Synthesis, characterization, DFT study and antioxidant activity of (2-hydroxynaphthalen-1-yl) methyl 2-hydroxyphenyl amino phosphonic acid

Article abstract of DOI:10.1016/j.molstruc.2021.131322

New molecule α-aminophosphonate namely (2-hydroxynaphthalen-1-yl) methyl 2-hydroxyphenyl amino phosphonic acid (HMHP) was synthesized by condensation based on 2-amino phenol and 2-hydroxy naphthaldehyde. During the reaction, imine intermediate namely 2-hydroxyphenyl imino naphthalen-2-ol (HIN) was formed before adding acid phosphorous to the reaction mixture. It can be confirmed the concept of Kabachinik-Fields reaction, which it's perform the attachment of phosphorus atom to the carbon atom of imine function. Both compounds were identified using IR, ¹H, ¹³C and ³¹P NMR spectroscopic techniques. The evaluation of the antioxidant activity of HMHP and HIN was investigated by utilizing several in vitro assays: scavenging activity against DPPH radical and H₂O₂ non radical, β-carotene/linoleic acid against linoleic peroxidation, reducing power against ferric oxidation and phosphomolybdate (TAC) against molybdate ion oxidation. Results indicate that HMHP reflects greater antioxidant potency compared to HIN in most tests, where it is apt to offer the hydrogen radical very well at IC₅₀ (37.64 ± 1.43) in DPPH test. For the other tests the inhibition percent at 50 % are relatively closed between them. Except the H₂O₂ scavenging, while HIN exhibited excellent activity at IC₅₀ (24.7306 ± 0.71785). The quantum chemical calculations were performed using density functional theory (DFT) to study the effects of the transfer electronic and proton transfer on the antioxidant activities of the synthesized compounds. Also, theoretical FT-IR was calculated. The experimental results are in comfort with those calculated theoretically.

Full text of DOI:10.1016/j.molstruc.2021.131322

Journal of Molecular Structure 1247 (2022) 131322
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Synthesis, characterization, DFT study and antioxidant activity of
(2-hydroxynaphthalen-1-yl) methyl 2-hydroxyphenyl amino
phosphonic acid
a,b
a
a,∗
c
a
Noudjoud Houas , Salah Chafaa , Nadjib Chafai , Samira Ghedjati , Meriem Djenane ,
Siham Kitounia,b
a
Laboratory of Electrochemistry of Molecular Materials and Complex (LEMMC). Department of Process Engineering, Faculty of Technology, University of
Ferhat ABBAS Setif-1, El-Mabouda Campus, Sétif 19000, Algeria
b
Department of Chemistry, Faculty of Sciences, University of Ferhat ABBAS Setif-1, Setif 19000, Algeria
c
Laboratory of Phytotherapy Applied to Chronic Diseases Faculty of Natural and Life sciences, University of Ferhat ABBAS Setif-1, Setif 19000, Algeria
a r t i c l e i n f o
a b s t r a c t
Article history:
New molecule α-aminophosphonate namely (2-hydroxynaphthalen-1-yl) methyl 2-hydroxyphenyl amino
phosphonic acid (HMHP) was synthesized by condensation based on 2-amino phenol and 2-hydroxy
naphthaldehyde. During the reaction, imine intermediate namely 2-hydroxyphenyl imino naphthalen-2-ol
Received 25 June 2021
Revised 13 August 2021
Accepted 15 August 2021
Available online 17 August 2021
(
HIN) was formed before adding acid phosphorous to the reaction mixture. It can be confirmed the con-
cept of Kabachinik-Fields reaction, which it’s perform the attachment of phosphorus atom to the carbon
atom of imine function. Both compounds were identified using IR, 1H, 13 C and 31 P NMR spectroscopic
techniques. The evaluation of the antioxidant activity of HMHP and HIN was investigated by utilizing
several in vitro assays: scavenging activity against DPPH radical and H O non radical, β-carotene/linoleic
Keywords:
Synthesis
α-aminophosphonates
Imines
2
2
acid against linoleic peroxidation, reducing power against ferric oxidation and phosphomolybdate (TAC)
against molybdate ion oxidation. Results indicate that HMHP reflects greater antioxidant potency com-
pared to HIN in most tests, where it is apt to offer the hydrogen radical very well at IC50 (37.64 ± 1.43) in
DPPH test. For the other tests the inhibition percent at 50 % are relatively closed between them. Except
the H2O2 scavenging, while HIN exhibited excellent activity at IC50 (24.7306 ± 0.71785). The quantum
chemical calculations were performed using density functional theory (DFT) to study the effects of the
transfer electronic and proton transfer on the antioxidant activities of the synthesized compounds. Also,
theoretical FT-IR was calculated. The experimental results are in comfort with those calculated theoreti-
cally.
Antioxidant activities
DFT calculations
©
2021 Elsevier B.V. All rights reserved.
1
. Introduction
Antioxidants are defined as any substances that at low concen-
trations significantly delay or prevent oxidation of oxidizable sub-
strate [6]. It is known that antioxidant activity promotional pro-
cesses fought against numerous diseases. Furthermore, they are
used as additives in the food industry, pharmaceutical and cos-
metic [7,8]. Synthesized Antioxidants are an interesting alterna-
tive in view of their variety of structures and chemical interac-
tions, as well as the several biological activities they can perform.
Intensive research activities are currently being carried out on
α-aminophosphonate and imine (Schiff base) compounds. While
α-aminophosphonates are known through R N-CP(O)(OR) sub-
Free radicals and non-radical hydrogen peroxide (H O ) are
2
2
highly reactive and unstable compounds. They are produced con-
tinuously during normal physiological events but their overproduc-
tion can affect and damage essential biomolecules [1]. All organ-
isms have antioxidant defenses, including antioxidant enzymes and
dietary components (Vitamin E, Vitamin C and beta-carotene) that
are used to remove or repair damaged molecules [2,3]. If the living
cells remain damaged, they may lead to serious diseases includ-
ing Alzheimer’s, cancer, atherosclerosis, diabetes, hypertension and
aging [4,5].
2
2
stratum characterized by a stable, covalent carbon to phospho-
rous bond. Their structures are analoges to the well-known amino
polycarboxylates, they are used innumerous biological techniques
[
9,10]. Thus in medicine, they can be used to treat various diseases
∗
Corresponding author.
of bone and calcium metabolism [11]. On the other hand, Schiff
E-mail address: n.chafai@univ-setif.dz (N. Chafai).
https://doi.org/10.1016/j.molstruc.2021.131322
0
022-2860/© 2021 Elsevier B.V. All rights reserved.

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
bases play a particular role in conferring wide exploitation of bi-
ological activities [12,13]. They are prepared by easy condensation
of an aldehyde with primary amines. The main formula for this
2.2. General procedure for the synthesis of α-aminophosphonates
(HMHP) and imine (HIN)
ꢀ
ꢀꢀ
typical categories RN=CR R substratum, it is an active site ampli-
fies in the process of electronic transfer and in the development
of coordination chemistry. This is clearly explained by numerous
publications showing the electrochemical properties of these com-
pounds [14,15].
2.2.1. Synthesis of (2-hydroxy naphthalen-1-yl) methyl
2-hydroxyphenyl amino phosphonic acid (HMHP)
In a modification of the already described procedure [45]. To a
stirred solution of 2- hydroxy naphtaldehyde, (0.001 mol, 0.172 g)
in toluene (10 mL) was added 2-aminophenol (0.001 mol, 0.109 g),
the mixture was heated progressively up to 100 °C and left for 1 h
at this temperature. After the phosphorous acid was added drop-
wise at room temperature and stirred for 24 h until completion of
the reaction as indicated by analytical thin layer chromatography
(TLC). Then the solvent has evaporated, the crud solid were fur-
ther washed with diethyl ether several times and afford the corre-
sponding α-aminophosphonate in good yield (Scheme 1).
In the context of this concept, chemical products in these bases
are actually used as antioxidants species, they allow the oxidizing
species to be affected by a reduction process that can be expressed
as largely positive sites within their structure [16].
Several in vitro methods have been developed to measure
antioxidant capacities of synthesized compounds, an extract or
other biological matrices (polyphenols, plants, fruits, . . .) [17,18].
The most commonly used antioxidant capacity assays were 1,1-
diphenyl-2-picrylhydrazyl radical (DPPH) and H O scavenging
2.2.2. Synthesis of imine: 2-hydroxyphenyl imino naphthalen-2-ol
(HIN)
2
2
models [19–21], ferric reducing antioxidant power assay [22],
phosphomolybdate (Total Antioxidant Activity) test [23] and β-
carotene bleaching assay [24,25]. These methods differ from each
other in terms of their mechanisms principles and reaction condi-
tions. Single test is inadequate to characterize the evolution of the
antioxidant activity. Although it is advisable to perform more than
one test and combine with them, to collect all antioxidant activity
of samples [26].
It is interesting to suggest that the product imine is already
documented [46]. However in this work, we have inspired through
the previously article [45] another method of synthesis, which
leads to formation of imine intermediate. Practically in equimo-
lar mixture of aniline derivative (2-amino phenol) and 2- hydroxyl
naphtaldehyde were reacted in refluxing toluene, to give HIN with
an important yield at short duration (Scheme 2).
Various synthetic methods for, α-aminophosphonates and Schiff
bases have been reported [27–29]. Of them, Kabachinik-Fields re-
action [30–33] is one of the best known pathways targeted for the
formation of carbon-phosphorus bonds, carried out by the nucle-
ophilic substitution of the phosphorus atom on imine entity.
The quantum chemical calculations by DFT method estimate the
different energetic and electronic properties of molecules which
can be exploited widely for several spaces of fields such as in bio-
logical activities [34–39] and corrosion inhibitors [40–44].
This work aims at synthesizing (2-hydroxyphenyl) imino
naphthalen-2-ol (HIN) and new molecule namely: (2-
hydroxynaphthalen-1-yl) methyl 2-hydroxyphenyl aminophos-
phonic acid (HMHP) and at evaluating the antioxidant activity
in vitro by the means of several reducing analysis methods in
order to investigate their biologic inhibition power. The structures
of these compounds have been determined using spectroscopic
analysis: FT-IR and ( H, 13C, 31P) NMR. Density functional methods
have been increasingly used for modeling molecular properties
and vibrational frequencies with FT-IR spectrum. In addition,
HOMO, LUMO analysis has been used to elucidate the information
about charge transfer within the molecule. The local reactivity
descriptors like energy gap, softness and electrophilicity indices
are expressed according to the calculation related to the energy
of Frontier Molecular Orbitals and compared the theoretical data
with the experimental ones.
2.3. Spectroscopic studies
The treatment of 2-amino phenol and 2- hydroxy naphtalde-
hyde was performed with two steps; while the first one is easily
realizable for the preparation of imine intermediate. Hence adding
acid phosphorous to mixture a new phosphonate was produced,
this second step takes a long time to be completed. The struc-
tures of the α-aminophosphonate and imine compounds were elu-
cidated by FT-IR and ( H, 13C, 31P) NMR.
1
2.3.1. α-aminophosphonic acid (HMHP)
The HMHP product is a yellow solid, M. p (228 °C), 98.2 % yield.
Spectral analysis illustrates:
FT-IRν
(cm 1): ν(OH) (3670.66), ν(C-H)aromatic (2976),
−
ν(C-H)aliphatic (2891), νC-N (1052.7), νP-O (870.9), νP=O (1242),
1
δOH (1139.73). 1H NMR (400 MHz, DMSO-d ), chemical shift (δ)
6
ppm: 2.50 (s, CH-N), 6.78–8.40 (m, 10H, aromatic protons); 9.48,
10.30 (s, 2H, OH), 15.69–15.72 (s, 2H, P-OH). 13C NMR (400 MHz,
DMSO-d ), (δ) ppm: 108.2–150 (m, 16 C, aromatic carbons), 177.93
6
3
1
(N-C-P).
(Fig. 1).
P NMR (400 MHz, DMSO-d ), (δ) ppm: 1.32 (s, 1P)
6
2.3.2. Imine (HIN)
The HIN product is slightly dark yellow solid. M. p (262 °C),
9
9.6 % yield.
2. Materials and methods
Spectral analysis illustrates:
FT-IR ν (cm 1): ν(OH) (3661.58), νC-H(aromatic) (2993, 2903.74),
−
2
.1. Reagents and devices
ν
(1612.91), δOH (1236.42).
H NMR (400 MHz, DMSO-d ), (δ) ppm: 6.98–7.94 (m, 10H,
C=N
1
6
13
The reagents used in the synthesis are: 2- hydroxyl naphtalde-
aromatic protons); 8.37, 8.39 (s, 2H, OH), 9.50 (s, H, N=CH).
C
hyde (98%), 2-amino phenol (99%) and phosphorous acid (99%), all
the chemical compounds were purchased from Sigma-Aldrich and
Fluka and were utilized without further purification.
NMR (400 MHz, DMSO-d ), (δ) ppm: 108.2–149.9 (m, 16C, aro-
6
matic carbons), 178.06 (CH=N).
The reactions were monitored by thin–layer chromatography
2.4. Antioxidant activity test in vitro
(
TLC) carried out on 0.25–mm E Merck silica gel plates, the spots
were visualized by UV lamp tool. Melting points were determined
on a digital apparatus Koefler Banc. Infrared spectra were recorded
2.4.1. Phosphomolybdate assay (total antioxidant capacity)
The antioxidant capacity of the sample was measured spec-
trophotometrically through phosphomolybdenum method, based
on the reduction of Mo(VI) to Mo(V) by the sample test [47]. This
analysis method is quantitative one to determine the antioxidant
on FT/IR JASCO 300 E (4000–600 cm 1). The absorbance’s reading
was illustrated by UV-Vis spectrophotometer (UV- 680 JASCO dou-
ble beam spectrometer).
−
2

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
Scheme 1. Synthesis of (2-hydroxynaphthalen-1-yl) methyl 2-hydroxyphenyl amino phosphonic acid (HMHP).
Scheme 2. Synthesis of 2-hydroxyphenyl imino naphthalen-2-ol (HIN).
activity in terms of reduction the molybdate ions, the subsequent
2.4.3. Scavenging activity (DPPH)
formation of green color degradation phosphate/Mo(V) complex
was detected with a maximum absorption at 765 nm.
The DPPH assay is a simple, efficient method; it is a stable free
radical that is reduced by antioxidant molecules, by acceptation of
an electron or hydrogen [49,50]. In the initial radical form, DPPH
has a deep purple color, which changes to yellow in the reduced
form, this causes a decrease in the concentration of the radical
free. The more this color changes, the more DPPH is reduced and
the better the antioxidant activity of the sample is. DPPH has an
important absorption band in 517 nm, an easy spectrophotometry
tool measure the absorbance of analyzed species.
Experimental protocol. At various concentrations, an aliquot of
0.1 ml of the sample solution was shaken with 1 mL of reagent
solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM
ammonium molybdate). The mixtures were incubated in a water
bath at 95 °C for 90 min. After the mixtures were cooled to room
temperature, absorbance was measured at 765 nm. Ascorbic acid
was used as standard.
The antioxidant capacity is commonly expressed as the efficient
concentration (IC50), which represents the quantity of the sample
needed to decrease the initial concentration of the DPPH free rad-
ical at 50 %.
2
.4.2. Ferric reducing antioxidant power (FRAP)
The reducing power of α-aminophosphonate and imine is esti-
mated according to the method described by Ebrahimzadeh [48].
Experimental protocol. 250 μl of each product was dissolved in
Dimethyl sulfoxide DMSO at different concentrations and 500 μl
of the methanolic solution of DPPH (4 mg/100 ml), after shaking,
the mixture is left in the dark at room temperature for 30 min. The
reading was carried out by measuring the absorbance at 517 nm.
The radical scavenging activity (RSADPPH) or inhibitory capacity of
the tested compounds was calculated using the following equation
This technique was developed to measure the capacity of com-
pounds to reduce the ferric ion (Fe3 ) present in the K Fe (CN)6
+
3
complex (red color) in ferrous ion (Fe2 ) which turns blue or
+
green, the color change is proportional to the antioxidant activity.
3
+
−
2+
Fe + e Fe
[
51]:
Experimental protocol. A volume of 100 μl of compounds dissolved
in Dimethyl sulfoxide DMSO at different concentrations was mixed
with 100 μl phosphate buffer (0.2 M, pH 6.6) and 100 μl of 1%
potassium ferri cyanide [K Fe(CN) ], then the mixture was incu-
Acontrol − Asample
RSADPPH% =
× 100
(1)
A
control
3
6
Acontrol is the absorbance of the DPPH, namely control; Asample is
the difference between the sample absorbance and its correspond-
ing blank absorbance of each value of concentration.
bated at 50 °C in a water bath for 20 min and the reaction was
terminated by the addition of 250 μl of 10% trichloro acetic acid
(
TCA). The mixture was centrifuged for 10 min (3000 rpm). Finally,
50 μl of the supernatant solution was mixed with 250 μl of dis-
2
2.4.4. H O2 scavenging activity
2
+
tilled water and 500 μl of FeCl3 (0.1%) freshly prepared in water. A
blank without ferri cyanide ion is prepared in the same conditions.
The absorbance was recorded at 700 nm. The results were ex-
pressed as EC50 which means effective concentration at which the
absorbance is 0.5. Butylated hydroxyl toluene BHT and quercetin
were used as standards.
This assay is based on the reaction of ferrous ion (Fe 2) in Am-
monium iron sulfat with 1,10-phenanthroline. Ferrous ion specifi-
cally forms red-orange tri-phenanthroline complex which absorbs
maximally at 510 nm. It is known that H O2 will oxidize all the
2
ferrous ion to ferric ion if it is added before 1,10-phenanthroline,
while it became incapable of forming red-orange complex and a
3

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
Fig. 1. 1H NMR, 13 C NMR and 31 P NMR spectra of HMHP.
4

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
sharp reduction in λ510 can be seen [52]. This concept has been
2.5. Statistical analysis
exploited for determination of H O in the samples [53,54]. The
2
2
advantage of this assay is that after adding ferrous ion, the scav-
enger sample is added and followed by known amount of H O
All measurements were performed in triplicate, and the results
were expressed as mean ± standard deviation (SD). The treatment
of biological test data, when a significant difference is observed be-
tween the means of the inhibition percentages of standard com-
pounds and synthesized products was studied by ANOVA, this test
identifies the products that significantly differ from standard prod-
ucts. The smallest difference significant between the activities of
the products was set at p ≤ 0.05. If p ≤ 0.05 we conclude that
the difference is significant between the means of activities; on
the other hand when p ≥ 0.05, we conclude that the difference
between the activity average is not significant.
2
2
for few minutes, no conversation to ferric ion would occur, it is
detected by addition of 1,10-phenanthroline which leads to a com-
plex with ferrous ion.
Experimental protocol. H O assay was performed by adding the
2
2
test solution containing ferrous ammonium sulphate (1 mM), hy-
drogen peroxide (5 mM) along with different concentrations of
tested samples, after incubation in the dark for 5 min 1,10-
phenanthroline (1 mM) was added to reaction mixture and caught
again in the dark about 10 min, then the reading at λ510. The ex-
pression of scavenging activity was calculated proportionally by
varying the concentration of the samples using following formula:
The results were analyzed by using an ANOVA followed by Dun-
net’s test. These statistical analyzes were done with the software
Graph Pad Prism (version 5.01 for Windows). Figures were plotted
using GRAPH and Origin 6.0 softwares.
Atest − A
control
blank
%
H2O2 scavenging activity =
X 100
(2)
A
where:
2.6. Computational details
Atest is the absorbance of the solution containing ferrous ammo-
nium sulphate, hydrogen peroxide and 1,10-phenanthroline. Acontrol
is the absorbance of solution containing only ferrous ammonium
sulphate and 1,10-phenanthroline without sample; All the assays
were done in triplicate and the data were showed in the graph as
mean ± SD.
The entire calculations of structural parameters, geometry op-
timization, vibrational frequencies of the studied new compounds
were performed at B3LYP levels included in the Gaussian 09 pack-
age program [57] with 6–311G (d,p) basis set. We have utilized
the gradient corrected density functional theory with the three pa-
rameter hybrid functional (B3) [58] for the exchange part and the
Lee-Yang-Parr (LYP) correlation function to study the effect of the
changes of molecular and electronic structures on the antioxidant
activity of the present samples.
2
.4.5. β-carotene bleaching
The β-carotene bleaching assay is one of the common meth-
ods used to estimate the antioxidant activity of compounds in the
field of food chemistry [55]. The principle of the method based on
the discoloration of yellowish color of a β-carotene solution due to
the breaking of π-conjugation by addition reaction of lipid or lipid
peroxyl radical to a C=C double bond of β-carotene.
For an ideal approach to real results of the vibrational frequen-
cies, we have utilized the scale factor (0.9614) for the computation
of the data relating to the values of the wavenumbers [59].
The estimation of β-carotene reaction in the examined sys-
tems was performed by the method [56] slightly modified. β-
carotene/linoleic acid emulsion in water with generated peroxyl
3. Results and discussion
•
radicals (LOO ) is the main component of the reaction medium.
3
.1. FT-IR analysis
The changes of β-carotene concentration in a control sample and
in a sample containing an examined antioxidant are monitored
during the assay. The antioxidant activity of the tested substances
is evaluated by the spectrophotometric measurement at 490 nm.
The FT-IR spectra of the present compounds were characterized
−1
by the following absorption bands. A band at 870.9 cm
is due
to the stretching vibration of the P-O group. P=O stretching vibra-
aromatic/aliphatic
−1
tion was observed at 1242 cm , The bands of CH
Experimental protocol. The stock solution of β-carotene/linoleic
acid emulsion in water was prepared by dissolving 0.5 mg β-
carotene in 1 ml of chloroform, then 25 μl of the linoleic acid and
−1
were observed at 2976 and 2891 cm
successively, while the
−1
bands of OH/CN groups are located at 3670.66 and 1052.7 cm
,
−1
the band at 1139.73 cm
is attributed to the deformation of the
2
00 mg of Tween 40 were added to the mixture. Chloroform was
phenolic group (δ(O-H)) [60], are the characteristic bonds of the
evaporated at 40 °C using a rotavapor and then 100 ml of distilled
water saturated with oxygen was added to complete the reaction.
After that, a volume of 350 μl of each product (2 mg/ml) dissolved
in DMSO and standard BHT which is soluble in methanol, were
mixed with 2.5 ml of the emulsion. Under the same conditions
α-aminophosphonate compound. The IR spectrum of Schiff base
−1
shows a strong band appeared at 1612.91 cm
is assigned to υ
−1
(
C=N), The both peaks illustrated as bands at 2903–2993 cm are
aromatic .
assigned to CH
Finally υ(OH) and δ (OH) were detected at
−1
3
661.58 and 1236.42 cm (Figs. 2 and 3).
the negative process (methanol, H O and DMSO) is performed only
2
Set of theoretical and experimental data of the wavenumbers
by the emulsion without samples, while blank consists only the
samples. Finally the emulsion system was incubated in the dark at
room temperature, at regular time (t) of 0, 1, 2, 4, 6, and 24 h, the
absorbance in which is measured at 490 nm. The rate of bleaching
of β-carotene was calculated as antioxidant activity (AA) using the
equation:
of the main studied functions are summarized in (Table 1). A
new OH stretching vibration of phenolic entity was computed as a
−1
3
831.63 cm
of the HMHP, while the calculated peak of NH ap-
−1
pears at 3570.27 cm
with slight intensity but it is not found
on the experimental spectrum. The differences between the ex-
perimental and calculated values can be explained by considering
the theoretical calculations belonged to the gaseous phase while
the observed results are valid for the solid phase of the molecule.
Moreover the experimental results agree with those calculated. The
AA(%) = (Ae/Ac) × 100
(3)
Where
2
Ae: absorbance of β-carotene in the presence of samples and
linear correlation coefficient (R ) value was found to be 0.9945 and
Ac: control absorbance (BHT).
0.9981 for HMHP and HIN, successively.
5

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
Fig. 2. IR spectrum of HMHP: (A) experimental, (B) theoretical.
Fig. 3. IR spectrum of HIN: (A) experimental, (B) theoretical.
Table 1
Experimental and calculated IR results, with determination of the correlation coefficient.
HMHP
HIN
Experimental
Calculated
Experimental
Calculated
ν
ν
ν
ν
ν
ν
ν
ν
δ
ν
(OH)
(OH)
(C-H)aromatic
C-H
-
3831.63
3630.56
3156.48
3081.7
1021.6
894.17
1259.8
-
-
3670.66
2976
2891
1052.7
870.9
1242
3661.58
3820.84
2993 2903.74
3187.63 3142.41
-
-
C-N
-
-
P-O
-
-
P=O
C=N
OH
-
-
1612.91
1236.42
1685.76
1290.23
1139.73
-
1119.29
3570.27
0.9981
N-H
Correlation coefficient R2
0.9945
Scaling factor: 0.961 for B3LYP/6-311G (d,p)
3
.2. Evaluation of the antioxidant activity
mation of a green phosphate/Mo(V) complex which is mea-
sured spectrophotometrically. The obtained results are shown in
Fig. 4.
To investigate the antioxidant activity of the synthesized com-
pounds, several in vitro assays were used, the scavenging activities
H O and DPPH radical, the FRAP method, the TAC model and the
We suggest that the calculation of the percentage of inhi-
bition performed using the straight linear equation, is effec-
tively estimated at 50% (Fig. 5). The antioxidant activity of ascor-
bic acid, used as the standard product, was comparatively more
effective than that of HMHP and HIN products successively.
The results of % inhibition are evaluated and summarized in
Table 2.
2
2
β-carotene bleaching assays. Results of IC50 and EC50 values have
been determined graphically by linear calibration, were considered
to be correct when the coefficient R2 = 0.9 at least. From a single
sample were measured three times to test the reproducibility of
the assays.
In terms of evaluation the antioxidant capacities of the present
products. Absorbance results as a function of concentration were
displayed by the spectrophotometer apparatus during reduction re-
actions, have revealed that the two compounds HMHP and HIN
reflect an important reducing power, visually observed by color
degradation.
3.2.2. Reducing power (FRAP method)
The reduction of ferric cyanide complex to the ferrous form by
donating an electron was indicated by appearance of blue color in
the end of the reaction. The amount of Fe2 increases with increas-
+
ing of the concentration at 700 nm.
According to the graphical results, we see that the curves of
corresponding compounds are all plotted above the standard BHT
curve. At the first view, we can predict the sequence of the ana-
lyzed products and the references products in the order of reduc-
3
.2.1. Phosphomolybdate assay (total antioxidant capacity)
This assay is based on the reduction of phosphomolyb-
date ion in the presence of an antioxidant, resulting the for-
6

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
Fig. 4. Total antioxidant activity of HMHP, HIN and Ascorbic acid standard at different concentrations followed by their linear straight of % inhibition.
Table 2
Evolution of the percent inhibition (%) of the studied compounds depending on concentration and values of total antioxidant activity (IC50) expressed as means ±
SD.
concentrations of HMHP, HIN and Ascorbic acid (mg/ml)
HIN %
IC5 0 (μg/ml)
HMHP%
IC5 0 (μg/ml)
Ascorbic acid %
IC5 0 (μg/ml)
0
0
0
0
0
0
0
0
0
0
.80
.72
.64
.48
.32
.24
.16
.08
.032
.01
54.20
49.70
44.80
40.15
38.30
32.95
25.45
20.50
16.25
-
63.908 ± 0.0408
63.05
58.80
52.60
45.75
41.75
36.25
30.20
25.30
16.20
-
50.07 ± 0.3889
-
21.6477 ± 0.5845
-
-
93.2
73.6
68.6
48.6
31.5
16.6
3.70
Table 3
Inhibition of products in different concentrations and effective values EC50 of the reducing power of Fe at the absorbance 0.5 as means ± SD.
+
3
HIN
HMHP
BHT
Trolox
Quercetin
Inh/con
EC50 (μg/ml)
Inh/con
EC50 (μg/ml)
Inh/con
EC50 (μg/ml)
Inh/con
EC50 (μg/ml)
Inh/con
EC50 (μg/ml)
0
0
0
0
0
0
0
0
0
.71
.58
.51
.50
.34
.28
.43
.12
.07
12.818 ± 0.351
0.83
0.61
0.56
0.47
0.39
0.34
0.26
0.18
0.10
11.365 ± 0.389
0.63
0.54
0.51
0.36
0.30
0.21
0.18
0.12
0.10
20.518 ± 1.281
0.87
0.79
0.70
0.69
0.58
0.50
0.40
0.31
0.26
8.092 ± 0.112
0.83
0.75
0.64
0.55
0.54
0.46
0.38
0.28
0.18
2.509 ± 0.064
Inh/con: inhibition at various concentrations
+
3
ing efficiency of the Fe
ions from this graph (Fig. 6). After es-
3.2.3. Free radical scavenging activity (DPPH)
timating of EC50 (Fig. 7 and Table 3), our results show a strong
gap in front of the BHT, while HMHP relatively is the most ac-
tive compared to HIN sample. Finally, we can classify the results
of inhibitory capacity at the absorbance 0.5, under the order of a
following sequence: Quercetin> Trolox> >HMHP>HIN>BHT.
DPPH is widely used to assess the radical scavenging activity of
different antioxidant compounds. The results of IC50 values extrap-
olate from calibration line are illustrated in Fig. 8.
In order of best ranking of inhibition, α-aminophosphonate
(HMHP) sample exhibited an excellent scavenging activity than
7

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
Fig. 5. Histograms of IC50 of different compounds and Ascorbic acid standard (n = 3).
Fig. 6. Reducing capacity of synthesized compounds depending of concentration, followed by linear straight.
that of HIN imine and in front of the standard BHT, shown in Fig. 9.
Therefore, HMHP acid releases hydrogen radicals well via its chem-
ical composition richer in hydroxyl groups.
3.2.4. H O2 scavenging activity
2
The antioxidant efficiency of both products takes place colori-
metrically by the degradation of the brick red color through a se-
ries of concentration. (n = 3)
Table 4 below; collects the % inhibition, including IC50 values.
8

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
Fig. 7. Histogram of effective concentration of HMHP, HIN compounds and BHT, Trolox, Quercetin standards (n = 3).
Fig. 8. Radical Scavenging Activity of HMHP, HIN and BHT at different concentrations with linear straight of % inhibition of corresponding compound.
Table 4
Evolution of the percent inhibition (%) of the studied compounds depending on concentrations and values of radical scavenging activity (IC50) expressed as
means ± SD.
concentrations of HMHP and BHT (mg/ml)
HMHP (%)
IC50 (μg/ml)
BHT %
IC50 (μg/ml)
Concentration of HIN (mg/ml)
HIN (%)
IC50 (μg/ml)
0
0
0
0
0
0
0
.2
67.20
56.77
49.03
35.28
27.68
15.18
11.50
37.64 ± 1.43
54.14
44.13
39.13
37.02
23.23
11.43
5.47
57.31 ± 5.36
0.40
0.36
0.28
0.24
0.2
63.63
60.88
51.5
44.1
40.9
35.1
31.8
90.13 ± 0.15
.1
.08
.06
.04
.02
.01
0.16
0.12
9

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
Fig. 9. RSADPPH or IC50 of different compounds and BHT standard (n = 3).
Fig. 10. H2O2 scavenging activity of HMHP, HIN, BHT and Ascorbic acid used as standard samples at different concentrations combined to linear straight of % inhibition.
On the basis of results obtained from H O scavenging activity
the following order: IC50 Ascorbic acid > IC50 HIN > IC50 HMHP
> IC50 BHT, while the BHT standard exhibited a lowly antioxidant
power in this model of activity (Fig. 11 and Table 5).
2
2
assays (Fig. 10). In this case of model, imine is the more advanta-
geous on capacity reductive as signed at IC50 (24.73 ± 0.7).
Synthetic antioxidants such as butylated hydroxytoluene (BHT)
and vitamin C (Ascorbic acid) are often used in the food industry,
they are also known as standard reference compounds in the full
of antioxidant tests. However, the Ascorbic acid is the best one in
Following the analysis of Table 6, we notice that all products il-
lustrated the same significant level compared to standard products;
this theoretical comparison of finding results of EC50 and IC50 was
analyzed according to ANOVA program, followed by Dunnet’s test.
power scavenging H O . The corresponding IC50’s are classified in
2
2
10

Table 5
Evolution of the percent inhibition (%) of the studied compounds depending on concentrations and values of H2O2 scavenging activity (IC50) expressed as means ± SD.
Concentrations of HMHP, HIN and BHT (mg/ml)
HIN (%)
IC50 (μg/ml)
HMHP (%)
IC50 (μg/ml)
BHT (%)
IC50 (μg/ml)
Concentration of Ascorbic acid
Ascorbic acid (%)
IC50 (μg/ml)
0
0
0
0
0
0
0
0
0
0
0
.80
.72
.64
.48
.42
.32
.24
.16
.08
.01
.032
90.41
87.67
87.38
80.82
-
86.98
79.72
70.68
70.41
-
-
0.040
0.020
0.010
0.008
0.004
0.002
0.001
94.625
83.806
59.106
27.536
21.120
13.059
10.895
-
96.623
73.037
54.642
35.949
33.628
30.844
28.646
23.586
24.7306 ± 0.71785
138.957 ± 6.202113
4.9723 ± 0.219806
79.45
73.69
66.85
51.64
-
64.38
57.53
46.57
32.60
-
90.76 ± 0.83018
45.75
24.48

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
Fig. 11. Histogram of IC50 of HMHP, HIN, BHT and Ascorbic acid as standards.
Table 6
sify the results displayed below in Table 7, with ascending order
of AA% as follows: H O <MeOH< DMSO < BHT<HIN < HMHP.
Values of inhibition of total assays DPPH, FRAP, H2O2 and TAC
2
were analyzed by ANOVA program (p ≤ 0.05) compared to BHT,
Trolox, Quercetin and Ascorbic acid as standards.
3
.3. Comparative study of the antioxidant activities of suggestive
HMHP
HIN
products in the literature and those carried out in the present work
∗
∗
∗
∗
∗
∗
∗
∗∗
∗∗
∗∗
∗∗
∗∗
∗∗
∗∗
∗∗∗
∗∗∗
∗∗∗
∗∗∗
∗∗∗
∗∗∗
∗∗∗
DPPH test
FRAP test
Significant p≤ 0.05/BHT
significant p≤ 0.05/BHT
significant p≤ 0.05/Quercetin
Significant p≤ 0.05/Trolox
Significant p≤ 0.05/BHT
Significant p≤ 0.05/Vit C
Significant p≤ 0.05/Vit C
Few works on the antioxidant activities of chemical products
particularly α-aminophosphonates are argued. The comparison of
these activities intended to enrich and to give more originality and
value for this work. The Table 8 shows that all compounds contain-
ing at least one hydroxyl and Alkoxy entities are active in DPPH.
Even in the missing of these active groups, L4 can show activity
towards DPPH, we noted also that L5 exhibited the less potent ef-
H2O2 test
TAC test
fect and the better of them is L 1. However the compounds: L 3,
8
2
L 4, L 1, L 2 and L 3 displayed any antioxidant activity in DPPH as-
2
6
6
6
3
.2.5. β-carotene bleaching test
In terms of expanding the exploitation of the present products,
say. Probably, it is because of non-availability of hydrogen in these
bases that can readily be donated as H radical to DPPH due to
which this last acts through HAT (hydrogen atom transfer) and not
ET (electron transfer) mechanism.
another antioxidant activity strategy has been implemented in bi-
ological analysis such as β-carotene bleaching test. The emulsion
β-carotene/linoleic acid forms hydroperoxide free radicals dur-
ing the incubation [61]. These hydroperoxides change the color
of β-caroteneby an oxidation reaction. Unambiguously, the neg-
According to the EC50 parameter given in Table 8, the FRAP test
shows that the L4, L 1 and L 2 display excellent reducing power
8
8
of ferric ion (Fe3 ) following by HMHP and HIN where the abil-
ity of L3 is the lowest to donate electron. Additionally, β-carotene
bleaching assay, HMHP and HIN are the closer to BHT standard
they get well activity higher than L5 and L3, successively. More-
+
ative controls like DMSO, MeOH and H O without samples acti-
2
vate the β-carotene oxidation. The aqueous solvent H O exhib-
2
ited a low potency inhibitor compared to those of organic sol-
vents MeOH and DMSO which are used in dissolution of the stud-
ied products. They are often found below the positive BHT, while
this last shows percent inhibition fixed at 100% along of biological
treatment.
over, in TAC model, the L 1, L 2 and L 3 are closely comparable
6
6
6
to our studied products. Towards H O2 scavenging, HIN shows an
2
ability reductive more significant than α-aminophosphonates: L 1,
7
HMHP, L 3 and L 2, respectively.
7
7
After 24 h of preservation in the dark. The set of percent in-
hibition results processed with graphical curves and histograms
Figs. 12 and 13, show that the antioxidant efficiency of α-
aminophosphonate acid is more significant than the reference BHT
and relatively important in front of imine product. We can clas-
Generally the addition of phosphonic acid group (PO H ) to
3 2
Schiff base structure increases its activity to reduce free radicals.
This behavior is due to the presence of two hydroxyl groups (−OH),
responsible for the enhancement of acidity, which increases the
+
number of the liberated protons (two H ).
12

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
Fig. 12. Antioxidant activity of synthesized products using β-carotene bleaching method, compared to BHT as positive control and to different solvents as negative controls.
Fig. 13. Percent inhibition histogram AA% after 24 h, compared to BHT as positive control and to negative controls, p≤ 0.05 (n = 3).
Table 7
Percent inhibition AA% after 24h, estimated as means ± SD.
HIN
HMHP
BHT
DMSO
MeOH
H2O
AA% at 24h
107.2 ± 4.94
112.13 ± 1.55
100 ± 3.37
32.93 ± 1.45
22.362 ± 0.63
19.84 ± 0.68
3
3
.4. Calculation of quantum chemical parameters
.4.1. Study of frontier orbitals
HOMO and LUMO is related to the ionization potential and elec-
tron affinity, the gap energy ꢀE is the absolute difference in en-
ergy between the Frontier Molecular Orbitals which expressed the
reactivity of the compounds, that this activity became important at
small gap energetic [68]. Through the estimated energy of HOMO
and LUMO using DFT/6-311G (d, p) method, we can determine the
The energy of the highest occupied molecular orbital (HOMO)
and lowest unoccupied molecular orbital (LUMO) defined the pro-
cess of electron transfer such as electrons donor or acceptor unit,
13

Table 8
Comparison of the antioxidant activities of the referenced products and those of ours investigated.
β-carotene
H2O2
Molecular structures
HIN
DPPH μg/ml
FRAP μg/ml
bleaching
TAC μg/ml
scavenging
Schiff Bases
90.13 ± 0.15
500 ± 1.57
12.82 ± 0.35
107.2 ± 4.94
63.9 ± 0.04
24.73 ± 0.72
-
) 4-[(1E)-N-{2-[(Z)-benzylideneamino]
-
-
-
-
ethyl}ethanimidoyl]-benzene-1,3-diol (L1) [62]
R1 R2 [63]
117.67 ± 0.58
159.83 ± 0.72
none
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
H OH (L21)
CH3 OH (L22)
HCH2Ph (L23)
CH3 CH2Ph (L24)
none
ꢀ
ꢀ
-
) 2, 2 -((1E,1 E)-((4-methyl-1, 3-phenylene) bis
210.477 ± 31.6 73.24 ± 3.36
50.184
-
-
(
(
benzylidene)) bis (methylethylidene)) bis (4-methoxyphenol)
L3) [13]
-
) 4-(1H-Indol-3-ylmethyleneamino)-1,5-dimethyl-2-phenyl-
H-pyrazol-3(2H)-one (L4) [64]
) 2,2’-(((azanediylbis(propane-3,1-diyl)) bis(azanylylidene))
bis(methanylylidene))diphenol(L5) [65]
48.49 ± 0.13
4.084 ± 0.015
-
-
-
-
-
1
-
3.38 ± 0.01
mg/ml
-
69.72 ± 1.00
-
-
-
) N-(1-phenylethylidene)aniline (L61) [66]
None
24.11 ± 0.01
25.26 ± 0.02
25.26 ± 0.02
11.365 ± 0.39
-
46.50 ± 0.02
49.40 ± 0.03
55.25 ± 0.01
50.07 ± 0.4
-
) 2-Chloro-N-(1-phenylethylidene) aniline (L62)
) 2,4-dichloro-N-(1-phenylethylidene) aniline (L63)
None
-
-
None
-
-
α-aminophosphonates
HMHP
37.64 ± 1.43
112.13 ± 1.5
90.76 ± 0.83
R-NO2 (L71)
R-Br (L72)
31.88
-
-
-
38.63
206
151.2
-
-
-
R-H (L73) [67]
205.93
-
-
-
155.27
- -
-
) ((2-Hydroxy-5-methyl-
phenylamino)-phenyl-methyl)-phosphonic acid diethyl ester
L81) -) ((2-Hydroxy-5-nitro-phenylamino)-phenyl-
methyl)-phosphonic acid diethyl ester (L82) [35]
2.06 ± 0.01
26.72 ± 0.08
4.84 ± 0.75
7.78 ± 1.30
- -
- -
(

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
Fig. 14. Highest occupied and lowest unoccupied molecular orbitals of HIN (A) and HMHP (B) calculated at B3LYP/6-311G (d,p) Level.
Table 9
The calculated quantum parameters of HIN and HMHP.
EHOMO (ev)
ELUMO (ev)
EGAP (ev)
chemical potential μ
Hardness η
Electrophilicity ω
dipole moment (Debey)
Total energy (a.u)
HIN
-7.279
-5.726
-1.847
-1.237
5.432
4.489
-4.563
-3.480
2.716
2.244
3.833
2.698
3.1528
2.2200
-861.04
HMHP
-1430.04
2
values of the overall electrophilicity ω = μ /(2η) (4), from calcu-
clouds, red describe the regions of the most negative electrostatic
potential (highest density), blue represent the regions of the most
positive electrostatic potential (lowest density). Negative and pos-
itive regions of MEP are related to electrophilic and nucleophilic
reactivity [70]. The MEP map shows that the distribution of neg-
ative potential sites of two molecules are illustrated only on the
oxygen atom (O30) attached to the phosphorus atom, will positive
potential sites are located around the hydrogen atoms (H32, H34
and H36) of hydroxyl groups (HMHP) and (H30 and H32) of hy-
droxyl groups (HIN) Fig. 15. It can be considered that the positive
sites play an essential role in donating hydrogen atoms and elec-
trons to oxidizing agents.
lating the chemical potential μ = (E
+ E
)/2 (5), which is
− EHOMO)/2 (6)
HOMO
LU MO
a negative value and chemical hardness η = (E
LU MO
for these molecules [69].
The distribution of charge density of the HOMO and LUMO lev-
els for the studied molecules are shown in Fig. 14. HOMOs en-
tirely cover every molecule, of which LUMOs were mainly local-
ized throughout the molecular structure of HIN and HMHP except
for the hydroxy phenyl group and the hydroxyl group attached to
the phosphorous atom of the last. The lowest EGAP=4.489 ev, il-
lustrated the highest reactivity of the HMHP molecule agrees well
with biological experimental data except for H O scavenging ac-
2
2
tivity where HIN is the best one (Table 9).
The polarity of molecules is expressed in terms of dipole mo-
ment; this comprehension signifies the efficiency of dissolution
that the more polar one is able to dissolve easily. The highest
dipole moment (DM) of imine compound means that this com-
pound is the most polarizable than α-aminophosphonate com-
pound.
3
.4.3. Mulliken atomic charges for HMHP and HIN
Mulliken atomic charge tends to produce qualitative results at
best; it is very useful to estimate the partial atomic charges of
molecular system [71]. The DFT computed negative charge distri-
bution in the carbon atoms of the both compounds, where the
positive charges of HMHP were presented at C14 (0.1986e), C22
(
0.180445e), Otherwise the most negative charge C17 is attached
3
.4.2. Molecular electrostatic potentials (MEP)
to phosphorus atom. The large positive process is attributed to the
hydrogen atoms which linked to the hydroxyl groups of the phos-
phonyl function. It’s found also that all nitrogen and oxygen ele-
The Molecular electrostatic potential (MEP) is a visual technique
for providing the molecular shape presented with graduated color
15

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
Fig. 15. Molecular electrostatic potential maps of HIN and HMHP.
Table 10
ments accumulate negative charge partials. It is worthy mentioned
that the P29 has an immense positive charge (1.13408e) tend to
lose electrons in chemical reactions (may privilege reduction reac-
tions particularly in antioxidant activities).
Mulliken atomic charges of HIN and HMHP determined by DFT using B3LYP
levels with 6-311G (d, p) basis set.
HIN
HMHP
Atom
C14, C17 and C22 HIN’s charges are positive, they are bonded
with heteroatoms O32, N18 and O29 successively, while C17 is the
most positive that is belong to the linear chain of molecule. H30
and H32 are the greater positive charges; they are attached to hy-
droxyl groups. The charge noticed on the N18, O29 and O31 of
imine product are almost identical (≈ -0.37e).
Atom
Charge
Charge
C1
-0.0922530
-0.0398820
-0.0524190
-0.0530200
-0.0631820
-0.0851700
0.0918520
0.1272350
-0.1479970
-0.0415050
0.0800020
0.0897890
-0.0956110
0.1777990
0.0873150
0.0859500
0.1928480
-0.3738150
-0.1007550
-0.0929140
-0.1137010
0.1767960
0.0688960
-0.0628660
0.0895930
0.0909210
0.0824080
0.0983050
-0.3703270
0.2480470
-0.3647980
0.2507050
0.1117520
C1
-0.0883830
-0.0631660
-0.0465060
-0.0516140
-0.0702610
-0.1079910
0.0845850
0.0891640
0.0788580
-0.0456250
0.0001840
0.1486460
0.0941890
0.1986240
-0.0965340
0.0854770
-0.3676920
-0.4053520
-0.0906040
-0.0504490
0.0736890
0.1804450
-0.0943510
-0.0912270
0.0936990
0.1157640
0.0866350
0.0934620
1.1340840
-0.5750740
-0.5174420
0.3163190
-0.4438180
0.2727840
-0.3783680
0.2522800
-0.5304720
0.3029110
0.2158560
0.1972740
C2
C2
C3
C3
C4
C4
C5
C5
C6
C6
We suggested that the carbon C17 assigned to the imine func-
tion (HIN) and the amine function (HMHP) changes its ionic char-
acter from positive to negative process, it is oriented towards gain
electrons in chemical reactions. Therefore, the hydrogen charges
become more positive as they approach of the main functions
H7
H7
H8
H8
C9
H9
C10
H11
H12
C13
C14
H15
H16
C17
N18
C19
C20
C21
C22
C23
C24
H25
H26
H27
H28
O29
H30
O31
H32
H33
C10
C11
H12
H13
C14
C15
H16
C17
N18
C19
C20
C21
C22
C23
C24
H25
H26
H27
H28
P29
O30
O31
H32
O33
H34
O35
H36
O38
H39
H40
H41
(
imine, amine and phosphonate), these results required informa-
tion to define the most reactive sites within the molecule. The
Mulliken atomic charges in numerals are listed in Table 10.
4. Conclusion
In this work, we have synthesized a new α-aminophosphonate
acid (HMHP) and inspired through this reaction another easy oper-
ating mode in order to produce HIN imine before adding phospho-
rous acid to reaction. The identification of their structures was car-
ried out by spectroscopic methods FR-IR, and (1H, 13C, 31P) NMR.
Using the IR spectra, we have demonstrated the different principal
functions such as: C=N of HIN which is located at 1612.91 cm 1.
On the other hand this characteristic peak has disappeared from
the spectrum of HMHP due to substitution of phosphonic entity
on the carbon atom of imine function, while the phosphorus range
is detected in the fingerprint region spectrum. The NMR analysis
method can also detail the chemical formulas of molecules based
−
on the number of hydrogen atoms bounded to carbon atoms or to
heteroatoms using 1H NMR spectra, the carbon numbers detected
of molecules using 13C NMR spectra and the phosphorus num-
bers requires 31P NMR spectra. The interpretation of NMR spec-
tra is already discussed previously. Also, the antioxidant activity
of the synthesized compounds have been investigated in vitro us-
ing several methods such as DPPH and H O scavenging activities,
2
2
reducing power (FRAP), Phosphomolybdate (TAC) and β-carotene
bleaching. The valuable comparison of the inhibitory power of cor-
responding assays has the particularity to improve the antioxidant
activity of imine on which we can foresee expressed the effect
ciency of EC50 and AA% in FRAP and β-carotene tests, even eas-
ily converted the Mo(VI) into Mo(V) ions by reduction process.
We can also conclude from the finding results that the evolu-
tion of chemical structure of imine (HIN) to α-aminophosphonate
(HMHP) clearly affected well their antioxidant activities except in
of PO H2 substitutes on the carbon atom of imine function. Ef-
3
fectively, it has bring a considerable improvement in most tests,
view its richness in hydroxyl entity, HMHP become more inter-
esting through DPPH scavenging model, it readily released hydro-
gen radical to DPPH. However, it has slightly increased the effi-
the case of H O2 scavenging activity; PO H2 had no positive ef-
2
3
fect on the HMHP because HIN is showed the best inhibitor power.
Eventually, the results of IC50, EC50 and AA% of our investigated
16

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
compounds have high potential antioxidants compared to standard
compounds; where all have been attested by significant p ≤ 0.05
analyzing with ANOVA program. Practically, all these assays were
carried out in triplets for each operation in order to ensure good
reproducibility of results. The quantum chemical calculations in-
dicate that the α-aminophosphonate acid is the best compound to
reduce the oxidizable species by releasing hydrogen radical or elec-
tron through the positive sites of lower electronegativity, it’s visu-
ally detected by the MEP map as well as calculated according to
Mulliken atomic charges. Except HIN which has been taken the ad-
vantage in term of H O scavenging activity. Furthermore, we can
[10] W. Al Zoubi, M.P. Kamil, S. Fatimah, N. Nashrah, Y. Gun Ko, Recent advances in
hybrid organic-inorganic materials with spatial architecture for state-of-the-
art applications, Progress Mater. Sci. 112 (2020) 100663, doi:10.1016/j.pmatsci.
2020.100663.
[11] H. Fleisch, Bisphosphonates: a new class of drugs indiseases of bone and cal-
cium metabolism, Recent Results Cancer Res. 116 (1989) 1–28, doi:10.1007/
978-3-642-83668-8_1.
[
12] M. Mesbah, T. Douadi, F. Sahli, S. Issaadi, S. Boukazoula, S. Chafaa, Synthesis,
characterization, spectroscopic studies and antimicrobial activity of three new
Schiff bases derived from Heterocyclic moiety, J. Mol. Struct. 1115 (2018) 41–
4
8, doi:10.1016/j.molstruc.2017.08.098.
[
13] M.A. Abdelmadjid, D. Haffar, F. Benghanem, S. Ghedhti, L. Toukal, V. Dorcetd,
R. Bourzam, Synthesis, crystal structure, electrochemical, theoretical studies
and antioxidant activities of new Schiff base, J. Mol. Struct. 1227 (2020)
2
2
129368, doi:10.1016/j.molstruc.2020.129368.
conclude that the small energy gap (4.489 eV) as the greater the-
oretical reactivity conforms to most reducing activities of HMHP. A
good correlation was established between the theoretical FT-IR and
experimental spectra.
[
[
[
[
14] W.A. Zoubi, Y.G. Ko, Organometallic complexes of Schiff bases: Recent progress
in oxidation catalysis, J. Organomet. Chem. 822 (2016) 173–188, doi:10.1016/j.
jorganchem.2016.08.023.
15] W.A. Zoubi, Y.G. Ko, Schiff base complexes and their versatile applications as
catalysts in oxidation of organic compounds: part I, Appl. Organomet. Chem.
31 (3) (2016) e3574, doi:10.1002/aoc.3574.
16] W.A. Zoubi, A.A.S. Al-Hamdanib, M. Kaseema, Synthesis and antioxidant activ-
ities of Schiff bases and their complexes: a review, Appl. Organometal. Chem.
Declaration of Competing Interest
30 (2016) 810–817, doi:10.1002/aoc.3506.
17] N. Bougandoura, N. Bendimerad, Evaluation de l’activité antioxydante des ex-
traits aqueux et méthanolique de Satureja calamintha ssp.Nepeta(L.) Briq, Nat.
Technol. B Sci. Agrono. Biol. 09 (2013) 14–19.
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
[18] P. Comuzzo, F. Battistutta, M. Vendrame, M.S. Páez, G. Luisi, R. Zironi, Antioxi-
dant properties of different products and additives in white wine, Food Chem.
168 (2015) 107–114, doi:10.1016/j.foodchem.2014.07.028.
CRediT authorship contribution statement
[19] R. Uddin, M.R. Saha, N. Subhan, H. Hossain, I.A. Jahan, R. Akter, A. Alam, HPLC
analysis of olyphenolic compounds in gardenia jasminoides and determination
of antioxidant activity by using free radical scavenging assays, Adv. Pharm.
Bull. 4 (2014) 273–281, doi:10.5681/apb.2014.040.
Noudjoud Houas: Writing – original draft, Formal analysis,
Methodology, Project administration, Data curation, Formal analy-
sis, Methodology, Resources, Writing – review & editing, Resources,
Investigation. Salah Chafaa: Project administration, Writing – re-
view & editing, Supervision, Investigation, Funding acquisition, Val-
idation. Nadjib Chafai: Conceptualization, Methodology, Validation,
Writing – original draft, Supervision, Project administration, Re-
sources, Investigation, Writing – review & editing. Samira Ghed-
jati: Project administration, Visualization. Meriem Djenane: Writ-
ing – review & editing. Siham Kitouni: Investigation, Formal anal-
ysis.
[20] R. Romanet, C. Coelho, Y. Liu, F. Bahut, J. Ballester, M. Nikolantonaki,
R.D. Gougeon, The antioxidant potential of white wines relies on the chem-
istry of sulfur-containing compounds: an optimized DPPH assay, Molecules 24
(2019) 1353, doi:10.3390/molecules24071353.
[21] D. Mukhopadhyay, P. Dasgupta, D.S. Roy, S. Palchoudhuri, I. Chatterjee, S. Ali,
S.G. Dastidar, A sensitive in vitro spectrophotometric hydrogen peroxide scav-
enging assay using 1,10-phenanthroline, Free Radic. Antioxid. 6 (2016), doi:10.
5530/fra.2016.1.15.
[22] R. Pulido, L. Bravoand, F. Saura-Calixto, Antioxidant activity of dietary polyphe-
nols as determined by a modified ferric reducing/antioxidant power assay, J.
Agric. Food Chem. 48 (2000) 3396–3402, doi:10.1021/jf9913458.
[
23] M.K. Aadesariya, V.R. Ram, P.N. Dave, Evaluation of antioxidant activities by use
of various extracts from abutilon pannosum and grewiatenax in the kachchh
region, MOJ Food Process. Technol. 5 (2017) 216–230, doi:10.15406/mofpt.2017.
05.00116.
[
24] H. Ueno, S. Yamakura, R. Arastoo, T. Oshima, K. Kokubo, Systematic evalua-
tion and mechanistic investigation of antioxidant activity of fullerenols using
β-carotene bleaching assay Hindawi publishing corporation, J. Nanomater. 17
(2014) 2014, doi:10.1155/2014/802596.
Acknowledgments
This research was supported by the General Directorate for Sci-
entific Research and Technological Development (DGRSDT), Alge-
rian Ministry of Scientific Research, Laboratory of Electrochemistry
of Molecular Materials and Complex (LEMMC), Ferhat ABBAS Uni-
versity of Sétif.
[25] S. Saoudi, S. Khennouf, N. Mayouf, S. Amira, S. Dehamna, H. Karim, Phytochem-
ical screening and in vivo and in vitro evaluation antioxidant capacity of far-
gariaananassa, prunusarmeniaca and prunuspersica fruits growing in Algeria,
Progress Nutr. 22 (2020) 236–252, doi:10.23751/pn.v22i1.8011.
[26] J. Pérez-Jiméneza, S. Arranz, M. Tabernero, M.E. Diaz-Rubio, J. Serrano, I. Goni,
F. Saura-Calixto, Updated methodology to determine antioxidant capacity in
plant foods, oils and beverages: extraction, measurement and expression of,
Food Res. Int. 41 (2008) 274–285, doi:10.1016/j.foodres.2007.12.004.
References
[
27] K. Moedritzer, R.R. Irani, The direct synthesis of a-aminomethylphosphonic
acids. Mannich-type reactions with orthophosphorous acid, J. Org. Chem. 31
[
1] T. Ak, I. Gulcin, Antioxidant and radical scavenging properties of curcumin,
Chem. Biol. Interact. 174 (2008) 27–37, doi:10.1016/j.cbi.2008.05.003.
(1966) 1603–1607, doi:10.1021/jo01343a067.
[
28] M. Mehri, N. Chafai, L. Ouksel, K. Benbouguerra, A. Hellal, S. Chafaa, Synthe-
sis, electrochemical and classical evaluation of the antioxidant activity of three
α-aminophosphonic acids: experimental and theoretical investigation, J. Mol.
Struct. 1171 (2018) 179–189, doi:10.1016/j.molstruc.2018.05.074.
29] Z. Hussain, E. Yousif, A. Ahmed, A. Altaie, Synthesis and characterization of
Schiff’s bases of sulfamethoxazole, Org. Med. Chem. Lett. (2014) 1–4, doi:10.
[
2] B. Halliwell, Reactive oxygen species in living systems: source, biochemistry,
and role in human disease, Am. J. Med. 91 (1991) S14–S22, doi:10.1016/
0
002-9343(91)90279-7.
[
3] T. Stevanovic, N. Diouf, M. Garcia-Perez, Bioactive polyphenols from healthy
diets and forest biomass, Curr. Nutr. Food Sci. 5 (2009) 264–295, doi:10.2174/
[
157340109790218067.
1186/2191-2858-4-1.
[
4] G.M. Cole, G.P. Lim, F. Yang, B. Teter, A. Begum, Q. Ma, M.C. Harris-White,
Frautschy, a prevention of Alzheimer’s disease: omega-3 fatty acid and phe-
nolic antioxidant interventions, Neurobiol. Aging 26 (2005) S133–S136, doi:10.
[
30] E.K. Fields, The synthesis of esters of substituted amino phosphonic acids, J.
Am. Chem. Soc. 74 (6) (1952) 1528–1531, doi:10.1021/ja01126a054.
[
31] G. Keglevich, E. Bálint, The kabachnik-fields reaction: mechanism and synthetic
1016/j.neurobiolaging.2005.09.005.
use, Molecules 17 (2012) 12821–12835, doi:10.3390/molecules171112821.
32] E. Bálint, A. Tripolszky, Á. Tajti, G. Keglevich, Synthesis of α-
[
5] P. Codoñer-Franch, V. Valls-Belles, A. Arilla-Codoñer, E. Alonso-Iglesias, Oxidant
mechanisms in childhood obesity: the link between inflammation and oxida-
tive stress, Transl. Res. 158 (2011) 369–384, doi:10.1016/j.trsl.2011.08.004.
6] E.M. Becker, L.R. Nissen, L.H. Skibsted, Antioxidant evaluation protocols: food
quality or health effects, Eur. Food Res. Technol. 219 (2004) 561–571, doi:10.
[
aminophosphonates by the kabachnik-fields reaction and by the Pudovik
reaction, in: Organophosphorus Chemistry
de Gruyter GmbH, Germany, 2018, pp. 108–147, doi:10.1515/9783110535839-
06.
– Novel Developments, Walter
[
0
1007/s00217-004-1012-4.
[
[
33] M.I. Kabachnik, T.Y. Medved, New synthesis of aminophosphonic acids, Dokl.
Akad. Nauk SSSR 83 (1952) 689–692.
34] R. Damiche, S. Chafaa, Synthesis of new bioactive aminophosphonates and
study of their antioxidant, anti-inflammatory and antibacterial activities as
well the assessment of their toxicological activity, J. Mol. Struct. 1130 (2017)
[7] M. Suhaj, Spice antioxidants isolation and their antiradical activity: a review, J.
Food Compos. Anal. 19 (2006) 531–537, doi:10.1016/j.jfca.2004.11.005.
[8] J.L. Pousset, Politiques nationales: place des médicaments traditionnels en
Afrique, Méd. Trop. 66 (2006) 606–609.
[
9] E.A. Decker, B. Welch, Role of ferritin as a lipid oxidation catalyst in muscle
food, J. Agric. Food Chem. 38 (1990) 674–677, doi:10.1021/jf00093a019.
1009–1017, doi:10.1016/j.molstruc.2016.10.054.
17

N. Houas, S. Chafaa, N. Chafai et al.
Journal of Molecular Structure 1247 (2022) 131322
[
35] S. Zaout, S. Chafaa, A Hellal, O. Boukhemis, L. Khattabi, H. Merazig, N. Chafai,
C. Bensouici, L. Bendjeddou, Hydroxyphenylamine phosphonate derivatives:
Synthesis, X-ray crystallographic analysis, and evaluation of theirs anti-
Alzheimer effects and antioxidant activities, J. Mol. Struct. 1225 (2021) 129121,
doi:10.1016/j.molstruc.2020.129121.
[53] H. Afsar, R. Apak, I. Tor. Spectrophotometric determination of hydrogen perox-
ide using Tris(1,10-phenanthroline) iron (II) analyst.115 (1990) 99–103. doi:10.
1039/AN9901500099
[54] R. Bailey, D.F. Boltz, Differential spectrophotometric determination of hydrogen
peroxide using 1,10-phenanthroline and bathophenanthroline, Anal. Chem. 31
(1959) 117–119, doi:10.1021/ac60145a025.
[
36] A. Hellal, S. Chafaa, N. Chafai, L. Touafri, Synthesis, antibacterial screen-
ing and DFT studies of series of α-amino-phosphonates derivatives from
aminophénols, J. Mol. Struct. 1134 (2017) 217–225, doi:10.1016/j.molstruc.2016.
[55] M.S. Al-Saikhan, L.R. Howard, J.C. Miller, Antioxidant activity and total phe-
nolics in different genotypes of potato (Solanumtuberosum, L.), J. Food Sci. 60
(1995) 341–347, doi:10.1111/j.1365-2621.1995.tb05668.x.
12.079.
[
37] A. Hellal, S. Chafaa, N. Chafai, Synthesis, antibacterial and antifungal screen-
[56] M. Gulluc, F. Sahin, M. Sokmen, H. Ozer, D. Daferera, A. Sokmen, M. Polissiou,
A. Adiguzel, H. Ozkan, Antimicrobial and antioxidant properties of the essential
oils and methanol extract from Menthalongfolia L. ssp. Longifolia, Food Chem
103 (2007) 1449–1456, doi:10.1016/j.foodchem.2006.10.061.
ing of three new of α-aminophosphonic acids, Int. J. Sci. Eng. Res. 6 (2015)
1622–1627.
[
[
[
38] A. Hellal, S. Chafaa, N. Chafai, Synthesis, characterization and computational
studies of three α-amino-phosphonic acids derivatives from meta, Ortho and
Para aminophenol, J. Mol. Struct. 1103 (2016) 110–124, doi:10.1016/j.molstruc.
[57] M.J. Frisch, et al., Gaussian 09, Revision D.01, Gaussian Inc., Wallingford, CT,
2009.
2015.08.070.
[58] C. Lee, W. Yang, R.G. Parr, Phys. Rev. B37 (1988) 785–789, doi:10.1103/
PhysRevB.37.785.
[59] D. Young, in: Computational Chemistry: A Practical Guide for Applying Tech-
niques toReal-World Problems, Cytoclonal Pharmaceutics Inc, John Wiley &
Sons, Inc, 2001, p. 408.
39] K. Benbouguerra, N. Chafai, S. Chafaa, Y.I. Touahria, H. Tlidjane, New α-
hydrazinophosphonic acid: synthesis, characterization, DFT study and in sil-
ico prediction of its potential inhibition of SARS-CoV-2 main protease, J. Mol.
Struct. 1239 (2021) 130480, doi:10.1016/j.molstruc.2021.130480.
40] N. Chafai, S. Chafaa, K. Benbouguerra, D. Daoud, A. Hellal, M. Mehri, Synthe-
sis, characterization and the inhibition activity of a new a-aminophosphonic
derivative on the corrosion of XC48 carbon steel in 0.5 M H2SO4: experi-
mental and theoretical studies, J. Taiwan Inst. Chem. Eng. 70 (2017) 331–344,
doi:10.1016/j.jtice.2016.10.026.
[60] J. Losada, I. del Peso, L. Beyer, Electrochemical and spectroelectrochemical
properties of copper(II) Schiff-base complexes, Inorg. Chim.Acta 321 (2001)
107–115, doi:10.1016/S0020-1693(01)00511-4.
[61] I. Goze, A. Alim, A.S. Tepe., M. Sokmen, K. Sevgi, B. Tepe, Screening of the
antioxidant activity of essential oil and various extracts of origanumrotundi-
folium boiss. from Turkey, J. Med. Plants Res. 3 (2009) 246–254.
[
41] O. Moumeni, S. Chafaa, R. Kerkour, K. Benbouguerra, N Chafai, Synthesis, struc-
tural and anticorrosion properties of diethyl (phenylamino) methyl) phospho-
nate derivatives: Experimental and theoretical study, J. Mol. Struct. 1225 (129)
[62] I.P.
Ejidike,
Cu(II)
Complexes
of
4-[(1E)-N-{2-[(Z)-benzylidene-
amino]ethyl}ethanimidoyl] benzene-1,3-diol Schiff base: synthesis, spec-
troscopic, in-vitro antioxidant, antifungal and antibacterial studies, Molecules
23 (2018) 1581, doi:10.3390/molecules23071581.
(2021) 121, doi:10.1016/j.molstruc.2020.127693.
[
42] N. Chafai, S. Chafaa, K. Benbouguerra, A. Hellal, M. Mehri, Synthesis, spectral
analysis, anti-corrosive activity and theoretical study of an aromatic hydra-
zone derivative, J. Mol. Struct. 1181 (2019) 83–92, doi:10.1016/j.molstruc.2018.
[63] Y. Zhang, Y. Fang, H. Liang, H. Wangc, K. Huc, X. Liu, X.Y.Y. Peng, Synthe-
sis and antioxidant activities of 2-oxo-quinoline-3-carbaldehyde Schiff-base
derivatives, Bioorg. Med. Chem. Lett. 23 (2013) 107–111, doi:10.1016/j.bmcl.
2012.11.006.
12.073.
[
43] K. Benbouguerra, S. Chafaa, N. Chafai, M. Mehri, O. Moumeni, A. Hellal, Syn-
thesis, spectroscopic characterization and a comparative study of the corrosion
inhibitive efficiency of an α-aminophosphonate and Schiff base derivatives: ex-
perimental and theoretical investigations, J. Mol. Struct. 1157 (2018) 165–176,
doi:10.1016/j.molstruc.2017.12.049.
[64] H. Kizilkaya, B. Dag, T. Aral, N. Gen, R. Erenler, Synthesis, characterization, and
antioxidant activity of heterocyclic Schiff bases, J. Chin. Chem. Soc. (2020) 1–6,
doi:10.1002/jccs.202000161.
[65] N. Charef, F. Sebti, L. Arrar, M. Djarmouni, N. Boussoualim, A. Baghi-
ani, S. Khennouf, A. Ourari, M.A. AlDamen, M.S. Mubarak, D.G. Pe-
ters, Synthesis, characterization, X-ray structures, and biological activityof
some metal complexes of the Schiff base 2,2’-(((azanediylbis (propane-3,1-
diyl))bis(azanylylidene))bis(methanylylidene))diphenol, Polyhedron 85 (2015)
450–456, doi:10.1016/j.poly.2014.09.006.
[
44] M. Djenane, S. Chafaa, N. Chafai, R. Kerkour, A. Hellal, Synthesis, spec-
tral properties and corrosion inhibition efficiency of new ethylhydro-
gen[(methoxyphenyl)(ethyl amino)methyl]phosphonate derivatives: experi-
mental and theoretical investigation, J. Mol. Struct. 1175 (2018) 398–413,
doi:10.1016/j.molstruc.2018.07.087.
[
45] C. Sampath, K.V. Vani, Y. Kotaiah, N.H. Krishna, C.N. Raju, C.V. Rao, A facile and
efficient one-pot three component reaction (kabachinik-fields reaction) for the
synthesis of novel α-aminophosphonates by 1, 4-dimethylpiperazine as a new
catalyst, J. Chem. Pharm. Res. 4 (2012) 1375–1382.
[66] H. Mir, D. Ahmed, Synthesis of Schiff bases of acetophenone with aniline and
its different chloro-substituted derivatives, and study of their antioxidant, en-
zyme inhibitory and antimicrobial properties, J. Chem. Soc. Pak. 38 (05) (2016).
[67] S. Tellamekala, M. Gundluru, S. Sarva, M.R. Nadiveedhi, M. Sudileti, R. Alla-
gadda, A. Rao Chippada & S.R. Cirandur, Meglumine sulfate-catalyzed one-
pot green synthesis and antioxidant activity of α-aminophosphonates, Synth.
Commun. Int. J. Rapid Commun. Synth. Org. Chem., (2019) 1–13. doi:10.1080/
00397911.2018.1563795.
[68] D.F.V. Lewis, C. Loannides, D.V. Parke, Interaction of a series of nitriles with the
alcohol-inducible isoform of P450: computer analysis of structure-activity re-
lationships, Xenobiotica 24 (1994) 401–408, doi:10.3109/00498259409043243.
[69] R. Shahidha, S. Muthu, M. Raja, R. Raj Muhamed, B. Narayana, S.N. Prakash,
B.K. Sarojini, Spectroscopic (FT-IR, FT-Raman), first order hyperpolarizabili-
ties, NBO, Fukui function and molecular docking study of N-(4-Chloro-3-
methylphenyl)-2-phenylacetamide, Opt. Int. J. Light. Electron J.Ijleo 140 (2017)
1127–1142, doi:10.1016/j.ijleo.2017.03.120.
[
46] J. Matijevic’-Sosa, M. Vinkovic, D. Vikic, Topic’. NMR spectroscopy of 2-hy-
droxy-1-naphthylidene Schiff bases with chloro and hydroxy substituted ani-
line moiety croat, Chem. Acta 79 (2006) 489–495.
[
47] P. Prieto, M. Pineda, M. Aguilar, Spectrophotometric quantitation of antioxidant
capacity through the formation of a phosphomolybdenum complex: specific
application to the determination of vitamin E, Anal Biochem. 269 (1999) 337–
341, doi:10.1006/abio.1999.4019.
[
48] M.A. Ebrahimzadeh, S.F. Nabavi, S.M. Nabavi, B. Eslami, H. Asgarirad, In vitro
antioxidant and free radical scavenging activity of Leonuruscardiaca subsp.
Persicus, grammosciadiumplatycarpum and onosmademawendicum, African J.
Biotechnol. 9 (2010) 8865–8871, doi:10.5897/AJB10.782.
[
49] C. Popovici, I. Saykova, Evaluation de l’activité antioxydant des composés
phénoliques par la réactivité avec le radical libre DPPH, Revue de Génie Ind.
[70] M.K. Awad, M.F Abdel-Aal, F.M. Atlam, H.A. Hekal, Molecular docking, molec-
ular modeling, vibrational and biological studies of some new heterocyclic
α-aminophosphonates, Spectrochim.Acta Part A Mol. Biomol. Spectrosc. 206
(2019) 78–88, doi:10.1016/j.saa.2018.07.083.
4
(2009) 25–39.
[
50] C.M. Archana, J. Showmya, K. Harini, M. Pradeepa, N. Geetha, Radical scaveng-
ing and antioxidant activity of methanolic leaf extract of Chromolaenaodorata
(
l) king and robinson by in vitro assays, Int. J. Pharm. Sci. Bus. Manag. 3 (2015)
–12.
[71] I. Sıdır, Y.G. Sıdır, M. Kumalar, E. Tasal, Ab initio hartree–fock and den-
sity functional theory investigations on the conformational stability, molecu-
lar structure and vibrational spectra of 7-ace-toxy-6-(2,3-dibromopropyl)-4,8-
dimethylcoumarin molecule, J. Mol. Struct. 964 (2010) 134–151, doi:10.1016/j.
molstruc.2009.11.023.
1
[
52] B. Yardpiroon, S. Aphidech, S. Prasong, Phytochemical and biological activities
of the wild grape fruit extracts using different solvents, British J. Pharm. Res.
4
(2014) 23–36, doi:10.9734/BJPR/2014/5626.
51] I.M. Kolthoff, D.L. Leussing, T.S. Lee, Reaction of ferrous and ferric iron with
,10-phenanthroline. III. The ferrous monophenanthroline complex and the col-
orimetric determination of phenanthroline, J. Am. Chem. Soc. 72 (1950) 2173–
177, doi:10.1021/ja01161a083.
[
1
2
18