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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 225–230

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Spectroscopic and density functional theory studies

of 5,7,30 ,50 -tetrahydroxyflavanone from the leaves of Olea ferruginea
Muhammad Ali Hashmi a, Afsar Khan a,⇑, Khurshid Ayub a,b, Umar Farooq a
a
Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
b
Department of Chemistry, College of Science, King Faisal University, Al-Hafouf 31982, Saudi Arabia

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Spectroscopic and DFT study of

5,7,30 ,50 -tetrahydroxyflavanone.
 Experimental and theoretical IR, UV–
Vis, and NMR studies.
 IP, EA, HOMO, LUMO, and EN
simualations at DFT-B3LYP/6-311 G
(d, p).
 Use of water molecule as model for
hydrogen bonding interactions.

a r t i c l e i n f o a b s t r a c t

Article history: 5,7,30 ,50 -Tetrahydroxyflavanone (1) was isolated from the leaves of Olea ferruginea and a theoretical
Received 22 October 2013 model was developed for obtaining the electronic and spectroscopic properties of 1. The geometric and
Received in revised form 20 February 2014 electronic properties were calculated at B3LYP/6-311 G (d, p) level of Density Functional Theory (DFT).
Accepted 23 February 2014
The theoretical data was in good agreement with the experimental one. The optimized geometric param-
Available online 12 March 2014
eters of compound 1 were calculated for the first time.
The theoretical vibrational frequencies of 1 were found to correlate with the experimental IR spectrum
Keywords:
after a scaling factor of 0.9811. The UV and NMR spectral data computed theoretically were in good
Density functional theory
B3LYP/6-311 G (d, p)
agreement with the experimental data. Electronic properties of the compound i.e., ionization potential
DFT (IP), electron affinity (EA), coefficients of HOMO and LUMO were estimated computationally for the first
Olea ferruginea time which can be used to explain its antioxidant as well as other related activities and more active sites
Tetrahydroxyflavanone on it. The intermolecular interactions and their effects on IR frequencies, electronic and geometric param-
eters were simulated using water molecule as a model for hydrogen bonding with flavonoid hydroxyl
groups.
Ó 2014 Elsevier B.V. All rights reserved.

Introduction Khoona in Hazara Division, Pakistan. It is a 10–50 feet tall tree,

Olea is a small, widespread genus of family Oleaceae with 40
species occurring throughout warm temperate and tropical regions
of Southern Europe, Africa, Southern Asia, and Australasia [1]. Olea
ferruginea (Syn. Olea cuspidate/cuspidata) is locally called Kahoo or
⇑ Corresponding author. Tel.: +92 992 383591/2/3/4/5; fax: +92 992383441.
E-mail addresses: afsarhej@yahoo.com, afsarkhan@ciit.net.pk (A. Khan).
greyish green in color with evergreen leaves shining above [2]. It
is mainly distributed in Afghanistan, Pakistan, and Kashmir. In
Pakistan, it is mostly found in the hilly areas of Rawalpindi, Khyber
Pakhtunkhwa, and Azad Kashmir [3]. Locally, its stem bark is used
to treat fever [4]. The extract of fresh leaves of O. ferruginea is used
to treat toothache and bleeding of gums, dental problems, skeletal
muscular problems [5], and to cure skin diseases by the local
people [6]. The oil extracted from the fruits is used as massage in

http://dx.doi.org/10.1016/j.saa.2014.02.163
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
 Author's personal copy
226 M.A. Hashmi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 225–230
the treatment of rheumatism and dislocation of bones. The plant is
also reported to act as antiperiodic, astringent, uretic, antiseptic,
anthelmintic, anti-malarial, anti-leprosy, rubefacient, gonorrhea
tonic, and used to cure diabetes, asthma, mouth ulcer, and sour
throat [7–13]. Decoction of fresh leaves of O. ferruginea is used to
relieve bleeding gums and pimples [14].
Various parts of Olea europaea such as leaves, fruit, stem bark,
and roots contain different classes of secondary metabolites. The
lignans ()-olivil, (+)-cycloolivil, (+)-1-acetoxypinoresinol, (+)-1-
hydroxypinoresinol, (+)-1-acetoxypinoresinol-400 -O-methylether,
and (+)-1-hydroxypinoresinol-400 -O-methylether have been iso-
lated from the ether extract of O. europaea bark [15,16]. O. europaea
fruits contain a considerable amount of flavonoids and other
phenolic components like seco-irridoids and seco-irridoid glyco-
sides [17]. Oleoside dimethyl ester has also been isolated from
water extract of O. europaea leaves [18,19]. 5,7,30 ,50 -Tetrahydroxyf-
lavanone (1) has been reported from the leaves of Blumea
balsamifera [20], twigs of Broussonetia papyrifera [21], and from
the aerial parts of Thymus quinquecostatus var. japonica [22].
Compound 1 showed very good free radical scavenging [20] and
cytotoxic activities [23]. To the best of our knowledge, compound
1 has never been isolated from the genus Olea or family Oleaceae.
We are reporting it for the first time from O. ferruginea.
A detailed characterization of compound 1 by DFT in compari-
son with its UV–Vis, IR, and NMR spectroscopic studies is being
reported in the present work.
Methods
Experimental
Plant material
The leaves of the plant O. ferruginea were collected in the month
of May 2011, from Abbottabad, Khyber Pakhtunkhwa, Pakistan and
identified by Dr. Gul Jan (Taxonomist) at Botany Department, Haz-
ara University, Mansehra, Pakistan. The specimen of the plant was
submitted in the herbarium of the above Department under the
voucher number 3544.
Extraction and isolation
The shade dried and powdered leaves (16 kg) of O. ferruginea
were extracted with methanol for 30 days at room temperature.
The methanolic extract was concentrated under reduced pressure
which resulted in a brownish gum (1285 g). This crude residue
was suspended in water and successively partitioned between n-
hexane, CHCl3, EtOAc, and n-BuOH.
The EtOAc soluble fraction (162 g) was subjected to column
chromatography (Silica gel, 230–400 mesh, E-Merck) with a
gradient of CHCl3/MeOH (100:0–0:100) to give 14 major fractions
(Fr. A1–A14). Fr. A4 (30 g) was further subjected to silica gel col-
umn chromatography and eluted with CHCl3/MeOH (95:5–0:100)
to produce 9 subfractions (A5-i–A5-ix). Subfraction A5-iii was sub-
jected to recycling preparative HPLC and eluted with CH3CN/H2O
(1:1) on a RP-18 column to afford compound 1 (5.1 mg) as off-
white powder.
General experimental procedure
Melting point was determined by using glass capillaries in
Büchi melting point apparatus. IR (KBr) spectrum was recorded
on JASCO IRA-1 spectrometer. UV spectrum was recorded on Shi-
madzu UV 240 Hitachi U-3200 spectrophotometer. Electron impact
mass spectrum was recorded on a Finnigan MAT 311 spectrometer,
coupled with PDP 11/34 computer system. 1H NMR spectra were
recorded at 600 MHz on Bruker Avance AV-600 nuclear magnetic
resonance spectrometer using TMS as an internal reference while
13
C NMR spectra were scanned with the same instrument at
150 MHz. All the spectra were scanned using methanol as solvent.
Column chromatography was performed on silica gel 60 (0.063–
0.200 mm, Merck). TLC was carried out on pre-coated aluminum
sheets (silica gel 60 F254, Merck).
Computational details
All the computational studies were carried out using the
Density Functional Theory (DFT) methods implemented in
Gaussian 09 suite of programs [24]. Some calculations were per-
formed in gas phase and some in different solvents. The results
were visualized using Gabedit [25]. The molecular properties of
compound 1 were computed by DFT using the standard 6-311G
(d, p) basis set. In the DFT calculations the Lee, Yang and Parr cor-
relation function [26] was used together with Becke’s three param-
eters [27] exchange function B3LYP. Conformational analysis of the
molecule was performed to get an idea about the lowest energy
conformer of the molecule. More specifically, rotational potential
energy profiles were calculated in relation with the torsion angles
between the rings B and C defined by the (C3AC2AC10 AC20 ) atoms
(Fig. 1). The torsion angles were scanned in steps of 10° without
constraints using B3LYP/6-311 G (d, p) level of theory. The mini-
mum energy conformation thus obtained, was further subjected
to geometry optimization at the B3LYP density functional theory
with the same basis set [26–28]. Vibrational frequencies from the
optimized structures were computed at the same level of theory
to confirm the true minima on potential energy surface. UV spec-
troscopic transition energies were calculated using time-depen-
dent density functional theory (TD-DFT) and the 6-311 G (d, p)
basis set in gas phase and in MeOH as solvent (see Fig. 2). NMR
calculations were performed using GIAO WP04/6-311 G (d, p)
and WC04/6-311 G (d, p) basis sets with TMS as internal standard
and MeOH as solvent.
Other electronic properties such as ionization potential (I. P),
electron affinitiy (E. A), co-efficient of highest occupied molecular
orbital (HOMO), co-efficient of lowest unoccupied molecular orbi-
tal (LUMO), band gap, electronegativity (E. N), hardness (n), and
softness (S) were simulated at B3LYP/6-311 G (d, p) level of theory.
Band gap was estimated as a difference in the energies of LUMO
and HOMO.
Results and discussion
Optimized geometric structure of 5,7,30 ,50 -tetrahydroxyflavanone
Compound 1 was isolated as an off-white amorphous powder
and its molecular formula was assigned as C15H12O6 based on
HREI-MS at m/z 288.0665 (calculated 288.0630 for C15H12O6). Its
structure was assigned on the basis of its 1H NMR, 13C NMR, and
2D NMR techniques. The minimum energy optimized structure of
compound 1 at DFT-B3LYP/6-311 G (d, p) level of theory is given
Fig. 1. Structure of compound 1 with rotational potential energy surface scan
between C3AC2AC10 AC20 .
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M.A. Hashmi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 225–230 227

Fig. 2. UV spectrum of compound 1 calculated at TD-B3LYP/6-311 G (d, p).

in Fig. S2 in supporting information. Different geometric features and 727 cm1 are due to CAH wagging movements and are in very
including important bond lengths, bong angles and dihedral angles good agreement with the experimental data (Table 2).
of the optimized structure are listed in Table 1. The ring C exists in
half chair form in its lowest energy state. The dihedral angle UV–Vis spectroscopic study
between C3AC2AC10 AC20 is 85.04°.
Ultraviolet spectra of compound 1 have been investigated
experimentally as well as by theoretical calculations. The experi-
Infra-red spectral characteristics mental UV spectrum was recorded in methanol while theoretically
it has been scanned both in gas phase as well as in methanol at TD-
The experimental IR spectrum of compound 1 showed charac- B3LYP/6-311 G (d, p) level of theory (Fig. 3). The density of states
teristic absorption bands for ketonic, OH, and ACH functional (DOS) with HOMO and LUMO diagrams was also calculated as
groups. A few prominent bands of experimental and computed IR shown in Fig. 3. Calculations of the molecular orbital geometry
spectra are given in Table 2 along with their approximate assign- revealed that the visible absorption maxima of the molecule
ments. The simulated IR spectrum at B3LYP/6-311 G (d, p) level correspond to the electronic transitions between frontier orbitals
of theory was scaled with a factor of 0.9811 to correlate it with such as translation from HOMO to LUMO. The energy of HOMO
the experimental one. The whole IR spectra (computed and exper- describes the ability of electron donation while the energy of
imental) may be divided into 10 prominent bands. The OAH LUMO describes the ability of accepting electrons, and the gap be-
stretching band appeared at 3259 cm1 which was in good agree- tween HOMO and LUMO characterizes the chemical stability of the
ment with the computed spectrum where this band appeared at molecule [29]. The band gap is a critical parameter in determining
3238 cm1 in the case of isolated molecule and at 3296 cm1 when electrical transport properties of the molecule because it is a mea-
the molecule was H-bonded with water molecules. The other two sure of electron conductivity (Fig. S4 in Supplementary informa-
prominent peaks at 2977 and 2951 cm1 were due to ACH2 and tion). The two important molecular orbitals, i.e. HOMO and
ACH stretching, respectively, and showed good correlation with LUMO were examined for compound 1 by drawing surfaces for
the respective observed bands 2974 and 2924 cm1. The absorp- the frontier orbitals in order to understand the bonding scheme.
tion bands at 1659, 1588, and 1424 cm1 have little differences It is obvious from Fig. S4 (Supplementary information) that the
from the experimental data, and are believed to be due to C@O charge density is localized mainly on the ring B of compound 1
stretching, AOH scissoring, and ACH2 scissoring, respectively. in its HOMO while LUMO is characterized by a charge distribution
The simulated band peak at 1188 cm1 showed correlation with on ring A and the carbonyl functionality of ring C. Both experimental
1182 cm1 which was assigned to ACH2 bending. The absorption and calculated absorption maxima (kmax, nm) have been presented
band at 1091 cm1 is due to CAH bending, while those at 832 in Table 3. The spectrum showed three prominent absorption bands
at 208, 288, and 330 nm. A shoulder can also be observed at 226 nm.
Each of these bands correspond to a number of overlapping transi-
Table 1 tions. The computed absorption maxima values are in agreement
Selected optimized geometric parameters of compound 1 (atomic labels are with
reference to Fig. 1).
with the experimental ones. The deviation between the experiment
and theory may result from the solvent effects as the solvent makes
Atoms Bond lengths (Å) Atoms Bong angles (°) the chemical environment of the molecule in the simulation very
Geometric parameters complex. It may also be explained as in theory, a single molecule
O1AC2 1.44 C3AC2AC10 112.85 is considered while in experiment there are infinite number of
C2AC10 1.51 O1AC2AC10 108.54
molecules of the compound under study.
C2AH2 1.10 O1AC2AC3 110.10
C2AC3 1.53 C2AC10 AC20 118.77
C3AC4 1.52 Dihedral angles NMR studies of compound 1
C4AC10 1.45 Atoms Angles (°)
C9AC10 1.42 C3AC2AC10 AC20 85.04 The NMR spectra of compound 1 were taken in CD3OD on a
O1AC9 1.36 O1AC2AC10 AC20 152.65 600 MHz NMR spectrometer. The theoretical NMR calculations
were run at two different levels of theory with different basis sets.
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228 M.A. Hashmi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 225–230

Table 2
IR data calculated at B3LYP/6-311 G (d, p).

S. no Experimental IR (cm1) Calculated frequency at B3LYP/6-311 G (d, p) Approximate assignments

Gas phase Solvated by water
Scaled (cm1) Unscaled (cm1) Scaled (cm1) Unscaled (cm1)
1 3259 3238 3301 3233 3296 OAH stretching
2 2974 2977 3035 2976 3034 ACH2 stretching
3 2924 2951 3008 2949 3006 CAH stretching
4 1675 1659 1691 1655 1687 C@O stretching
5 1596 1588 1619 1598 1629 OAH scissoring
6 1435 1424 1452 1429 1457 ACH2 scissoring
7 1182 1188 1211 1184 1207 ACH2 bending
8 1084 1091 1113 1090 1112 CAH bending
9 829 832 849 816 832 CAH wagging
10 729 727 742 730 745 CAH wagging

Fig. 3. The density of states (DOS) with HOMO and LUMO diagrams for compound 1.

Table 3
UV–Vis data of compound 1 calculated at TD-B3LYP/6-311 G (d, p) level of theory. (IEFPCM) [33,34]. Even the WP04 and WC04 functions were
Experimental Calculated
described exclusively for chloroform as solvent, yet they showed
very good results using methanol as solvent. The chemical shifts
k (nm) k (nm) E (eV) f
of all the contributing conformers were averaged to get the final
208 221.9 5.59 0.0268 calculated d values. In case of 13C NMR, the chemical shift values
226 249.1 4.98 0.0376
288 296.8 4.18 0.0108
of WP04 and WC04 functions were taken into consideration and
330 316.9 3.91 0.0358 averaged to get the values which are in a much better agreement
with the experimentally determined 13C NMR chemical shifts.
The results of experimental and theoretical NMR are shown in
Tables 4 and 5 for 1H and 13C NMR. It is evident from the data that
The calculation of NMR at B3LYP/6-311 G (d, p) level of theory the computed chemical shifts are in very good agreement with the
resulted in an inconsistent pattern. Some chemical shifts were in experimentally determined ones without applying any scaling
a good agreement with the experimental data whereas some factor. There is one fact which is still unclear to us that these the-
showed large deviations. Then the Cramer’s reparametrized func- oretical methods provide results with a deviation from the exper-
tions (WP04 and WC04) [30] were used to calculate NMR using imental data for the aromatic carbon having a hydroxyl group and
the polarized continuum model available in Gaussian 09 [24]. So its neighboring carbons. We applied different methods and basis
the other level of theory used was WP04/6-311 G (d, p) using sets but the same results were obtained for these type of carbons.
methanol as a solvent and TMS as reference.
Compound 1 is a flexible molecule which may adopt different Inter-molecular interactions of compound 1
conformations, so the optimized structure and its conformers
within 2.5 kcal/mol range were included in NMR calculations as In order to study the inter-molecular interactions water mole-
their role is significant in determining more accurate chemical cule was chosen as a model hydrogen bonding molecule. This small
shifts. For each conformer, the NMR was calculated using the gauge molecule was selected to minimize the computational cost which
independent atomic orbital (GIAO) formalism [31,32] and includ- would be too high if we have applied the same molecules of
ing the effects of methanol solvation via the polarizable continuum compound 1 to study hydrogen bonding. The inter-molecular
model (PCM) using the integral equation formalism variant interaction energy between water molecule and compound 1 was
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M.A. Hashmi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 225–230 229

Table 4 Table 6
Experimental and theoretical 1H NMR data. Electronic properties of compound 1 from optimized geometries.

Carbon Experimental 1H Theoretical 1H NMR Theoretical 1H NMR Properties Isolated H-bonded to

no. NMR chemical chemical shifts (d, chemical shifts (d, molecule water
shifts (d, ppm) ppm) at GIAO B3LYP/ ppm) at GIAO WP04/
Ionization potential IP = EHOMO (eV) 6.25 5.54
6-311 G (d, p) 6-311 G (d, p)
Electron affinity EA = ELUMO (eV) 1.78 1.29
2 5.26 (dd, J = 3.0, 4.95 5.15 Electronegativity EN = (IP + EA)/2 4.02 3.42
14.4 Hz) (eV)
3 2.68 (dd, J = 3.0, 2.26 2.41 Hardness n = (IP–EA)/2 (eV) 2.23 2.13
17.4 Hz) Softness S = 1/2n 1.12 1.06
3.05 (dd, J = 12.6, 2.46 2.73
16.8 Hz)
6 5.85 (d, J = 2.4 Hz) 5.54 5.80
8 5.87 (d, J = 2.4 Hz) 5.95 6.00 for obtaining the electronic and spectroscopic properties of com-
20 6.77 (br s) 6.15 6.21 pound 1. The DFT theoretical model was adopted at B3LYP/6-311
40 6.78 (br s) 5.99 6.18
G (d, p) level of theory. Bond lengths and bong angles of the opti-
60 6.90 (d, J = 1.2 Hz) 6.39 6.55
mized geometry of compound 1 have been calculated for the first
time. The simulated IR spectrum at the same level of theory was
scaled with a scaling factor of 0.9811 and the scaled IR absorptions
Table 5 showed very good correlation with the experimental values.
13
Experimental and theoretical C NMR data. UV–Vis spectra calculated at TD-B3LYP/6-311 G (d, p) level of
Carbon Experimental Theoretical 13C NMR Theoretical 13C NMR theory were also in agreement with the experimental spectra
no. 13
C NMR chemical shifts (d, ppm) chemical shifts (d, recorded in methanol. The NMR studies have also been done using
chemical shifts at GIAO B3LYP/6-311 G ppm) at GIAO a variant of B3LYP, i.e. Wx04/6-311 G (d, p) with methanol solva-
(d, ppm) (d, p) WC04 + WP04/6-311
tion via the polarizable continuum model (PCM) using the integral
G (d, p)
equation formalism variant (IEFPCM) which proved to be a very
2 80.50 83.95 79.97
good method for studying the NMR spectra of conformationally
3 44.10 48.10 46.53
4 197.60 196.68 197.44 flexible molecules especially flavanones. The electronic properties
5 165.50 171.84 171.02 such as IP, EA, HOMO, LUMO, EN, hardness, and softness of
6 97.10 96.17 93.09 compound 1 were estimated for the first time.
7 168.5 170.20 170.43
8 96.20 96.39 92.94
9 164.8 169.25 169.15 Acknowledgements
10 103.2 104.66 100.71
10 131.7 149.24 148.62 One of us (M.A.H) acknowledges funding from the HEC of
20 116.2 106.22 103.97
Pakistan for his PhD studies.
30 146.9 164.32 162.47
40 119.2 100.98 98.95
50 146.5 164.51 162.84
Appendix A. Supplementary material
60 114.6 103.02 101.79

Supplementary data associated with this article can be found, in

calculated to be 32.76 kcal/mol. This interaction energy indicates
hydrogen bonding interactions between compound 1 and water
molecules. The partial bond length of hydrogen bond was found
to be 1.84 Å. Frontier molecular orbitals of water attached with
compound 1 are given in supplementary information. IP and EA
of the water bonded compound 1 were estimated to be 5.54 and
1.29 eV, respectively.
Electronic properties
Although, electronic properties of compound 1 have not yet
been studied experimentally, the computed electronic properties
can give us apparent clue of the activity and reactivity of com-
pound 1. Electronic properties such as ionization potential (IP),
electron affinity (EA), HOMO, LUMO, electronegativity (EN), hard-
ness (n), and softness (S) were calculated from the optimized struc-
tures. The IP and EA of compound 1 were found to be 6.25 and
1.78 eV, respectively. Electronegativity of compound 1 was calcu-
lated to be 4.02 eV. Energies of HOMO and LUMO were computed
as 6.25 and 1.78 eV, respectively. Hardness and softness were
calculated to be 2.23 and 1.12, respectively (Table 6).
Conclusion
We have carried out a comprehensive theoretical and experi-
mental study of 5,7,30 ,50 -tetrahydroxyflavanone isolated from the
leaves of O. ferruginea. A suitable theoretical model was developed
the online version, at http://dx.doi.org/10.1016/j.saa.2014.02.163.
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