Antioxidant activities of 4-methylumbelliferone derivatives

Article abstract of DOI:10.1371/journal.pone.0156625

The synthesis of derivatives of 4-Methylumbelliferone (4-MUs), which are structurally interesting antioxidants, was performed in this study. The modification of 4-Methylumbelliferone (4-MU) by different reaction steps was performed to yield the target compounds, the 4-MUs. The 4-MUs were characterized by different spectroscopic techniques (Fourier transform infrared; FT-IR and Nuclear magnetic resonance; NMR) and micro-elemental analysis (CHNS). The in vitro antioxidant activity of the 4-MUs was evaluated in terms of their free radical scavenging activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH), Nitric oxide radical scavenging activity assay, chelating activity and their (FRAP) ferric-reducing antioxidant power, which were compared with a standard antioxidant. Our results reveal that the 4-MUs exhibit excellent radical scavenging activities. The antioxidant mechanisms of the 4-MUs were also studied. Density Function Theory (DFT)-based quantum chemical studies were performed with the basis set at 3-21G. Molecular models of the synthesized compounds were studied to understand the antioxidant activity. The electron levels, namely HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital), for these synthesized antioxidants were also studied.

Full text of DOI:10.1371/journal.pone.0156625

RESEARCH ARTICLE
Antioxidant Activities of 4-Methylumbelliferone
Derivatives
Yasameen K. Al-Majedy1, Ahmed A. Al-Amiery1*, Abdul Amir H. Kadhum1, Abu
Bakar Mohamad2
1 Department of Chemical and Process Engineering, University Kebangsaan Malaysia (UKM), Bangi,
Selangor 43000, Malaysia, 2 Fuel Cell Institute, University Kebangsaan Malaysia (UKM), Bangi, Selangor
43000, Malaysia
* dr.ahmed1975@gmail.com
Abstract
a11111
The synthesis of derivatives of 4-Methylumbelliferone (4-MUs), which are structurally inter-
esting antioxidants, was performed in this study. The modification of 4-Methylumbelliferone
(4-MU) by different reaction steps was performed to yield the target compounds, the 4-MUs.
The 4-MUs were characterized by different spectroscopic techniques (Fourier transform
infrared; FT-IR and Nuclear magnetic resonance; NMR) and micro-elemental analysis
(CHNS). The in vitro antioxidant activity of the 4-MUs was evaluated in terms of their free
radical scavenging activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH), Nitric oxide radi-
cal scavenging activity assay, chelating activity and their (FRAP) ferric-reducing antioxidant
power, which were compared with a standard antioxidant. Our results reveal that the 4-MUs
exhibit excellent radical scavenging activities. The antioxidant mechanisms of the 4-MUs
were also studied. Density Function Theory (DFT)-based quantum chemical studies were
performed with the basis set at 3-21G. Molecular models of the synthesized compounds
were studied to understand the antioxidant activity. The electron levels, namely HOMO
(highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital), for
these synthesized antioxidants were also studied.
OPEN ACCESS
Citation: Al-Majedy YK, Al-Amiery AA, Kadhum
AAH, Mohamad AB (2016) Antioxidant Activities of 4-
Methylumbelliferone Derivatives. PLoS ONE 11(5):
e0156625. doi:10.1371/journal.pone.0156625
Editor: Ing-Feng Chang, National Taiwan University,
TAIWAN
Received: January 15, 2016
Accepted: May 17, 2016
Published: May 31, 2016
Copyright: © 2016 Al-Majedy et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Introduction
Umbelliferone derivatives (Us) are a family of coumarins that are recognized to have anti-
inflammatory, antithrombotic, enzyme inhibitor and antioxidant properties [1–4]. The
potency of various substituents on Us as antioxidants were deeply investigated by researchers,
who found that the number and nature of the hydroxyl, methoxy or methyl substituents as
electron-donating groups are the most important factors responsible for adjusting the antioxi-
dant activities of Us. Generally, 4-MUs have excellent antioxidant properties based on the
nature and number of substituted hydroxyl groups [5–9]. Coumarins inhibit the proliferation
of various tumor cell lines, they retard the development of renal and prostate-carcinoma [10–
12], in addition to prevent the repetition of melanoma [13]; Umbeliferons show cytotoxic
impacts against the lung-carcinoma cell lines [14–16]. These compounds have undergo clinical
Data Availability Statement: All relevant data are
within the paper.
Funding: This study was supported by Universiti
Kebangsaan Malaysia under the DIP-2012-02 Grant.
The funder had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
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Antioxidant of 4-Methylumbelliferones
attempts for therapy of the lymphedema subsequent breast-cancer therapy moreover therapy
of kidney and lung carcinoma, that had been utilized jointly [17] in isolate and in combined
with cimetidine, as an anti-neoplastic therapy [10,18, 19]. Based on the medicinal applications
of Us and as a continuation of previous studies [20–23], we focus here on the design of our
approach to increase the antioxidant activity based on a conjugated system and applied the the-
oretical studies to associate the antioxidant activities with the electronic structures. There was a
good relationship between H atom abstraction and unpaired electron delocalization. To under-
stand the relation between delocalization and radical reactivity, one can investigate the electron
distribution in the HOMO and LUMO. The main aim of this study was to optimize the struc-
tures of all of the studied 4-MUs to explain the structure-antioxidant relationship. We had also
been concerned with the calculation of antioxidant descriptors, such as the Dipole moment,
Ionization potential (IP), Electron affinity (EA), Hardness (η), Softness (S), and Electronegativ-
ity (μ), for the 4-MUs and the standard compound (Trolox). In addition, we determined the
preferred antioxidant mechanism and calculated the HOMO and LUMO energies and Band
gap. The structure-antioxidant relationships of the synthesized antioxidants and Trolox were
investigated using the DFT-based quantum chemical method together with the 3-21G basis set.
Based on the obtained results, we conclude that the N-H, O-H, and S-H groups are responsible
for antioxidant abilities. The quantum chemical calculations confirmed the high antioxidant
activity of the 4-MUs. Initially, we used 4-MU as the starting material, and all of the synthe-
sized 4-MUs are shown in Fig 1.
Fig 1. Synthesis of the 4-MU compounds
doi:10.1371/journal.pone.0156625.g001
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Antioxidant of 4-Methylumbelliferones
Materials and Methods
2.1 General Information
All chemicals were supplied by Sigma-Aldrich (Selangor, Malaysia). The FT-IR spectra were
obtained on a Nicolet 6700 FT-IR spectrophotometer (Thermo-Nicolet-Corp., Madison, WI,
USA) in cm−1. The NMR spectra were recorded using an AVANCE III 600 spectrometer (Bru-
ker, Billerica, MA, USA) using DMSO-d6 and are expressed in δ ppm. Elemental microanalysis
was performed using an Elemental Vario El Iii, Carlo Erba 1108 Elemental analyzer.
2.1.1 Synthesis of 4-MUs 1–8. Compounds (1–8), namely ethyl 2-((4-methyl-2-oxo-2H-
chromen-7-yl)oxy)acetate (1), 2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetohydrazide (2),
potassium (2-(2-((4-methyl-2-oxo-2Hchromen-7-yl)oxy)acetyl)hydrazinecarbonothioyl)
amide (3), 7-((5-mercapto-4H-1,2,4-triazol-3-yl)methoxy)4-methyl-2H-chromen-2-one (4),
7-((5-mercapto-1,3,4-oxadiazol-2-yl)methoxy)4-methyl-2H-chromen-2-one (5), 7-(2-
(3,5-dimethyl-1H-pyrazol-1-yl)-2-oxoethoxy)4-methyl-2H-chromen-2-one (6), 7-hydroxy-
2-oxo-2H-chromene-4-carbaldehyde (7) and 4-(benzo[d]thiazol-2-yl)-7-hydroxy-2H-chro-
men-2-one (8), were synthesized using the procedures described by Al-Amiery in 2014 [24].
2.2 Antioxidant Activity
2.2.1 DPPH radical scavenging activity assay. The DPPH radical scavenging activity
assay [25] was performed with trivial adjustments. Various concentrations of the prepared
compounds (250, 500, 750 and 1000 mg/mL) were used. The DPPH solution was prepared by
dissolving 6.0 mg of this compound in 100 mL of the solvent (methanol). Then, 1 mL of each
concentration was added to 2 mL of the prepared DPPH solution. Finally, the control was pre-
pared by adding 1 mL of methanol to 2 mL of DPPH. Trolox was used as the standard. The
mixture was shaken vigorously and incubated in the dark for 30 min. The absorbances of the
resulting solutions were spectrophotometrically examined at 517 nm. The scavenging activities
of each prepared concentration of the DPPH radicals were estimated using Eq (1).
A
Aꢀ
DPPHscavenginig effect % ¼ Aꢀ ꢁ ꢂ 100
ð1Þ
where A° is the absorbance of the control reaction and A is the absorbance in the presence of
the samples or standards. All samples were prepared and measured in triplicate.
2.2.2 Ferric reducing/antioxidant power assay (FRAP). The FRAP assay was performed
as previously described [26]. The FRAP reagent was prepared from acetate buffer (pH 3.6), a
10 mM TPTZ solution in 40 mM HCl and a 20 mM iron (III) chloride solution in a ratio 10:1:1
(v/v), respectively. A total of 50 μl of the various concentrations of the prepared compounds
were added to 1.5 ml of the FRAP reagent and mixed well. The concentrations were measured
in three replicates. A standard curve was prepared using a similar procedure. The results were
expressed as μmol Fe (II)/100 g of the synthesized compounds. Trolox was used as the
standard.
2.2.3 Nitric oxide radical scavenging activity assay. Aqueous solution Sodium nitroprus-
side at physiological pH generates nitricoxide spontaneously and interact with oxygen to yield
nitrite ion that could be calculated utilizing reaction named Griess-Ilosvay [27]. In this study,
Griess-Ilosvay reagent was adjusted utilizing naphthylethylenediaminedihydro chloride (0.10%
w/v) rather than 1-naphthyl-amine (5.0%). Reaction mixture (3 mL) having sodium nitroprus-
side (10.0 mM, 2.0 mL) in addition to phosphate buffered saline (0.50 mL) and various concen-
trations of the prepared compounds (250, 500, 750, and 1000 _g/mL) or a standard solution
(0.50 mL) that incubate at 25°C for 150.0 minute. After incubated, 0.50 mL of mixture having
nitrite mixed with 1.0 mL of sulfanilic acid reagent that allows standing for 5.0 minutes to
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Antioxidant of 4-Methylumbelliferones
accomplishment diazotization process. Following, 1.0 mL of naphthylethylenediamine dihy-
drochloride (1.0%) was mixed, and left for 30 minutes. The absorbance had been measured at
540.0 nm versus the proper blank. The scavenging activities of each prepared concentration of
the nitric oxide percentage were estimated utilizing Eq (1).
2.2.4 Ferrous ions chelating activity. Chelating activity of the synthesized compounds
toward Fe+2 was investigated as described in the reference [28]. Various concentrations of the
prepared compounds (250, 500, 750, and 1000 _g/mL) or a standard solution (1.0 mL) were
equally mixed with FeCl2 (0.05 mL), and ferrozine solution (0.2 mL), shaken and of incubation
at room temperature. Absorbance was measured at 545 nm. EDTA had been utilized as positive
control.
2.3 Quantum Studies
The molecular representation of the reference compound was plotted using ChemBioOffice
2010 software. All of the quantum chemical calculations were performed using the DFT meth-
odology with the 3–21G basis set.
2.4 Statistical Analysis
The results were expressed as the means standard deviation, and the statistical significance of
the differences was determined utilizing one-way analysis of variance (ANOVA) with the SPSS
17.0 statistical software program. Differences were considered significant at P < 0.05. The val-
ues are presented as the means SD (n = 3).
Results and Discussion
3.1 Antioxidant activities
3.1.1 DPPH radical scavenging activity assay. The DPPH 1,1-diphenyl-2-picrylhydrazyl
radical is a steady radical that could share an electron or hydrogen and transfer it to a stable
compound. The methanolic DPPH solution showed a strong absorption band at 517 nm due
to the single-electron (ode-electron), and if this solution was mixed with suitable reducing
agents, the result would be pairing of the electron and a lack of color. The color is stoichiomet-
rically reduced as the number of electrons taken up increases, and the decrease in the absor-
bance could be directly measured and compared with the standard (Trolox). Fig 2 shows the
high DPPH radical scavenging activities compared with the Trolox standard.
3.1.2. Proposed antioxidant mechanisms of 4-MUs 4 and 8. Based on the chemical
structures, we can assume that the antioxidant activities of the 4-MUs, as shown in Figs 3 and
4, depend on the hydrogen atoms of the hydroxyl and/or amino groups, which were under the
impact of resonance and/or inductive effects. Resonance and inductive effects facilitate the
reduction of the hydrogen group, making the molecule more stable. 4-MUs have scavenging
activities due to the stability of the intermediate radicals of 4-MUs. The elimination of a hydro-
gen group from an amine or hydroxyl group may occur easily [29]. Heterocyclic residues and
steric hindrance enhance the antioxidant activities [30, 31].
3.1.3 Nitric oxide radical scavenging activity assay. Nitric oxide radical (NO•),
exclaimed as the " molecule of year" in 1992 by the Science journal [32], has a significant part
in the control of various physiological and pathophysiological forms [33]. The Griess interac-
tion is repeatedly utilized for evaluation of NO• generation by entire cells or enzyme [34]. Its
applications to in-vitro limitation of NO• scavenging capacity is continual. For this situation,
the nitric oxide staying after interaction with the sample is measured as nitrite. It is significant
to confirm that nitrate can be formed, hence it ought to be diminished to nitrite before
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Antioxidant of 4-Methylumbelliferones
Fig 2. Antioxidant activity of different concentrations of the synthesized compounds (1–8) and Trolox
in the DPPH radical scavenging assay.
doi:10.1371/journal.pone.0156625.g002
determination [35]. The derivative of azo chromophor was formed from nitrite after Griess
reaction determined spectrophoto-metrically at 540 nm. Standard curve produced utilizing
sodium nitrite and result was demonstrated as percentage change from control reaction.
Fig 3. The reaction scheme between DPPH free radicals and compound 4.
doi:10.1371/journal.pone.0156625.g003
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Antioxidant of 4-Methylumbelliferones
Fig 4. The reaction scheme between DPPH free radicals and compound 8.
doi:10.1371/journal.pone.0156625.g004
Compared with different strategies, is not direct, requiring the expansion of a several enzymatic
reagents [36]. From Fig 5, it can be shown that compounds 3,4, 6 and 8 have the highest antiox-
idant activities.
Fig 5. Antioxidant activity of different concentrations of the synthesized compounds (1–8) in the Nitric
oxide radical scavenging assay
doi:10.1371/journal.pone.0156625.g005
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Antioxidant of 4-Methylumbelliferones
Fig 6. Fe2+ chelating activities of synthesized compounds 1–8
doi:10.1371/journal.pone.0156625.g006
3.1.4 Chelating activity. Amongst the significant mechanism of action of secondary anti-
oxidant has been chelation of pro-oxidant metal. Iron enhances oxidation by expressible as cat-
alyst of free-radical reaction. Iron has transfer single electron through replacements in
oxidation state. Complexation of Iron with organic compounds decrease the pro-oxidant
impact by reduce it redox potential and stabilize the oxidizing form of the Iron [37]. All the
studied compounds had the ability to complexation with Iron. Iron complexation impacts of
the synthesized compounds (1–8) were conditioned by concentrations and straight increased
with the tested compound concentration raised. The relationship as in Fig 6, of compounds
1,2,5 and 6 to Fe+2 had been relatively-low as compare with EDTA. However the activity of
compounds 3,4,7 and 8 were the like as the EDTA. superior effectiveness of the studied com-
pounds 1–8 might also include the utilize of the synthesized compounds as iron chelators, simi-
lar to other compounds of that class [38,39].
3.2 Molecular Modeling Studies
To determine the scavenging activity and the electron levels (HOMO "highest occupied molec-
ular orbital" and LUMO "lowest unoccupied molecular orbital") of the prepared scavengers (1–
8), DFT-based quantum chemical investigations were performed with the basis set 3-21G. The
EHOMO and ELUMO were reported as energies in eV (electron volt) values in Fig 7. The
4-MUs with higher scavenging activities could be established using the values of the HOMO
and LUMO energies. In this study, we used the DPPH method, which obviously showed that
the scavenging activities of 4-MUs 7 and 8 were higher than the others due to resonance effects
of the aromatic ring and electron withdrawal. Theoretically, it was conceivable that the HOMO
energy is a valid indicator for scavenging activity and that this scavenging activity was not
based on LUMO energy. The differences in the scavenging activities of the 4-MUs (1, 2, 3, 5, 6,
7 and 8), as represented by the HOMO values, essentially refer to the delocalization of pi-elec-
trons, leading to the stability of the radicals acquired after hydrogen abstraction such that the
delocalized pi-electrons in the MUs (1, 2, 3, 5, 6, 7 and 8) (Fig 7) also occur in the
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Antioxidant of 4-Methylumbelliferones
Fig 7. Highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital of
compounds 1, 2, 3, 5, 6, 7 and 8.
doi:10.1371/journal.pone.0156625.g007
corresponding radical. The HOMO electron density could be considered to understand the
relation between the pi-electrons and scavenging activity [40].
The HOMOs (highest occupied molecular orbitals) of 4-MUs (1–8) are delocalized over the
entire molecules, which agree with the unshared electrons. The spin densities of the free radi-
cals that had been generated from 4-MUs (1–8) were compared. Increased delocalization indi-
cates that the generation of free radicals was easier. The spin density seems to be slightly more
delocalized for the radicals created from 4-MUs (7 and 8) than for the other 4-MUs. The
HOMO energies of the 4-MUs (1–8) and the reference compound are computed as -12.084 eV,
-7.051 eV, -7.401 eV, -9.748 eV, -9.735 eV, 9.788 eV, 11.380 eV and -5.279 eV. Moreover, the
LUMO energies for the 4-MUs (1–8) and reference compound are estimated as -4.062 eV,
3.876 eV, -3.883 eV, -3.875 eV, -3.875 eV, -3.872 eV, -4.398 eV and -3.983 eV. The energy gaps
for the 4-MUs (1–8) and the control were recorded as 8.022 eV, 3.715 eV, 3.118 eV, 5.837 eV,
5.806 eV, 5.916 eV, 5.98 eV, and 1.296 eV due to the shift of absorption toward the blue region.
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Antioxidant of 4-Methylumbelliferones
Delocalization of the heterocyclic ring and carbonyl group for the 4-MUs 7 and 8, respectively,
were responsible for the variations between the HOMOs and LUMOs of the 4-MU scavengers
(1–8). A comparison of the potential according to the band gaps for 4-MUs (1–8) and control
clearly showed that the highest band gap was observed for the control (reference), followed by
4-MUs 7 and 8, and this is highly compatible with the results of the experiment shown in Fig 2.
The Dipole moment values of the 4-MUs (1–8) and control indicated that they are polar com-
pounds and may be soluble in polar solvents. The estimated IP (Ionization potential) could
provide an understanding of the initial energy required to release an electron from the com-
pounds [41], which implied an inverse relation between the scavengers and IP (Eq 2).
IP ¼ ꢁEHOMO
2
EA (Electron affinity) is the amount of energy launched when an electron is absorbed by a
molecule (Eq 3). Higher EA values allow the compound to easily absorb electrons. In other
words, the compound has a positive relation with the antioxidant activity.
EA ¼ ꢁELUMO
3
η (Hardness) is charge transfer resistance, and S (softness) is the measure of the capacity of
an atom to receiving an electron (Eqs 4 and 5, respectively).
1
Z ¼ ꢁ ðEHOMO ꢁ ELUMO
Þ
4
5
2
2
S ¼ ꢁ
ðEHOMO ꢁ ELUMO
Þ
μ (electronegativity) is defined as the capacity to attract electrons (Eq 6) in the chemical
bond.
1
m ¼ ꢁ ðEHOMO þ ELUMO
Þ
6
2
Table 1 obviously shows the potential values of the studied parameters, and these parame-
ters could support the high scavenging activities. The theoretical parameters (using DFT, with
the basis set 3-21G) and experimental findings were compared. A good correlation between the
experimental and calculated data was observed. In addition, the HOMOs and LUMOs of the
4-MUs (1–8) were investigated using corresponding methods with the 3-21G basis set. The cal-
culated HOMO-LUMO energies were used to calculate some of the properties of title
molecule.
Table 1. The Electronic Properties of antioxidants 1–8 were obtained using the DFT method with the 3-21G basis set.
Parameters
Comp. 1
Comp. 2
Comp. 3
Comp. 4
Comp. 5
Comp. 6
Comp. 7
Comp. 8
Trolox
IP; eV
EA; eV
η
12.084
4.062
4.011
0.249
8.073
12.084
4.062
8.022
7.051
3.876
1.812
0.538
5.463
7.051
3.876
3.715
7.401
3.883
1.559
0.641
5.642
7.401
3.883
3.118
9.748
3.875
2.918
0.342
6.811
9.748
3.875
5.837
9.730
3.875
2.403
0.344
6.802
9.730
3.875
5.806
9.877
3.872
2.958
0.388
6.874
9.877
3.872
5.916
11.380
4.398
2.990
0.334
7.889
11.380
4.398
5.98
5.279
3.983
0.658
1.543
4.631
5.279
3.983
1.296
10.215
0.704
5.107
0.210
5.459
10.215
0.704
9.509
S
μ
EHOMO
ELUMO
Band gap
doi:10.1371/journal.pone.0156625.t001
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Antioxidant of 4-Methylumbelliferones
Fig 8. Linearity of the FRAP assay (dose–response line) for standard solutions.
doi:10.1371/journal.pone.0156625.g008
3.3 FRAP assay
The FRAP assay measures the capability of a molecule to diminish the ferric 2,4,6-tripyridyl-s-
triazine complex to the colored ferrous-complex [42]. FRAP significances are obtained by com-
paring the absorbance shift to 593 nm in the examined reaction mixtures with that containing
a recognized concentration of ferrous-ions. The linearity of the FRAP assay for standard solu-
tions is shown in Fig 8. The results of the FRAP assay are reported in Table 2. The scavenger
activities were expressed as the concentrations of antioxidants having a ferric-reducing ability
equivalent to that of 1 mM of FeSO4, and the results showed that three of the 4-MUs (3, 7 and
8) exhibited higher antioxidant capacity.
Conclusions
4-Methylumbelliferons (4-MUs) were successfully synthesized and characterized using spec-
troscopic techniques (FT-IR and NMR) and micro-elemental analysis (CHNS). The antioxi-
dant activities were evaluated by DPPH and FRAP assays, and the results indicated that these
compounds have good scavenging activities. The antioxidant mechanisms of the synthesized
4-MUs were also studied. DFT-based quantum chemical studies were performed with the basis
set 3-21G, and molecular modeling was performed for the synthesized 4-MUs to understand
their antioxidant activities. The electron levels, namely the HOMO (highest occupied molecu-
lar orbital) and the LUMO (lowest unoccupied molecular orbital), were also studied. We were
also concerned with the calculation of the following antioxidant descriptors for the synthesized
4-MUs and the reference compound: Dipole moment; Ionization potential (IP), Electron affin-
ity (EA), Hardness (η), Softness (S), and Electronegativity (μ).
Table 2. Antioxidant activity of (1–8) compounds using the FRAP assay.
Compounds
Inhibition (%)
1
2
3
4
5
6
7
8
50%
20%
75%
55%
40%
25%
73%
77%
doi:10.1371/journal.pone.0156625.t002
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Antioxidant of 4-Methylumbelliferones
Acknowledgments
This study was supported by the University Kebangsaan Malaysia under the DIP-2012-02
Grant.
Author Contributions
Conceived and designed the experiments: AA-A. Performed the experiments: YA-M. Analyzed
the data: AA-A AAHK ABM. Contributed reagents/materials/analysis tools: AAHK ABM.
Wrote the paper: AA-A.
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