for the Suzuki reaction is shown below,
where a carbon-carbon single bond is formed by coupling a halide (R1-X) with an
organoboron species (R2-BY2) using a palladium catalyst and a base.

13. AMINE SYNTHESIS
In the beginning the nitrile 3 was
synthesized with bromination,
elimination, and cyanation reactions
respectively. Then, cyclohepta-2,4,6-
trien-1-ylmethanamine 4 was obtained
from previous product 3 with reduction
via LiAlH4.

14. REACTION CONDITIONS OPTIMIZATION

15. Amine was reacted with benzaldehyde and the yields of reactions were determined with GC. According to the previous
table it has been concluded that polar protic solvents better than polar aprotic ones and the temperature increase
improved the yield.

16. Several substrates through the reactions of amine 4 and
different aldehydes 7a–h were conducted, and obtained new
Schiff base series with high yield 8a–h. All structures have been
identified and confirmed by using NMR, FT-IR spectroscopy,
and GC–MS technics. The OH band was not observed in the
FTIR spectra due to the hydrogen bonding between the CHN
and the OH groups. Literature shew that when analysis with
similar function groups has been set up, the OH bond can’t
feature in the FT-IR spectrum.

17. OPTIMIZATIONS FOR SUZUKI CROSS-
COUPLING REACTION
In this assay, the Suzuki reaction was achieved with phenylboronic acid and 4-
bromoacetophenone in the presence of our previous products as ligand and
palladium chloride as catalyst using different substrates for optimizing the reactions
conditions see the scheme below as example of one of them.

19. CATALYTIC ACTIVITIES
The results reported that polar protic solvents improve the catalytic activity better
than nonpolar solvents. In addition, organic bases enhance the reactions efficiency.

21. CATALYTIC ACTIVITIES
The catalytic activity of all synthesized Schiff bases was investigated by reactions
between various aryl bromides and phenylboronic acid. In the literature, besides the
steric effect, substituted groups in aryl bromide significantly affect the reaction
yields. Electron-donating groups and steric barriers reduce reaction efficiency, while
electron-withdrawing groups increase it. As we can note that all Schiff bases show
moderate and good catalytic activity, it can be said that the best catalytic activity in
general is achieved with 8g and the worst catalytic activity with 8h Schiff base [see
the table above.]

22. CATALYTIC MECHANISM (MERCURY TEST)
Considering the results obtained in all Suzuki C-C coupling reactions with
synthesized Schiff bases, it is seen that Schiff bases substituted with halogen
groups that are deactivating show less catalytic activity than Schiff bases
substituted with activating OH groups. For assimilating this phenomenon, the
mercury test was realized, and the results were examined by GC, it has been
observed that there was no conversion in all reactions. Thus, this means Schiff
bases catalyze Suzuki coupling reactions with heterogeneous catalyst by
stabilizing Pd formed in the reaction medium.

23. DPPH PRINCIPLE
The ability of a molecule to be active in this method depends on its ability to deliver
protons. When the molecule transfers protons to the DPPH radical, the solution of
purple-colored DPPH in ethanol turns into yellow-colored DPPH-H. Thus, DPPH,
which absorbs at 517 nm, decreases the absorbance and the antioxidant capacity
(TAC) of the molecule is calculated according to this decrease.
%DPPH Scavenging =
𝐴0−𝐴
𝐴0
×100 where Ao is the absorbance of the control; As
is the absorbance of the sample at 517 nm.

24. ANTIOXIDANT ACTIVITIES
In the third part of that article, It has been recalled the examination of antioxidant
activities through the DPPH method which is worldwide used as an efficient assay
during the antioxidant evaluation by UV. In the beginning, the highest concentration
(800 μg/ml) of all synthesized molecules were prepared for specify of active
compounds in method DPPH using for investigation of antioxidant capacity (see
Table 5).

25. DPPH SCAVENGING ACTIVITY
When we observe the table 5 in the
colon of DPPH inhibition ratio, we will
remark that the molecules with OH
groups present the highest while the
molecules bond with the halides show
the lowest. Hence the OH can increase
the antioxidant activity while the halide
can decrease it.

26. HALF MAXIMAL INHIBITORY CONCENTRATION
(IC50)
The half maximal inhibitory concentration
(IC50) is a measure of the potency of a
substance in inhibiting a specific
biological or biochemical function. IC50 is
a quantitative measure that indicates how
much of a particular inhibitory substance
(e.g., drug) is needed to inhibit, in vitro, a
given biological process or biological
component by 50%.
When we take a look on the figure 1 and
table 6, the activity order is BHA > 8e >
BHT > 8c > 8a > 8d > 6 > 8g > 8h which
confirms the previous activity.

27. FERROUS IONS (FE2+) CHELATING ACTIVITY
Chelation is a type of bonding of ions and molecules to metal ions. It involves the
formation or presence of two or more separate coordinate bonds between a polydentate
(multiple bonded) ligand and a single central metal atom. These ligands are called
chelants, chelators, chelating agents, or sequestering agents. In this work, Chelation
was performed using the iron as a metal and our Schiff bases as our ligands. The
principle is based on the Fe2+ reduction and the percentage of Ferrous ions Fe2 +
chelating activity by the following formula:
% Ferrous ions Fe2 + chelating activity =
𝐴0−𝐴𝑠
𝐴0
×100
Ao is the absorbance of the control; As is the absorbance of the sample at 562 nm.

28. When we analyze the table 5, we can conclude that the
double bond and the halide increase the Ferrous ions
chelating activity while the OH group decreases it. In the one
hand, the other molecules show low iron activity because of
their bond with OH and adjacent CN groups, in the other
hand the BHT and BHA show the contrast. Through the
table bellow, the iron chelating activity decreases in the
following order 8g > BHT > 8f > BHA > 8b > 8h > 6.

29. CONCLUSION
In this presentation work, it has been spoken on the antioxidant generality and
activities, the Schiff bases and their synthesis and the Suzuki reaction. It has been
explained the Schiff bases synthesis and their optimization, catalytic and
antioxidants activities for this article.

30. THANK YOU FOR
YOUR KIND
ATTENTION