Strategy for the vibrational circular dichroism study of a glycosylflavonoid evaluated as its peracetate. The case of bioactive 7-O-β-D-glucopyranosylchrysin

Article abstract of DOI:10.1016/j.molstruc.2020.129147

As it is difficult to study polyhydroxylated natural products by vibrational circular dichroism (VCD) due to their very limited solubility in infrared transparent solvents, 7-O-β-D-glucopyranosylchrysin (1), isolated from Mimosa rosei, was acetylated to provide 7-O-(2??,3??,4??,6??-tetraacetyl-β-D-glucopyranosyl)-5-acetylchrysin (2). Density functional theory (DFT) VCD spectra of 2 were calculated using the B3LYP, B3PW91, and PBEPBE functionals and the DGDZVP basis set to ascertain a good level of theory for calculating this kind of compounds. Statistical comparison of the experimental and calculated VCD spectra revealed that the frequently employed B3LYP and B3PW91 functionals provided good confidence levels with similar statistical results, allowing band assignments for the pyranoside portion of 2. This is the first report on a satisfactory statistical validation of a VCD measurement for such a glycopyranoside. In addition, in vitro cytotoxicity studies of 1 and 2 against the human colon cell line HT-29 and the human melanoma cell line UACC-62 by the MTT method were assayed. Moreover, the immunomodulatory response of 1 and 2 was evaluated by measuring TNF-α, IL-1β, and IL-10 levels by the ELISA technique. The results revealed low cytotoxicity of both compounds against cancer cells. However, flavonoid 1 upregulated the IL-1β production in lipopolysaccharide (LPS)-induced THP-1 macrophage, thus suggesting the potential of this molecule as an immunostimulant for cancer treatment.

Full text of DOI:10.1016/j.molstruc.2020.129147

Journal of Molecular Structure 1225 (2021) 129147
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Strategy for the vibrational circular dichroism study of a
glycosylflavonoid evaluated as its peracetate. The case of bioactive
7-O-β-D-glucopyranosylchrysin
Lirenny Quevedo-Tinocoa, Gabriela Rodríguez-Garcíaa, Rosa E. del Ríoa, Elena Talero b,
Virginia Motilvab, Carlos M. Cerda-García-Rojasc,∗, Pedro Joseph-Nathanc,
Mario A. Gómez-Hurtadoa,∗
a Instituto de Investigaciones Químico Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Ciudad Universitaria, Morelia, Michoacán 58030,
Mexico
b Facultad de Farmacia, Universidad de Sevilla, Profesor García González No. 2, Sevilla 41012, Spain
c Departamento de Química, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Apartado 14-740, Mexico City 07000, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
As it is difficult to study polyhydroxylated natural products by vibrational circular dichroism (VCD) due
to their very limited solubility in infrared transparent solvents, 7-O-β-D-glucopyranosylchrysin (1), iso-
lated from Mimosa rosei, was acetylated to provide 7-O-(2ʹʹ,3ʹʹ,4ʹʹ,6ʹʹ-tetraacetyl-β-D-glucopyranosyl)-5-
acetylchrysin (2). Density functional theory (DFT) VCD spectra of 2 were calculated using the B3LYP,
B3PW91, and PBEPBE functionals and the DGDZVP basis set to ascertain a good level of theory for cal-
culating this kind of compounds. Statistical comparison of the experimental and calculated VCD spectra
revealed that the frequently employed B3LYP and B3PW91 functionals provided good confidence levels
with similar statistical results, allowing band assignments for the pyranoside portion of 2. This is the
first report on a satisfactory statistical validation of a VCD measurement for such a glycopyranoside. In
addition, in vitro cytotoxicity studies of 1 and 2 against the human colon cell line HT-29 and the hu-
man melanoma cell line UACC-62 by the MTT method were assayed. Moreover, the immunomodulatory
response of 1 and 2 was evaluated by measuring TNF-α, IL-1β, and IL-10 levels by the ELISA technique.
The results revealed low cytotoxicity of both compounds against cancer cells. However, flavonoid 1 up-
regulated the IL-1β production in lipopolysaccharide (LPS)-induced THP-1 macrophage, thus suggesting
the potential of this molecule as an immunostimulant for cancer treatment.
Received 21 February 2020
Revised 5 August 2020
Accepted 24 August 2020
Available online 25 August 2020
Keywords:
7-O-β-D-glucopyranosylchrysin
7-O-(2ʹʹ,3ʹʹ,4ʹʹ,6ʹʹ-tetraacetyl-β-D-
glucopyranosyl)-5-acetylchrysin
Mimosa rosei
Vibrational circular dichroism
Density functional theory
Immunostimulant
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
compounds. The sensibility of VCD for the study of glycosides was
evaluated half a century ago [2] and the use of VCD for sugars was
The structure elucidation of glycoside flavonoids frequently in-
volves hydrolysis of the natural product followed by optical rota-
tion analysis of the glycosidic portion requiring chiral characteri-
zation [1]. However, when limited amounts of a natural product
are available, chemical degradation is not the best structure elu-
cidation strategy as further studies, like biological tests, cannot be
performed. Therefore, non-destructive approaches should be devel-
oped for the absolute configuration (AC) determinations in such
cases. For this goal we considered vibrational circular dichroism
(VCD) as the tool of choice for the AC determination of this kind of
proposed [3] associating strategic VCD bands, in particular those
in the 3000-2800 cm−1 region owing to the anomeric stereogenic
center, while ignoring the fingerprint region of the spectrum [4–8].
The use of density functional theory (DFT) calculations to cor-
relate specific bands of VCD spectra of sugars with their confor-
mational preferences improved the understanding of this method-
ology [5,9–11], although it soon became clear that the presence of
hydroxy groups, which favor intramolecular associations by forma-
tion of hydrogen-bonds, causes significant spectra variations [6,12].
In order to diminish this limiting factor, simple derivatives of hy-
droxy groups, like ethers [9,10] or acetates, were successfully ex-
plored [13]. Furthermore, to provide cogent evidence for a success-
ful comparison of experimental and calculated VCD spectra, the
use of statistical methods is of prime importance [12,14,15], al-
∗
Corresponding authors.
E-mail
addresses:
ccerda@cinvestav.mx
(C.M.
Cerda-García-Rojas),
mario.gomez@umich.mx (M.A. Gómez-Hurtado).
https://doi.org/10.1016/j.molstruc.2020.129147
0022-2860/© 2020 Elsevier B.V. All rights reserved.

2
L. Quevedo-Tinoco, G. Rodríguez-García and R.E. del Río et al. / Journal of Molecular Structure 1225 (2021) 129147
range due to the flavonoid skeleton and another set of signals in
the δ 5.15-3.17 range due to a glycoside moiety. The 13C NMR spec-
trum showed 21 carbons, highlighting those at δ 182.7 and δ 100.1
assigned to the carbonyl of the flavonoid portion and the anomeric
carbon of the glycosidic residue. The dominant peak in the EIMS
(m/z 254) was associated to the chrysin skeleton, while fragment
ion peaks at m/z 128, 124, 102, and 85 were related to the glu-
cose ionization [24]. The combined physical and spectroscopic data
agreed for 7-O-β-D-glucopyranosylchrysin (1) [22,25,26].
Fig. 1. Formulas of glucosides 1 and 2.
Acetylation of 1 in pyridine with acetic anhydride gave 7-O-
(2ʹʹ,3ʹʹ,4ʹʹ,6ʹʹ-tetraacetyl-β-D-glucopyranosyl)-5-acetylchrysin (2) in
85% yield as colorless needles whose HREIMS showed m/z 649.1525
[M + Na]+ (calcd for C31H30O14 + Na+, 649.1528). Its 1H NMR
spectrum showed the characteristic chrysin pattern with signals at
δ 7.85 (2H, apparent dd, J = 8.0, 1.6 Hz, H-2ʹ and H-6ʹ), 7.53 (3H,
m, H-3ʹ, H-4ʹ, and H-5ʹ), 7.02 (1H, d, J = 2.4 Hz, H-8), 6.70 (1H,
d, J = 2.4 Hz, H-6), 6.63 (1H, s, H-3). The glycoside resonances
appeared in the δ 5.40-3.90 region and the singlets of the acetyl
groups became evident at lower frequencies. The 13C NMR spec-
trum showed the expected signals for the glycosidic flavonoid, to-
gether with ten signals attributed to five acetate groups. The 1H
and 13C NMR assignments were confirmed by 2D NMR experi-
ments (see Supplementary Data). Although 2 was described as de-
rived from 1 [27,28], only the elemental analysis and melting point
were reported at those times. Thus, the NMR spectra, IR, specific
rotation, and HREIMS data of 2 are herein reported for the first
time.
beit the AC determination of sugars by VCD is only described for
furanose derivatives [16]. Since the determination of the AC of a
molecule possessing many stereocenters could be a very challeng-
ing task if one lacks of independent evidences for the relative con-
figuration of several stereocenters, as is usually derived from nu-
clear magnetic resonance measurements, the main purpose of this
paper is to test a VCD strategy using a glucopyranose derivative of
known AC that can be handled in an IR transparent solvent at the
required concentrations.
In the present work 7-O-β-D-glucopyranosylchrysin (1) was
used as a sugar containing molecular model for AC studies by
VCD, including statistical validations to explore the use of com-
mon DFT calculation levels. Flavonoid 1, isolated from Mimosa ro-
sei B.L.Rob., was reacted providing 7-O-(2ʹʹ,3ʹʹ,4ʹʹ,6ʹʹ-tetraacetyl-β-D-
glucopyranosyl)-5-acetylchrysin (2) which turned out to be ade-
quate for VCD measurements. The VCD calculations of 2 were done
using the B3LYP, B3PW91, and PBEPBE functionals and the DGDZVP
basis set followed by statistical validation using the CompareVOA
software [17] from where it follows the popular B3LYP/DGDZVP
level of theory is suitable for this task [12]. The search for proper
levels to calculate VCD spectra is relevant since it has been shown
[18] that different DFT levels of theory produce different calculated
spectra since, among other facts, the number of conformers to be
considered in a given energy gap changes with the used level of
theory.
For the calculation of the VCD spectra of 2 a molecular model
was constructed in the Spartan’04 software. As a starting confor-
mation, the glucose ring was assembled as a chair and the acetate
groups of the sugar residue were assumed with C–O–C=O dihe-
dral angles of 0°. A conformational population search, without any
angles restriction, using the Monte Carlo protocol and the Merck
Molecular Force Field (MMFF) [29] provided 150 conformers in the
0-10 kcal/mol energy window. Those 88 conformers found in the
initial 5 kcal/mol were submitted to single point energy calcula-
tion at the B3LYP/6-31G(d,p) level of theory using the same soft-
ware. The resulting 35 conformers, found in the initial 3 kcal/mol,
were energy optimized at the B3LYP/DGDZVP level of theory, with
the Gaussian 16 software, and finally, the nine conformers found in
the initial 2 kcal/mol (Fig. S15 and Table S1) were used to calculate
the IR and VCD spectra (Fig. S16). A CompareVOA software evalu-
ation of the calculated and experimental spectra revealed a poor
confidence level of only 51%, the corresponding data being shown
in the Supporting Information section (Table S3).
An alternative methodology for the AC determination of organic
molecules is electronic circular dichroism (ECD), whose measure-
ments are performed in the UV and visible regions of the electro-
magnetic spectrum, instead using the IR radiation required for VCD
measurements. ECD is advantageous over VCD since it requires a
few orders of magnitude less sample, but it requires the presence
of a well absorbing chromophore in the neighborhood of stere-
ogenic centers, a situation not being fulfilled in the present case.
The antioxidant, anti-inflammatory, and anti-cancer effects of
chrysin are known [19,20], while 1 has shown to be a potential
diuretic and hypotensive agent [21], and also showed antioxidant
and antimicrobial activity [22]. We now evaluated the biological
profile of 1 and 2 for their cytotoxicity against the human colon
cell line HT-29 and the human melanoma cell line UACC-62, as
well as their immunomodulatory activity. The results revealed low
cytotoxicity of the compounds against cancer cells, but 1 upreg-
ulated IL-1β production in LPS-induced THP-1 macrophages sug-
gesting its potential use as an immunostimulant for cancer treat-
ment.
As the next trial, a similar calculation procedure, in which the
C–O–C=O dihedral angles of the glucose acetates at the C-2ʹʹ, C-
3ʹʹ, and C-4ʹʹ positions were fixed at 0° during the conformational
search, was performed. The conformational restrictions were re-
moved for the 105 conformers found in the initial 5 kcal/mol
which were submitted to single point energy calculations using
the Spartan’04 software at the B3LYP/6-31G(d,p) level of theory.
The 45 conformers found in the initial 3 kcal/mol were energy op-
timized at the B3LYP/DGDZVP level of theory and the eight con-
formers (Fig. S17 and Table S2) that remained in the same energy
gap were then used to calculate the IR and VCD spectra (Fig. S18)
in the Gaussian 16 suit. This calculation procedure provided a con-
fidence level of 76% after CompareVOA evaluation, which is better
than that obtained in the first calculation trial, but clearly unsatis-
factory (Table S3).
2. Results and discussion
Compound 1 was isolated from the methanol extracts of the
leaves of Mimosa rosei B.L.Rob, as detailed in the Experimental Sec-
tion, and its molecular structure, according to the prefered π-bond
order [23], is showed in Fig. 1. It was obtained as yellow amor-
phous powder, whose IR spectrum showed bands at νmax 3398 and
1653 cm−1 attributed to hydroxy and carbonyl groups, respectively.
The 1H NMR spectrum showed one set of signals in the δ 8.08-6.47
From the above two calculations, it seems that the software is
generating a large number of redundant conformers. Therefore, re-
stricted rotations of the acetate groups of the three acetates at the
sugar ring were imposed by fixing the respective Hʹʹ−Cʹʹ−O−Csp2
dihedral angles to 0° according to the well-known electronic stabil-
ities of this kind of atom arrangements [30,31]. A conformational

L. Quevedo-Tinoco, G. Rodríguez-García and R.E. del Río et al. / Journal of Molecular Structure 1225 (2021) 129147
3
Table 1
Thermochemical analysis of compound 2.
a
b
c
d
e
f
g
f
h
f
Conf
ꢀEMMFF
%
ꢀEDFT
%
ꢀGOPT
%
ꢀGOPT
%
ꢀGOPT
%
OPT
MMFF
DFT
OPT
OPT
2a
2b
2c
2d
2e
2f
3.06
2.51
2.96
3.05
4.95
3.83
4.82
3.65
4.59
0.89
0.00
1.31
1.38
0.47
2.77
0.43
2.64
3.87
3.79
1.18
3.15
2.85
2.38
2.29
3.06
0.20
0.52
0.24
0.21
0.01
0.05
0.01
0.07
0.01
8.17
37.06
4.04
3.60
16.57
0.34
17.91
0.01
0.05
0.06
5.02
0.17
0.29
0.66
0.76
0.21
0.76
1.03
0.89
0.94
2.07
1.13
2.75
0.99
1.65
0.20
0.00
2.40
2.34
0.43
4.81
0.30
1.37
1.19
1.08
2.06
1.50
2.25
1.67
1.64
2.31
4.68
2.98
3.77
3.47
0.51
2.52
0.16
3.19
1.04
12.01
17.06
0.29
0.32
8.19
0.01
10.25
1.66
2.28
2.74
0.52
1.35
0.37
1.01
1.05
0.34
0.00
0.18
0.31
1.02
1.04
1.32
1.52
1.59
1.77
1.80
1.86
1.99
31.98
23.81
18.91
5.78
5.56
3.51
2.49
2.23
1.65
1.56
1.38
1.13
0.00
0.21
0.50
0.90
0.77
1.00
1.36
1.76
1.17
1.08
16.49
11.59
7.12
3.63
4.52
3.03
1.67
0.85
2.29
2.67
0.68
0.50
1.10
1.00
1.26
1.39
3.90
5.26
1.90
2.26
1.46
1.17
2g
2h
2i
1.28
0.98
0.03
0.00
0.22
0.23
0.25
1.41
2.34
11.60
12.23
8.37
8.24
7.93
2j
2k
2l
0.30
0.25
0.44
0.45
0.66
1.38
1.40
1.90
1.94
9.96
10.76
7.83
7.63
5.42
1.60
1.56
0.68
0.62
2m
2n
2o
2p
2q
2r
0.63
0.35
1.33
1.32
0.08
0.80
1.39
1.44
1.47
1.88
4.24
6.75
1.31
1.32
10.69
3.19
1.18
1.08
1.02
0.52
2s
2t
2u
2v
2w
2x
2y
a
Molecular mechanics energies relative to 2k, EMMFF = 63.303 kcal/mol.
b
c
d
e
f
Population in % calculated from the MMFF energies according to ꢀEMMFF ≈ −RT ln K.
Single-point B3LYP/6-31G(d) energies relative to 2k, E6-31G(d) = −1413522.070 kcal/mol.
Population in % calculated from B3LYP/6-31G(d) energies according to ꢀE6-31G(d) ≈ −RT ln K.
Gibbs free energies relative to 2a for B3LYP/GDGDZVP = −1413384.882 kcal/mol.
Population in % calculated from Gibbs free energies according to ꢀG = −RT ln K.
Gibbs free energies relative to 2a for B3PW91/DGDZVP = −1412802.596 kcal/mol.
Gibbs free energies relative to 2k for PBEPBE/GDGDZVP = −1411776.794 kcal/mol.
g
h
Table 2
population search using the Monte Carlo protocol and the Merck
Molecular Force Field (MMFF) provided 100 conformers in the 0-
10 kcal/mol energy window. The reasoning behind this momentary
conformational restriction at the MMFF calculation stage is that
if each acetate just would generate two conformers, instead three
conformers, we would have 8 conformers, and if it would generate
three conformers, we would have a total of 27 conformers. It has to
be remembered that MMFF calculations do not consider electronic
effects at all. No real conformer is ignored by this momentary sim-
plification since it is well known that an ester essentially only ex-
ists in one conformational orientation, as this is the basis for the
very popular Mosher ester methodology used for the AC determi-
nation of a stereogenic center having a secondary hydroxyl group.
The conformational restrictions for the 38 conformers found in the
0-5 kcal/mol energy gap were released and these conformers were
submitted to energy optimization at the B3LYP/6-31G(d) level of
theory. In order to search for an adequate level of theory that equi-
librates the quality of the calculated data and the calculation time,
the 25 conformers found in the 0-3 kcal/mol energy gap were se-
lected for conformational optimizations using the B3LYP, B3PW91,
and PBEPBE functionals and the DGDZVP basis set. These levels of
theory are commonly used for VCD calculations, although the last
one has been used more when aromatic rings are present in the
studied molecules [12]. These three levels of calculation revealed
significant differences for 2, as the B3LYP/DGDZVP level of theory
gave 12 conformers, the B3PW91/DGDZVP level provided 19, and
the PBEPBE/DGDZVP level gave 23 conformers, always in the 0-2
kcal/mol gap (Table 1), as was the case for perezone [18]. These
conformers were used for the IR and VCD spectra calculations at
the same levels of theory. Conformer population weighting was
done according to the ꢀG = −RT ln K equation to generate the
respective Boltzmann-averaged IR and VCD spectra. The most rel-
evant conformers calculated with the B3LYP functional, represent-
Confidence level data for the IR and VCD spectra of 2 calculated
using the DGDZVP basis set and three functionals.
b
c
d
Compound
anHa
SIR
SE
S
ESIe
Cf (%)
−E
B3LYP
0.979
0.969
1.090
97.0
97.3
88.3
74.4
70.1
51.8
19.7
20.0
18.1
54.7
50.1
33.7
100
100
40
B3PW91
PBEPBE
a
Anharmonicity factor.
IR spectra similarty.
b
c
d
e
f
VCD spectra similarity for the correct enantiomer.
VCD spectra similarity for the incorrect enantiomer.
Enantiomer similarity index calculated as SE − S
.
−E
Confidence level for the configurational assignments.
ing 89.3% of the total population are shown in Fig. 2, where the
conformational preferences of the acetate groups at C-2ʹʹ, C-3ʹʹ, and
C-4ʹʹ are in close agreement with those of the starting molecular
conformation.
The experimental IR and VCD spectra of 2 were compared to
the calculated ones at the three levels of theory using the statistic
correlation provided by the CompareVOA program [17]. The corre-
sponding numeric confidence level data are summarized in Table 2,
where in the case of the B3LYP functional the optimal anharmonic-
ity factor (anH) was 0.979. The VCD spectroscopic similarity for the
correct enantiomer (SE) was 74.4, while that for the incorrect enan-
tiomer (S−E) was 19.7. The enantiomer similarity index (ESI), ob-
tained by the SE − S−E difference was 54.7, and the data indicated
a 100% confidence level (C) for the AC of compound 2. In turn, the
B3PW91 functional provided statistical values close to those calcu-
lated with the B3LYP functional (Table 2) and therefore the AC de-
termination of glucopyranosides like 2 is also feasible at this level
of theory, although the number of conformers at the B3PW91 level
is larger (Table 1) and therefore longer calculation times for this

4
L. Quevedo-Tinoco, G. Rodríguez-García and R.E. del Río et al. / Journal of Molecular Structure 1225 (2021) 129147
Fig. 2. Low-energy conformers of 7-O-(2ʹʹ,3ʹʹ,4ʹʹ,6ʹʹ-tetraacetyl-β-D-glucopyranosyl)-5-acetylchrysin (2) accounting for 89.3% of the conformational population at the
B3LYP/DGDZVP level of theory.
generalized gradient approximations (GGA’s) method [32] are re-
quired.
nate situation is also consequent to the fact that the C–O bands
are not overlapped in the VCD spectrum of the sugar portion of 2.
A great advantage of the CompareVOA software [17] is its con-
ception as a tool that allows ascertaining the AC of a molecule re-
gardless which enantiomer has been selected for DFT calculations.
That means, if the calculated enantiomer agrees with the measured
molecule, then the correct enantiomer (SE) will show a large value,
the incorrect enantiomer (S−E) will be a small number, and the
enantiomer similarity index (ESI) will be positive. Contrary, if the
calculated and measured data correspond to opposite enantiomers,
the (SE) value will be small, the (S−E) value will be large, and the
(ESI) value will be negative. VCD is also a good method to distingue
diastereoisomers and many examples have been reviewed [12].
The PBEPBE functional provided poor spectra similarities as can
be observed from the values included in Table 2. This result might
be related to the underestimated vibration frequencies (around 20
cm−1) inherent to this functional, as has already be highlighted
[33–35], although in the case of a natural compound in which the
stereogenic center is directly attached to the aromatic ring [15] this
calculation methodology provided cogent results for the AC deter-
mination.
Consequently, the B3LYP functional in combination with the
DGDZVP basis set provide an adequate theoretical tool for the VCD
calculations of pyranoside derivatives like 2. In this case a fortu-

L. Quevedo-Tinoco, G. Rodríguez-García and R.E. del Río et al. / Journal of Molecular Structure 1225 (2021) 129147
5
Fig. 4. Relative amplitudes of selected vibrational modes (in cm−1 shown under
each structure) for the lowest energy conformer of 2. The vibrational modes are:
1432 cm−1, rocking vibration between O-7−C-1ʹʹ−H-1ʹʹ; 1373 cm−1 and 1186 cm−1
,
scissoring vibrations between Csp2−C−H and Osp2−Csp2−C from the acetate at C-5;
1329 cm−1, scissoring vibrations and 1246 cm−1, wagging between O−C-2ʹʹ−H-2ʹʹ,
O−C-3ʹʹ−H-3ʹʹ, O−C-4ʹʹ−H-4ʹʹ, and O−C-5ʹʹ−H-5ʹʹ; 1213 cm−1, scissoring vibrations
between Osp2−Csp2−C and Csp2−C−H from acetates at C-2ʹʹ, C-3ʹʹ, and C-4ʹʹ; 1172
cm−1, scissoring vibrations between C-5ʹʹ−O-1ʹʹ−C-1ʹʹ; 1051 cm−1, scissoring vibra-
tions between O-1ʹʹ−C-1ʹʹ−O-7. Note the small arrows pointing to the atoms of in-
terest for a particular transition, drawn using the GaussianView software package.
Fig. 3. Comparison of the experimental and DFT B3LYP/DGDZVP calculated IR and
VCD spectra of 7-O-(2ʹʹ,3ʹʹ,4ʹʹ,6ʹʹ-tetraacety-β-D-glucopyranosyl)-5-acetylchrysin (2).
negative band at 1230 cm−1 [10] and therefore our results comple-
ment the VCD data for sugars.
Once the comparison of the experimental and calculated IR and
VCD spectra of 2 was quantitatively supported, assignment of sev-
eral relevant vibration bands of the sugar portion of 2 can be done
by use of the GaussView software (Fig. 3). The negative vibration at
1432 cm−1 (1) is originated by the O-7−C-1ʹʹ−H-1ʹʹ rocking vibra-
tion, the negative band at 1373 cm−1 (2) and the positive band at
1186 cm−1 (6) were assigned to the Csp2−C−H and Osp2−Csp2−C
scissoring vibrations, respectively, of the acetate at C-5, the posi-
tive vibration at 1329 cm−1 (3) was assigned to all O−C−H scis-
soring vibrations of the glucose moiety, while their corresponding
wagging vibrations were associated to the negative band at 1246
cm−1 (4). The most intense negative band at 1213 cm−1 (5) was
attributed to the Osp2−Csp2−C and Csp2−C−H scissoring vibrations
of the acetate groups at C-2ʹʹ, C-3ʹʹ, and C-4ʹʹ, the positive band
at 1172 cm−1 (7) is due to the C-5ʹʹ−O-1ʹʹ−C-1ʹʹ scissoring vibra-
tions, and the O-1ʹʹ−C-1ʹʹ−O-7 portion of the sugar moiety showed
its scissoring vibrations as a negative band at 1051 cm−1 (8). The
above vibrational modes are depicted in Fig. 4. The anomeric C−O
vibration of aromatic O-α-D-glucosides was already described as a
Since the biological properties of 1 or its derivatives have been
poorly evaluated, we explored the in vitro cytotoxic effects of 1
and 2 against the colon adenocarcinoma cell line HT-29 and the
human malignant melanoma cell line UACC-62 by using the MTT
method (Fig. 5). The cytotoxicity graphs for both compounds are
presented in Fig. 5C, where it can be compared to that of the anti-
cancer drug oxaliplatin. Compounds 1 and 2 exhibited slight cyto-
toxicity against HT-29 cells (Fig. 5A) with IC50 values above 100 μM
(Fig. 5C). Compound 1 also showed slight cytotoxic activity against
the UACC-62 cell line (IC50 > 100 μM) (Fig. 5B), while 2 showed
low but measurable cytotoxicity towards UACC-62 cells (IC50 = 61.5
μM).
The profile of 1 and 2 in inflammatory processes was also in-
vestigated by quantification of the proinflammatory cytokines TNF-
α and IL-1β, as well as the anti-inflammatory cytokine IL-10 in
the human acute monocytic leukemia cell line THP-1, differenti-
ated into macrophages. The cytotoxic effect of 1 and 2 on THP-1
macrophages was determined using the MTT assay. It turned out

6
L. Quevedo-Tinoco, G. Rodríguez-García and R.E. del Río et al. / Journal of Molecular Structure 1225 (2021) 129147
Fig. 5. Effect of 1 and 2 on the viability of HT-29 (A) and UACC-62 (B) cell lines after treatment for 48 h. Oxaliplatin was used as the positive reference compound. Results
obtained by the MTT assay are reported as percentage of viable cells (mean SEM), IC50 (μM). (C) Mean values SEM of three experiments.
Fig. 6. Effect of 1 and 2 on lipopolysaccharide (LPS)-induced TNF-α (A), IL-10 (B), and IL-1β (C) production by THP-1 macrophages. Cells were incubated with 1 at 10, 50, and
100 μM, and 2 at 1, 5, and 10 μM for 1 h and then stimulated with LPS (1 μg/mL) for 24 h. Cytokines were quantified in supernatants using the ELISA assay. Dexamethasone
(Dexa) was used as the positive reference compound at 1 μM. Data are means
SEM from three independent experiments. Statistical significance of any difference in each
parameter between several groups was evaluated by one-way analysis of variance (ANOVA) followed by Bonferroni test. +++p <0.001 vs. Control; ∗p <0.05, ∗∗∗p <0.001 vs.
LPS.
that 1 did not affect cell viability, with an IC50 value above 100
μM after 24 h, while 2 is more cytotoxic on THP-1 cells, display-
In contrast, compound 1 substantially increased IL-1β levels at
the highest used concentration (p <0.05) (Fig. 6C) suggesting its
potential use for anti-tumor therapy [37], since IL-1β has shown a
beneficial role on the resolution of acute inflammation and in tu-
mor suppression via natural killer and T cell-mediated cytotoxicity
[38].
ing an IC50 value of 36.2
2.8 μM. Therefore, to rule out cytotoxic
effects, 1 was tested at 10, 50, and 100 μM while 2 was tested at
1, 5, and 10 μM. To evaluate the effects of 1 and 2 on the produc-
tion of cytokines, THP-1 macrophages were pretreated with both
molecules for 1 h and then stimulated with LPS (1 μg/mL) for 24
h. Levels of TNF-α, IL-1β, and IL-10 were determined using ELISA
kits.
3. Experimental
TNF-α is a potent pro-inflammatory cytokine produced by im-
mune cells at the inflammation site and plays a crucial role in local
and systemic inflammatory responses [36]. As shown in Fig. 6, LPS
induced a significant increase in the production of this cytokine
in THP-1 cells as compared with unstimulated control cells (p
<0.001). The anti-inflammatory reference compound dexametha-
sone markedly reduced LPS-induced TNF-α levels. Neither of the
assayed compounds induced significant changes in TNF-α produc-
tion (Fig. 6A), and compounds 1 and 2 did not influence IL-10 pro-
duction as can be seen in Fig. 6B.
3.1. General experimental procedures
Melting points (uncorrected) were determined on a Fisher-Johns
apparatus. Optical rotations were recorded in CHCl3 or pyridine
solutions at room temperature on a Perkin Elmer 341 polarime-
ter. 1D and 2D NMR spectra were measured at 300 or 400 MHz
for 1H and at 75.4 or 100 MHz for 13C on Varian Mercury 300 or
400 spectrometers from CDCl3 or DMSO-d6 solutions using tetram-
ethylsilane as the internal reference. Chemical shift values are re-
ported in parts per million and coupling constants (J) are in Hz.
LRMS were recorded on a Varian Saturn 2000 spectrometer, while

L. Quevedo-Tinoco, G. Rodríguez-García and R.E. del Río et al. / Journal of Molecular Structure 1225 (2021) 129147
7
HRMS data were acquired on a Waters Synapt G2 spectrometer at
3.6. Vibrational circular dichroism calculations
the Department of Biochemistry, University of Colorado, Boulder,
CO, USA. Silica gel 230−400 mesh (Merck) was used for column
chromatography.
The in silico constructed molecular model of 2 was subjected
to Monte Carlo search protocols in a 10 kcal/mol energy gap using
the Merck Molecular Force Field (MMFF94) as implemented in the
Spartan’04 program. The searches were carried out from a model
with conformational restricted acetate groups at C-2ʹʹ, C-3ʹʹ, and C-
3.2. Plant material
4ʹʹ. For this purpose, the Hʹʹ−Cʹʹ−O−Csp dihedral angles were fixed
2
Specimens of Mimosa rosei B.L.Rob. were collected from La Hua-
to 0°, providing 100 conformers. The resulting 38 conformers in
a 0-5 kcal/mol energy gap were submitted to single-point energy
calculations using the DFT B3LYP/6-31G(d) level of theory in the
Spartan’04 program. Those conformers in the 0-3 kcal/mol range
were geometry optimized by DFT calculations using the B3LYP,
B3PW91, and PBEPBE functionals and the DGDZVP basis set em-
ploying the Gaussian 09 program. The structures accounting for
89.3% of the global minimum energy profile at the B3LYP/DGDZVP
level of theory are depicted in Fig. 2. The minimized structures at
the B3LYP/DGDZVP, B3PW91/DGDZVP, and PBEPBE/DGDZVP levels
of theory, within the first 2 kcal/mol (see Supplementary Data),
were used to calculate the thermochemical parameters and the
IR and VCD frequencies at 298 K and 1 atm. All minimum en-
ergy structures were verified for the absence of imaginary frequen-
cies and their relative free energies were employed to calculate
their Boltzmann population. The Boltzmann-weighted IR and VCD
spectra were calculated considering Lorentzian bands with half-
widths of 6 cm−1. Molecular visualization was accomplished us-
ing the GaussView 6.0 program. Geometry optimization and vibra-
tional calculations required some 20-30 minutes of CPU time per
conformer when using a processing node having 20 Cores at 2.3
GHz with 128 Gb RAM.
ꢀ
ꢀ
cana, Michoacán, Mexico (18° 57 09.1" N, 101° 53 28.3" W) 480 m
above the average sea level during July and August 2017. The plant
material was identified (No. 30189) by Prof. Rosa Isabel Fuentes
Chávez and Prof. Norma Patricia Reyes Martínez at Facultad de Bi-
ología, Universidad Michoacana de San Nicolás de Hidalgo.
3.3. Extraction and isolation of 7-O-β-D-glucopyranosylchrysin (1)
Dried leaves (300 g) were macerated with MeOH (3 × 2 L)
at room temperature for 48 h. The combined filtered and evapo-
rated extracts yielded 50 g (16.6%) of a dark residue which was
column-chromatographed using 250 mL of CH2Cl2-MeOH mix-
tures, 1:0, 9:1, 7:3, 1:1, 3:7, and 0:1 (F1-F6, respectively). F3
(9 g) was column-rechromatographed using CH2Cl2-MeOH-H2O
(90:10:1) obtaining 5 mL fractions. 7-O-β-D-glucopyranosylchrysin
(1) (100 mg) was obtained from fractions 15-25 as yellow amor-
phous powder, m.p. 208-210°C. Lit. 218-221°C [22] and 213-215°C
[25]; [α]589 −65, [α]578 −69, [α]546 −78 (c 0.05, pyridine). EIMS
m/z 254 [M − Glc]+, Glc pattern: 128 (0.8), 124 (13), 102 (4), 85
(1). IR, 1H and 13C NMR data are in agreement with published val-
ues [22,25,26].
3.7. Cell cultures
3.4. 7-O-(2ʹʹ,3ʹʹ,4ʹʹ,6ʹʹ-Tetraacetyl-β-D-glucopyranosyl)-5-acetylchrysin
(2)
The human colorectal adenocarcinoma cell line HT-29, human
malignant melanoma cell line UACC-62, and human acute mono-
cytic leukemia cell line THP-1 (European Collection of Cell Cul-
tures) were incubated in humidified air containing 5% CO2 at 37°C.
HT-29 was cultured in McCoy’s 5A medium (PAA, Austria), UACC-
62 and THP-1 were cultured in RPMI 1640 media with 2 mM L-
glutamine and 25 mM HEPES (Gibco, USA). All culture media were
supplemented with 10% heat-inactivated fetal bovine serum, 100
U/mL, penicillin, and 100 mg/mL streptomycin (PAA, Austria).
A solution of 1 (10 mg) in pyridine (0.5 mL) and Ac2O (1 mL)
was kept on a steam bath for 4 h, poured over ice−H2O, and ex-
tracted with EtOAc. The organic layer was washed with aqueous
10% HCl, H2O, aqueous NaHCO3, and H2O, dried over anhydrous
Na2SO4, filtered, and evaporated. The residue yielded 2 (12.7 mg,
85%) as colorless needles, m.p. 193-195°C. Lit. 197°C [27], and 197-
198°C [28]; [α]589 −26, [α]578 −28, [α]546 −32, [α]436 −59 (c 0.6,
CHCl3); IR νmax (KBr, cm−1) 2947, 1755, 1642, 1613 cm−1 1H NMR
.
(400 MHz, DMSO-d6) δ (ppm), 7.85 (2H, apparent dd, J = 8.0, 1.6
Hz, H-2ʹ, H-6ʹ), 7.53 (3H, m, H-3ʹ, H-4ʹ, H-5ʹ), 7.02 (1H, d, J = 2.4
Hz, H-8), 6.70 (1H, d, J = 2.4 Hz, H-6), 6.63 (1H, s, H-3), 5.40-3.90
(m, sugar protons), 2.45 (3H, s, Ac-5), 2.08, 2.07, 2.07, 2.05 (Ac from
sugar); 13C NMR (100 MHz, DMSO-d6), δ (ppm) 176.3 (C-4), 170.5
(Ac at C-5), 170.1, 169.6, 169.3, 169.2 (Ac from sugar), 162.4 (C-7),
159.9 (C-2), 158.4 (C-5), 150.7 (C-9), 131.7 (C-4ʹ), 131.1 (C-1ʹ), 129.1
(C-3ʹ, C-5ʹ), 126.1 (C-2ʹ, C-6ʹ), 112.9 (C-10), 109.3 (C-6), 108.5 (C-3),
102.6 (C-8), 98.1 (C-1ʹʹ), 77.4 (C-5ʹʹ), 75.4 (C-3ʹʹ), 70.8 (C-2ʹʹ), 68.1
(C-4ʹʹ), 61.9 (C-6ʹʹ), 29.7 (Ac C-5), 21.1, 20.6, 20.5 (Ac from sugar).
HREIMS m/z 649.1525 [M + Na]+ (calcd for C31H30O14 + Na+,
649.1528).
3.8. Cytotoxicity assay
The in vitro cytotoxicity was determined using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetraazolium bromide (MTT, Cal-
biochem, Germany) dye uptake assay [39] by triplicate. Briefly,
THP-1, HT-29, and UACC-62 cells were individually seeded into 96-
well plates (100 μL/well and 104 cells/well). Phorbol 12-myristate
13-acetate (PMA, Sigma-Aldrich) were added for THP-1 (q.s. 8 nM)
to promote its differentiation into macrophages. Incubation for 72
h (THP-1) or 24 h (HT-29 and UACC-62) were done. Cells were then
washed with phosphate saline buffer (PBS, 4 °C), and incubated
(48 h) as above, adding the assay compounds and controls (sep-
arately) freshly dissolved in DMSO (0.1% v/v). Cells were washed
with PBS prior to the addition of 100 μL/well of 0.25 mg/mL MTT
solution, and were incubated for 4 h. Replacement of the MTT so-
lution by DMSO (100 μL) was done and absorbance at 550 nm was
measured on a Multiskan EX microplate reader (Thermo Scientific,
USA) for establishing the 50% inhibitory concentration (IC50).
3.5. Vibrational circular dichroism measurements
The data were obtained on a BioTools dualPEM ChiralIR FT spec-
trophotometer using a solution of 3.6 mg of 2 in 150 μL of 100%
D atom CDCl3 which was placed in a BaF2 cell with a path-length
of 100 μm. The data were acquired at a resolution of 4 cm−1 for
6 h and the base-line was provided by subtracting the spectrum of
the solvent acquired under identical experimental conditions. The
stability of the sample was monitored by 1H NMR measurements
immediately before and after the VCD measurement.
3.9. Inflammatory response evaluation
Differentiation of THP-1 cells into macrophages (15
×
103
cells/well) was done as in Section 3.8. The cells were washed with

8
L. Quevedo-Tinoco, G. Rodríguez-García and R.E. del Río et al. / Journal of Molecular Structure 1225 (2021) 129147
ice-cold PBS twice and incubated (1 h) adding, separately and dis-
solved in DMSO, non-lethal concentrations of 1 (10, 50, and 100
μM), 2 (1, 5, and 10 μM), and dexamethasone (1 μM) as the
positive reference. Lipopolysaccharide (LPS) from Escherichia coli
(1 μg/mL) was added and incubated for 24 h (n = 3). Control
groups were included. Supernatants were collected and stored at
−80 °C until cytokines (TNF-α, IL-1β, and IL-10) measurements us-
ing ELISA kits (Diaclone GEN-PROBE, France), and read at 450 nm
on a Thermo Scientific Multiskan-EX microplate reader.
[7] M. Paterlini, T. Freedman, L. Nafie, Ring current enhanced vibrational circular
dichroism in the CH-stretching motions of sugars, J. Am. Chem. Soc. 108 (1986)
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[9] T. Taniguchi, K. Monde, Spectrum-structure relationship in carbohydrate vibra-
tional circular dichroism and its application to glycoconjugates, Chem. Asian J.
2 (2007) 1258–1266. https://doi.org/10.1002/asia.200700180.
[10] T. Taniguchi, I. Tone, K. Monde, Observation and characterization of a specific
vibrational circular dichroism band in phenyl glycosides, Chirality 20 (2008)
446–453. https://doi.org/10.1002/chir.20465.
[11] K. Jovan, D. Beckett, K. Raghavachari, Vibrational circular dichroism spectra for
large molecules through molecules-in-molecules fragment-based approach, J.
Chem. Theory Comput. 11 (2015) 4238–4247. https://doi.org/10.1021/acs.jctc.
5b00647.
3.10. Statistical analysis
Numerical values are expressed as arithmetic means standard
error of the mean (SEM). Data were evaluated with the GraphPad
Prism® Version 6.00 software (GraphPad Software, Inc., San Diego,
CA, USA). Statistical significance between the two control groups
was determined by Student’s test. The statistical significance of any
difference in each parameter between several groups was evalu-
ated by one-way analysis of variance (ANOVA) followed by Bonfer-
roni test. P values of <0.05 were considered statistically significant.
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configuration determination of natural products, in: A.D. Kinghorn, H. Falk,
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[14] H.M. Arreaga-González, J.C. Pardo-Novoa, R.E. del Río, G. Rodríguez-García,
J.M. Torres-Valencia, J.J. Manríquez-Torres, C.M. Cerda-García-Rojas, P. Joseph-
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Author statement
All authors contributed equally to this work.
Declaration of Competing Interest
[16] T. Taniguchi, K. Nakano, R. Bab, R. Monde, Analysis of configuration and confor-
mation of furanose ring in carbohydrate and nucleoside by vibrational circu-
lar dichroism, Org. Lett. 19 (2017) 404–407. https://doi.org/10.1021/acs.orglett.
6b03626.
[17] E. Debie, E.D. Gussem, R.K. Dukor, W. Herrebout, L.A. Nafie, P.A. Bultinck, A
confidence level algorithm for the determination of absolute configuration us-
ing vibrational circular dichroism or Raman optical activity, ChemPhysChem.
12 (2011) 1542–1549. https://doi.org/10.1002/cphc.201100050.
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.
Acknowledgments
[18] T. Rojo-Portillo, E. Reyes-López, E. Hernández-Huerta, B. Quiroz-García,
P. Joseph-Nathan, M. Sánchez-Castellanos, F. Cuétara-Guadarrama, G. Cuevas, Is
the VCD spectrum a fingerprint of the conformational population? The confor-
mation of perezone in the spotlight, J. Mol. Struct. 1202 (2020) 1–13 127273.
https://doi.org/10.1016/j.molstruc.2019.127273.
[19] M. Zeinali, S.A. Rezaee, H. Hosseinzadeh, An overview on immunoregula-
tory and anti-inflammatory properties of chrysin and flavonoids substances,
Biomed. Pharmacother. 92 (2017) 998–1009. https://doi.org/10.1016/j.biopha.
2017.06.003.
[20] E.R. Kasala, L.N. Bodduluru, R.M. Madana, A.K. V, R. Gogoi, C.C. Barua, Chemo-
preventive and therapeutic potential of chrysin in cancer: mechanistic per-
spectives, Toxicol. Lett. 4 (2015) 214–225. https://doi.org/10.1016/j.toxlet.2015.
01.008.
Partial financial support from CIC-UMSNH and CONACYT-
Mexico grant 284194 is acknowledged. L.Q.T., is grateful to
CONACYT-Mexico for scholarship 278567. We thank Prof. Rosa Is-
abel Fuentes Chávez and Prof. Norma Patricia Reyes Martínez, Fac-
ultad de Biología, UMSNH, for identifying the plant material, and
to Centro de Investigación, Tecnología e Innovación, Universidad
de Sevilla for providing technical assistance. We thank Prof. Miguel
López Lázaro (Departmento de Farmacología, Facultad de Farmacia,
Universidad de Sevilla) for providing the UACC-62 cell line, and to
Prof. Francisco Muriana (Instituto de la Grasa, CSIC, Sevilla) for pro-
viding the THP-1 cell line.
[21] K. Cherkaoui, M. Lachkar, M. Wibo, N. Morel, A.H. Gilani, B. Lyoussi, Pharma-
cological studies on hypotensive, diuretic and vasodilator activities of chrysin
glucoside from Calycotome villosa in rats, Phytother. Res. 22 (2008) 356–361.
https://doi.org/10.1002/ptr.2322.
Supplementary materials
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F. Yang, Flavonoids from Halostachys caspica and their antimicrobial and an-
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Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2020.129147.
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