Y. Zaoui, Y. Ramli, S.L. Tan et al.
Journal of Molecular Structure 1234 (2021) 130177
CCDC 2035086 contains the supplementary crystallographic
bridge Crystallographic Data Centre, 12, Union Road, Cambridge
CB2 1EZ, UK; fax: +44 1223 336033).
Where A is the central molecule, B are the molecules surrounding
A, and RAB is the separation of the molecular centroids within a
radius of RAB defined by the molecular centroids [49].
2.5. Biological assays
2
.5.1. Enzyme inhibitory activity: α-Glucosidase inhibition assay
The α-glucosidase inhibitory activity was performed in phos-
2
.3. Quantum chemical calculations
phate buffered saline PBS (0.1 M KH PO –K HPO , pH 6.7), us-
The gas-phase geometry optimisation calculations were per-
2
4
2
4
ing 4-nitrophenyl-α-D-glucopyranoside (ρNPG) as the substrate
according to the method described by Kee et al. [50], with some
modifications. All tested extracts were dissolved in phosphate
buffered saline (PBS) to a series of different concentrations. Briefly,
a mixture of 150 μL of the sample and 100 μL of phosphate
buffered saline (PBS) containing the enzyme α-glucosidase solu-
tion (0.1 U/mL) were incubated at 37°C for 10 min. Then, 200 μL
formed with Gaussian 16 [32] using Stewart’s semi-empirical PM7
method [33] followed by the DFT-B3LYP exchange-correlation func-
tional coupled with Ahlrichs’ valence triple-zeta polarization ba-
sis sets (def2-TZVP) [34] with tight SCF convergence criteria ap-
plied. Having established the optimised structures, the NBO analy-
sis [35], molecular electrostatic potential (MEP) and frontier molec-
ular orbital (FMO) energies were computed for the optimised
structures using the same basis set and level of theory in which
the corresponding outputs were analysed and interpreted through
GaussView6 [36]. The condensed Fukui function [37] and relevant
dual descriptor [38,39] were calculated using the NPA charges de-
rived from the Gaussian program [32].
4
-nitrophenyl-α-D-glucopyranoside (1 mM) was added to the mix-
ture to initiate the reaction. After further incubation at 37°C for 30
min, 600 μL Na CO3 (0.1 M) was added and the absorbance was
2
measured at 405 nm on a UV/vis spectrophotometer.
Two concentrations were tested for 1: 45.0 and 22.5 mM/l. The
standard, Acarbose, was used as the positive control.
The results were expressed as percentage inhibition and calcu-
lated using the following formula (2):
2
.4. Computational modelling of intermolecular interactions
ꢁ
ꢂ
ꢃ
ꢄ
The intermolecular interactions and contacts were analysed
Inhibition(%) = 1 − A
− Absample /(Acontrol − Abcontrol
)
sample
through CrystalExplorer17 [40] using the methodologies as de-
×
100
(2)
scribed previously [41]. Briefly, the distances of atomic surface
points to the nearest nucleus inside (d ) and outside (de) the sur-
where:
i
face were computed and the resulted normalised contact distances
-
-
Acontrol refers to the absorbance of control (enzyme and buffer)
(
dnorm) were mapped onto the Hirshfeld surface in the range -
.0081 to 1.0105 arbitrary units. Contact distances shorter than the
Abcontrol refers to the absorbance of control blank (buffer with-
0
out enzyme)
sum of van der Waals radii are highlighted in red, while distances
equal to or longer than the sum of van der Waals radii are, respec-
-
-
Asample refers to the absorbance of sample (enzyme and in-
hibitor)
tively, shown in white and blue [42]. The combination of d and de
i
Absample is the absorbance of sample blank (inhibitor without
enzyme)
˚
in intervals of 0.01 A resulted in the plotting of two-dimensional
fingerprint plots, where different colours on the fingerprint plots
represent the probability of occurrence, ranging from blue (few
points) to green (moderate) and red (many points) [43]. All hydro-
gen atom bond lengths were normalised to the standard neutron
values prior to the analysis.
2
.5.2. In vitro anti-inflammatory study
Preparation of erythrocyte suspension. A suspension of erythrocytes
was prepared by the method described by Shinde et al. [51] with
some modifications.
Upon the identification of close contacts in the Hirshfeld sur-
face analysis, various qualitative and quantitative computations
were then performed to study the strength of all identified pair-
wise interacting molecules. The qualitative analysis was achieved
through NCIPLOT [44] by plotting the reduced density gradient as
a function of density across the molecules. The computed density
derivatives were mapped as iso-surfaces which correspond to any
favourable or unfavourable interactions as determined by the sign
of the second density Hessian eigenvalue times the density [45].
The program VMD Molecular Graphics Viewer [46] was used to vi-
sualise the non-covalent interaction index. As for the quantitative
analysis, the interaction energies were computed using the opti-
mised CE-B3LYP/6-31G(d,p) model as available in CrystalExplorer17
A sample of fresh whole human blood was obtained from a
healthy human volunteer at the National Blood Transfusion Cen-
ter of Rabat, Morocco, in January 2019. Serological tests were per-
formed and proved negative.
Whole human blood was transferred to heparinised centrifuge
tubes, centrifuged at 3000 rpm for 5 min and the supernatants
(
plasma and leucocytes) were carefully removed while the packed
red blood cell was washed three times with equal volume of nor-
mal fresh saline (0.9% w/v NaCl).
The volume of the blood was measured and reconstituted as a
4
0% (v/v) suspension with isotonic buffer solution (10 mM sodium
phosphate buffer pH 7.4).
The composition of the buffer solution (g/l) was NaH PO (0.2),
2
4
[
40], in which the corresponding energy was obtained by summing
Na HPO4 (1.15) and NaCl (9.0) [52].
2
up four energy components comprising the electrostatic (Eele), po-
larization (Epol), dispersion (Edis) as well as exchange-repulsion
Heat-Induced haemolysis assay. This test was carried out as de-
scribed by Okoli et al. [53] and Ranasinghe et al. [54]. The isotonic
buffer solutions (5 mL) containing 1000, 500 and 250 μg/mL of
(
Erep) energies with scale factors of 1.057, 0.740, 0.871 and 0.618,
respectively [47,48]. A similar method was applied in the simula-
tion of energy frameworks and calculation of the lattice energy for
the crystals. The energy frameworks comprising Eele, Edis and Etotal
were generated for a cluster of 2 × 2 × 1 unit-cells with the en-
ergy cut-off being set to 1.9 kcal/mol, whereas the lattice energy
1
were put in 5 sets of centrifuge tubes (per concentration). The
negative control tubes contained 5 mL of the vehicle and positive
control tubes contained 100 μg/mL of Indomethacin in 5 mL total
volume. The erythrocyte suspension (0.05 mL) was added to each
tube and gently mixed. A pair of the tubes was incubated at 54°C
for 20 min. in a regulated water bath. At the end of the incubation,
the reaction mixture was centrifuged at 1300 g for 3 min and the
˚
was calculated for a cluster of molecules within a 25 A radius from
a selected central molecule using equation (1):
ꢀ
AB
total
Elattice = 1/2
E
(1)
3