Journal of Molecular Structure 1200 (2020) 127061

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Journal of Molecular Structure

journal homepage: http://www.elsevier.com/locate/molstruc

A new cerebroside from the entomopathogenic fungus Ophiocordyceps

longiissima: Structural-electronic and antioxidant relations.
Experimental and DFT calculated studies
Abdelsamed I. Elshamy a, b, *, Tatsuro Yoneyama a, Nguyen Van Trang e, Ninh The Son a, c, **,
Yasuko Okamoto a, Sayaka Ban d, Masaaki Noji a, Akemi Umeyama a
a
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima, 770-8514, Japan
b
Chemistry of Natural Compounds Chemistry Department, National Research Centre, Dokki, 12622, Giza, Egypt
c
Department of Bioactive Products, Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet,
Caugiay, Hanoi, Viet Nam
d
Medical Mycology Research Center, Chiba University, Japan
e
Institute for Tropical Technology, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Viet Nam

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

Article history: The chemical characterization of n-butanol soluble extract of the fungus Ophiocordyceps longiissima NBRC
Received 11 June 2019 106965 afforded a new cerebroside, 1-O-(b-D-glucopyranosyl)-(2S,3R,4E,8E)-2-[(20 R)-20 -hydrox-
Received in revised form yheptadecanoylamino]-9-methylheptadeca-4,8-diene-1,3-diol, trivially named ophiocordylongiiside A
2 September 2019
(1), in addition to 22E,24R-24-methylcholest-5,22-diene-3-O-b-D-O-glucopyranoside (2). The chemical
Accepted 10 September 2019
Available online 13 September 2019
structure of 1 was established based upon the by modern spectroscopic and chemical techniques.
Compound 1 exhibited strong DPPH-free radical scavenging with the IC50 value of 55.72 mg/mL. The
biological result can be explained by density functional theoretical (DFT) calculation, in which antioxi-
Keywords:
Ophiocordyceps longiissima
dant activity is mostly controlled by the OeH bond dissociation enthalpy (BDE) following hydrogen atom
Cerebroside transfer (HAT) mechanism. In all studied environments, the lowest BDE values are found in 20 -OH.
DFT © 2019 Elsevier B.V. All rights reserved.
Antioxidant

1. Introduction sinensis (Syn. Cordyceps sinensis) is widely distributed used fungus

With natural product substances, fungi are always promising
sources of unusual secondary metabolites [1]. Ophiocordyceps
Abbreviations: HR-TOF-ESIMS, Time-of-flight mass spectrometry; 1D and 2D-
NMR, One and Two-dimensional nuclear magnetic resonance; HMBC, Hetero-
nuclear Multiple Bond Correlation; DFT, Density functional theory; TD-DFT, Time
dependent density functional theory; HOMO, Highest occupied molecular orbital;
LUMO, Lowest unoccupied molecular orbital; BDE, Homolytic bond dissociation
enthalpy; PDE, Heterolytic bond dissociation enthalpy; IP, Ionization potential; PA,
Proton affinity; ETE, Electron transfer enthalpy; HAT, Hydrogen atom transfer; SET-
PT, Single electron transfer-proton transfer; SPLET, Sequential proton loss electron
transfer; DDPH, 1,1-diphenyl-2-picryl hydrazine; sc, synclincal; sp, synperiplanar; ac,
anticlincal; ap, antiperiplanar.
* Corresponding author. Faculty of Pharmaceutical Sciences, Tokushima Bunri
University, Yamashiro-cho, Tokushima, 770-8514, Japan.
** Corresponding author. Faculty of Pharmaceutical Sciences, Tokushima Bunri
University, Yamashiro-cho, Tokushima, 770-8514, Japan.
E-mail addresses: elshamynrc@yahoo.com (A.I. Elshamy), yamantson@gmail.
com (N.T. Son), yamantson@gmail.com (A. Umeyama).
as a Chinese herbal medicine with a long history of uses for cancer
and diabetes treatments, antioxidant, etc [1,2]. The genus Ophio-
cordyceps is one of the most important medicinal fungi belonging to
the family Ophiocordycipitaceae [3,4]. They are specific fungi due to
their parasitic habit on insects [4]. Numerous interesting metabo-
lites have been isolated from Ophiocordyceps species, comprising of
depsipeptides and nortriterpenoids [5]. Cerebrosides are widely
distributive class of compounds reported from fungi [6e9] that
accompanied with the crucial pharmacological properties, such as
antioxidant, antiinflammatory, anticancer, antiapoptotic, hep-
atoprotective and nephroprotective activities [10e12]. Up to now,
there are no reports concerning the bioactive constituents from
Ophiocordyceps longiissima.
Most the human diseases, like neurodegenerative disorders,
inflammation, or bacterial infections are mainly caused by the
cellular damage. These oxidative injuries appear when free radicals
attacked the cellular membrane [13]. Cerebrosides and relative
compounds played a key role in cell signaling because they
controlled assembly and the specific activities of plasma membrane

https://doi.org/10.1016/j.molstruc.2019.127061
0022-2860/© 2019 Elsevier B.V. All rights reserved.
2 A.I. Elshamy et al. / Journal of Molecular Structure 1200 (2020) 127061
proteins [14]. Although there have been various experimental
literature studying on X-ray structure of these cerebrosides [15], no
theoretically specific calculated accounts employing the DFT
calculation discussed antioxidant processes of cerebrosides to date.
Herein, we report the isolation and identification of one new
cerebroside (1) alongside one well known steroid glucoside (2)
from n-butanol soluble portion of entomopathogenic fungus
O. longiissima NBRC 106965, along with its role in antioxidant ac-
tivity. So far structural-bioactive relationship was supported by a
general view of DFT theoretical study.
2. Materials and methods
2.1. General experimental procedures
Optical rotations were recorded on a JASCO P-2300 polarimeter
(Tokyo, Japan). IR spectra were measured on a Shimazu FTIR-8400S
instrument (Columbia MD 21046, USA). 1D and 2D NMR spectra
were recorded on a Bruker 500 NMR spectrometer (MA, USA). The
chemical shifts were given in d (ppm), and coupling constants were
reported in Hz. HR-TOF-ESIMS spectra were obtained on a JEOL
JMS-700 instrument (Tokyo, Japan). Silica gel 60 (Merck, 230e400
mesh, Merck, Darmstadt, Germany) was used for column chroma-
tography. Precoated silica gel plates (Merck, Kieselgel 60 F254,
0.25 mm, Merck, Darmstadt, Germany) was used for TLC analysis.
All used reagents in the antioxidant assessment were of analytical
grade (Sigma). Isolera-one flash chromatography (Biotage; Suite C
Charlotte, NC; USA) was used for flash chromatography and
purification.
2.2. Fungus material
The fungus Ophiocordyceps longiissima NBRC 106965 was iso-
lated from single-ascospore of stroma that occurring on an un-
identified pupa and authorized by Dr. Sayaka Ban, Medical
Mycology Research Center, Chiba University. A voucher specimen
(NBRC 106965) was deposited at the Biological Resource Center,
NITE (NBRC), Japan.
2.3. Extraction and isolation
The fungus O. longiissima was cultured in potato sucrose me-
dium (at 25  C, 21 days). Mycelia were separated from the broth by
filtration and subsequently extracted with MeOH (2 times, 60 min)
in an ultrasonic bath. After concentrating under vacuum, MeOH
crude extract (13.2 g) was then dissolved in distilled H2O and
fractionated with EtOAc and then n-butanol (500 mL  3 times).
The n-butanol soluble portion was concentrated to afford a black
gum (2.3 g). This extract was subjected to address silica gel flash
column chromatography by using a gradient mobile phase
CHCl3eMeOH (3:1, v/v) to yield 3 fractions (OL-1:OL-3). Fraction
OL-2 (0.6 g) was subjected to perform chromatography over the
normal silica gel column using CHCl3eMeOH (1:1, v/v) as a solvent
system, afforded the two major sub-fractions (OL-2A:OL-2B). The
sub-fraction OL-2A (0.25 g) was chromatographed by utilizing
normal silica gel column and n-hexane-EtOAc (1:3, v/v) as a mobile
phase, afforded a major sub-fraction that then subjected to flash
chromatography yielded a new cerebroside (1; 23.6 mg) and known
steroid (2; 21.9 mg).
2.4. Methanolysis of cerebroside 1
Cerebroside 1 (4.0 mg) was refluxed with 1.5 mL of 0.9 N HCl in
82% methanol for 16 h, the reaction mixture was left to cool, dried
under vacuum, the residue was dissolved in a small amount of
distilled water and then fractionated with 10 mL n-hexane three
times. The n-hexane layer was concentrated afforded fatty acid
methyl ester that identified via EIMS analysis [16e18].
2.5. Determination of the configuration of sugar residues
Cerebroside 1 (3.0 mg) was dissolved in 2% H2SO4 solution
(1 mL) and heated under reflux for 12 h. The reaction solution was
extracted with EtOAc (5 mL  3) to remove the aglycone. The
filtered water solution of 1 was dried under reduced pressure to
give a residue (1.0 mg), which was dissolved in pyridine containing
L-cysteine methyl ester hydrochloride (1 mg) and heated at 60 C

for 1 h. Then, phenylisothiocyanate (10 mL) was added and the
mixture was heated at 60  C for another 1 h. The reaction mixture
was directly analyzed by reversed-phase HPLC-MS. The instrument
consisted of a HPLC 1100 system, a PDA detector, an automatic
sample injector and an Agilent 1100 series LC/MSD mass spec-
trometer (Agilent, Santa Clara, CA, USA) with an electrospray
ionization interface. For the MS, the following conditions were
used: Capillary voltage 4.0 kV; capillary exit voltage (fragmentor)
70 V. The drying temperature was set at 300  C, the drying flow at
10 L/min and the nebulizer pressure at 50 psi. The mass spec-
trometer was operated in the positive ion mode and with a scan
range m/z 100e1000 amu. The HPLC separation was performed on
the on a 5C18-MS-II (COSMOSIL, Nacalai testque, INC, Kyoto, Japan)
ø4.6  150 mm inner diameter, 5 mm octadecyl column. The flow
rate through the HPLC column was 0.5 mL/min and the entire
effluent was directed to the mass spectrometer. The binary gradient
consisting of water and acetonitrile (containing 0.1% trifluoroacetic
acid) was used as follows: 0 min, 30% acetonitrile; 0e12 min, 30%
acetonitrile; 12e18 min, 50% acetonitrile; 18e22 min, isocratic 70%
acetonitrile; 22e25 min, 90% acetonitrile; 25e30 min, 100% aceto-
nitrile [19,20].
2.6. DPPH antioxidant assay of cerebroside (1)
Free radical scavenging activity of cerebroside (1) was carried
out by 1,1-diphenyl-2-picryl hydrazyl (DPPH) [21]. In brief, 0.1 mM
solution of DPPH in ethanol was prepared. This solution (180 mL)
was added to 20 mL of different concentrations of 1 in ethanol
(100.0, 50.0, 10.0, 5.0, 1.0 mg/mL). The mixture was carried out by a
96-well plate at room temperature for 20 min. Then, absorbance
was measured at 517 nm by using spectrophotometer (UV-VIS
Shimadzu). The percent DPPH scavenging effect was calculated by
using the following equation: DPPH scavenging effect (%) or percent
inhibition ¼ [(AO - A1)/AO]  100. Where AO was the absorbance of
control reaction and A1 was the absorbance in the presence of test
or standard sample.
2.7. Theoretical methodology
All calculations were performed by Gaussian 09 software
package [22]. In order to obtain the optimization structures, B3LYP
exchange-correlation functional method without constraints was
used coupling with 6-31G(d,p) basis set in the gaseous phase
(dielectric constant, ε ¼ 1) and methanol solvent (ε ¼ 32.61), and
water (ε ¼ 78.35) [22,23]. Vibrational frequencies were calculated
at the same level of theory to correct zero-point energy (ZPE), and
also confirmed that the presence of ground states with the absence
of imaginary frequency. The self-consistent reaction field polariz-
able continuum model (SCRF-PCM) has been employed for esti-
mating solvent effects [22].
As mentioned in the literature, there have been three known
mechanisms HAT (H atom transfer), SET-PT (Single electron
transfer-proton transfer), and SPLET (Sequential proton loss
 A.I. Elshamy et al. / Journal of Molecular Structure 1200 (2020) 127061 3

electron transfer), which concern radical scavenging properties of

parent molecular (ReOH) [22]. Io z  EH (9)

1) HAT mechanical route (Eq. (1)) involves in OeH bond breaking A z  EL (10)
of ReOH, then transfers to radicals, and is often controlled by
homolytic bond dissociation enthalpy (BDE) (Eq. (2)). h z ðIo  AÞ = 2 zðEL  EH Þ = 2 (11)
(1)
c z ðIo þ AÞ = 2 z  ðEL þ EH Þ = 2 (12)

(2) m z  ðIo þ AÞ = 2zðEL þ EH Þ = 2 (13)

whereas EH and EL were energies of HOMO and LUMO, respectively.

H(ReO), H(H), and H(ReOH) are the enthalpies of ReO, The atomic charges for neutral molecular were restricted by
hydrogen radical atom, and the parent molecule, respectively. Mulliken population analysis (MPA) following the same framework
2) SET-PT pathway is recognized by two steps (Eq. (3)). In details, of B3LYP/6-31G(d,p).
the first step accounts for the process of lost an electron to form The global electrophilicity index u indicates the stabilization
molecular radical cation ReOHþ. After that, ReOHþ is depro- energy of a molecule system when being saturated by electrons
tonated. The first action is evaluated by the sum of the ionization from outside. Therefore, the higher value of uþ (electron accepting)
potential (IP), whereas deprotonation is characterized by het- shows the significant electrophilicity while the lower one of u
erolytic bond dissociation enthalpy (PDE) (Eqs. (4) and (5)). (electron donating) evidently exhibits the better nucleophilicity of
(3) a compound. These chemical indices were expressed following the
below functions (Eqs. (14)e(16)) [22].
. .h i .h i
(4) u ¼ m2 2hzðIo þ AÞ2 4ðIo  AÞ zðEL þ EH Þ2 4ðEL  EH Þ
(14)
(5) .
u ¼ ð3Io þ AÞ2 ½16ðIo eAÞ (15)

H (ReOHþ) presents the enthalpies of radical cation ReOHþ .

after electron abstraction of the original molecule. The calcu-
lated gaseous phase enthalpy values of 0.75 kcal/mol and
1.48 kcal/mol are normally used for H(ee) and H(Hþ), respec-
tively [22].
3) The third mechanical SPLET is briefly described when the parent
molecule is deprotonated to afford a typical anion R-Oe, and the
sequential electron transfer from this anion happens (Eq. (6)).
Proton affinity (PA), and the electron transfer enthalpy (ETE) are
two conceptual parameters which correspond to deprotonation,
and electron transfer, respectively (Eqs. (7) and (8)).
uþ ¼ ðIo þ 3AÞ2 ½16ðIo eAÞ (16)
As a general conceptual comprehension, the condensed Fukui
parameters evidently provide information on a selective property
in a chemical reaction. The atom coupled with the high electronic
population displays as the most reactive site when compared to the
surrounding atoms in a molecule [22]. Briefly, Fukui descriptors
have been shown to associate with nucleophilic (fþ k ), electrophilic
(fk e), and or radical attacks (f0k), and were possibly described by the
below equilibriums (Eqs. (17)e(19)) [22].

fk þ ¼ qk ðN þ 1Þeqk ðNÞ (17)

(6)
f
k ¼ qk ðNÞeqk ðNe1Þ (18)


f ok ¼ ½qk ðN þ 1Þeqk ðNe1Þ 2 (19)



PA ¼ H R  O þ H Hþ eHðR  OHÞ (7)
whereas qk(N): Electronic population of atom k in a neutral
molecule.
(8)
qk ðN þ 1Þ: Electronic population of atom k in an anionic

H(ReO ) is the enthalpy of molecular anion after proton molecule.
abstraction of the original molecule. qk ðN  1Þ: Electronic population of atom k in a cationic
molecule.
Antioxidant activities have been explained by DFT-based reac-
tivity descriptors [22], including energies of the highest occupied In general, the electrophilic property had been shown to be
molecular orbital (HOMO), and the lowest unoccupied molecular associated with Dfk ¼ fþ
k e fk > 0. While, Dfk < 0 was responsible for
e

orbital (LUMO), dipole moments, atomic charges, electron affinity nucleophilic character [24].
A, the ionization potential Io, the global hardness h, the electro-
negativity c, the chemical potential m, global electrophilicity index 3. Results and discussion
u, and Fukui chemical parameters.
Based on the theoretical approach of DFT, Janak's theorem, and 3.1. Structural elucidation of 1
the finite difference approximation, these descriptors can be pro-
posed by the following Eqs. (9)e(13) [22]. The chemical characterization of the n-butanol soluble extract of
4 A.I. Elshamy et al. / Journal of Molecular Structure 1200 (2020) 127061

the fungus O. longiissima NBRC106965 afforded a new cerebroside, Table 1

1-O-(b-D-glucopyranosyl)-(2S,3R,4E,8E)-2-[(20 R)-20 -hydrox- Theoretical and experimental 1H,13C NMR chemical shift values with respect to TMS
in MEOD solution of studied compound 1 at B3LYP/6-31G(d,p) level of theory.
yheptadecanoylamino]-9-methylheptadeca-4,8-diene-1,3-diol,
that trivially named ophiocordylongiiside A (1), and known steroid No dH in ppm, multiplicity, J in Hz a dC in ppmb
glycoside, 22E,24R-24-methylcholest-5,22-diene-3-O-b-D-O-glu- Theo. Exp. Theo. Exp.
copyranoside (2) [25] (Fig. 1).
1 4.29 4.12, dd, 10.4, 5.9 63.0 69.8
Cerebroside 1 was obtained as amorphous powder. The molec- 3.93 3.72, dd, 10.4, 3.5
ular ion peaks at m/z 750.5496 by positive TOF-ESI-MS (calcd for 2 3.33 4.02, dd, 7.3, 3.4 54.9 54.6
750.5495 [MþNa]þ) and at m/z 726.5511 by negative TOF-ESI-MS 3 4.78 4.15, t, 7.6 62.7 73.1
4 5.78 5.50, dd, 15.4, 7.6 117.9 131.1
(calcd for 726.5510 [M  H]e) deduced the molecular formula of 1
5 6.19 5.74, m 125.3 134.7
as C41H77NO9. In the FT-IR spectrum, the absorption band at 6 2.11 2.05, m 27.3 33.7
3413.4 cm1 was assigned to hydroxy group, whereas 1726.9, 1640 7 2.12 2.09, m 21.8 28.7
and 1587.1 cm1 attributed to secondary amide (eNHeCO) [13]. 8 5.55 5.16, t, 12.2 112.4 124.8
The 1H and 13C NMR spectra displayed spectral patterns of three 9 125.4 136.8
10 2.02 1.99, t, 14.9 34.4 40.8
methyl groups [17-CH3 and 170 -CH3 (dH 0.92, t, J ¼ 13.8 Hz, dC 14.5),
11 1.54 1.38, m 23.6 29.1
and 18-CH3 (dH 1.61, s, dC 16.1)], one oxygenated methylene group 12e14 1.42e1.47 1.26e1.35, m 24.6e24.6 30.4e30.9
[H-1 (dH 4.12, dd, J ¼ 10.4, 5.9 Hz, and 3.7, dd, J ¼ 10.4, 3.5 Hz), C-1 15 1.44 1.26e1.35, m 25.9 33.0
(dC 69.8)] and twenty three saturated methylenes, one nitrogenated 16 1.52 1.26e1.35, m 17.4 23.8
17 1.10 0.92, t, 13.8 6.6 14.5
methine group [H-2 (dH 4.02, dd, J ¼ 7.3, 3.4 Hz), C-2 (dC 54.6)], two
18 1.72 1.61, s 6.3 16.1
oxygenated methines [H-3 (dH 4.15, t, J ¼ 7.6 Hz), C-3 (dC 73.1); H-2' 10 162.6 177.2
(dH 3.99, dd, J ¼ 7.6, 3.9 Hz), C-2' (dC 72.9)], two double bonds [H-4 20 4.86 3.99, dd, 7.6, 3.9 63.1 72.9
(dH 5.50, dd, J ¼ 15.4, 7.6 Hz), C-4 (dC 131.1); H-5 (dH 5.74, m), C-5 (dC 30 1.55 1.56, m 26.2 34.4
134.7); H-8 (dH 5.16, t, J ¼ 12.2 Hz), C-8 (dC 124.8), and C-9 (dC 1.99 1.73, m
40 1.43 1.41, m 19.2 26.2
136.8)], as well as one carbonyl group (C10, dC 177.2). Also, one
50 -150 1.36e1.51 1.26e1.35, m 19.2e25.9 30.4e30.9
glycosyl unit was characteristic with signals dH 3.18e3.87 ppm and 160 1.52 1.26e1.35, m 17.4 33.0
dC 62.6e78.0 ppm, especially in terms of anomeric atom [H-100 (dH 170 1.11 0.92, t, 13.8 6.6 14.5
4.29, d, J ¼ 7.8 Hz), and C-100 (dC 104.7)] (Table 1). The 1H and 13C 100 4.97 4.29, d, 7.8 95.9 104.7
200 4.15 3.21. dd, 9.1, 7.8 64.4 75.0
NMR spectra of 1 displayed spectral patterns similar to those of
300 4.12 3.37, dd, 9.1, 4.5 58.9 77.9
cordycerebroside A and sarcoehrenoside B [6,26]. The structure of 1 400 4.18 3.18, m 57.0 71.5
was confirmed by 2D-NMR spectral data (HSQC, HMBC, COSY and 500 4.34 3.30, m 67.1 78.0
NOESY) (Fig. 1). COSY correlations of H-1/H-2 and H-2/H-3 600 4.78 3.87, dd, 11.7, 1.0 53.6 62.6
confirmed the 2-amino-1,3-dioxygenated fragment [26]. COSY 4.25 3.68, dd, 11.7, 1.0
correlations H-3/H-4 and H-4/H-5, together with HMBC key cor- a
500 MHz.
relations of 18-CH3/C-8 (J3), 18-CH3/C-9 (J2), and 18-CH3/C-10 (J3)
b
125 MHz.
proved the appearance of D4,5, and D8,9 double bonds in main alkyl
chain. Similarly, serial COSY cross peaks in alkyl side chain from H-
established by NOESY correlations of CH3-18/H-7 [6,26,27]. The
30 to CH3-170, along with HMBC correlations of H-3'/C-10, H-3'/C-20 ,
glycoside moiety was recognized to be glucopyranose by the
H-2/C-10 were a key evidence to determine the presence of alkyl
further fragment ion at m/z 559.1307 in the positive TOF-ESI-MS
amidyl unit as well as its 2-position. The sharp downfield shift of C-
and its b-configuration was further deduced with the remarkable
1 at dC 69.8 by about 7 ppm [16e18] along with HMBC correlation of
coupling constant (J ¼ 7.8 Hz) of the anomeric proton [7,26]. Addi-
H-1''/C-1 confirmed the localization of the sugar moiety at C-1. The
tionally, HPLC-MS of the acid hydrolysis reaction mixture of 1
E geometry of D4,5 was deduced depending upon the vicinal
yielded D-glucopyranoside (tR 9.81) by comparing with D- and/or L-
coupling constant J ¼ 15.4 Hz. Otherwise, E geometry of D8,9 was
authentic glucopyranosides [7,20,26]. Furthermore, the

Fig. 1. The structures of 1 and 2, methanolysis product (1a), and the selective COSY, HMBC and NOESY correlations of new cerebroside (1).
 A.I. Elshamy et al. / Journal of Molecular Structure 1200 (2020) 127061
methanolysis of 1 yielded methyl-2-hydroxyheptadecanoate (1a)
that exhibited EIMS molecular ion peak at (m/z 301 [MþH]þ) cor-
responding to molecular formula C18H36O3. The D-erythro form
(2S,3R) between C-2 and C-3, and R-configuration of C-20 were
established depending upon the 13C NMR chemical shifts of these
three carbons that confirmed by the positive optical rotation
½a17
D þ58.6 (c 1.0, MeOH) of 1 that similar to those of sarcoehre-
nosides A-B [26]. In addition, our DFT calculated evidence, at B3LYP/
6-311G(d,p) level of theory, has resulted in the same optical rota-
tion as (½a17
D þ59.0 (c 1.0, MeOH) [28]. Based on these findings, 1
was established as 9-methylated cerebroside derivative 1-O-(b-D-
glucopyranosyl)-(2S,3R,4E,8E)-2-[(20 R)-20 -hydrox-
yheptadecanoylamino]-9-methylheptadeca-4,8-diene-1,3-diol,
which trivially named ophiocordylongiiside A (1).
Nuclear magnetic resonance (NMR) spectroscopy has been
extensively employed for structural analysis of isolated and syn-
thetic organic compounds. A positive signal was that the combi-
nation of the quantum chemistry calculation DFT method and
experimental NMR data was recognized to be an exceptional tool to
determine the structure and elucidate molecular conformation.
Nowadays, available methods such as IGLO (Individual gauge
localized orbital), have been applied to calculated chemical shift,
but GIAO (Gauge independent or invariant or including atomic
orbital) that gave the high correction with different nuclei [29]. In
the current paper, GIAO procedure will provide the 1H, 13C NMR
computational spectra of optimized structure 1 in MeOD, using
corresponding TMS shielding to predict chemical shifts as
d (ppm) ¼ dcalc(TMS) e dcalc (s is the absolute shielding constant)
[30]. At B3LYP/6-31G (d,p) level. The NMR calculated results were
generally in a good agreement with the observed experimental
ones (Table 1). For example, 13C NMR chemical shift of C-2 reached
to 54.9 ppm in comparison with the experimental value at dC
54.6 ppm. DFT calculated study on NMR data of the optimized
structure 1 also successfully identified non-equivalent proton
phenomenon with three pairs of methylene proton at dH 3.93 and
4.29; dH 1.55 and 1.99; dH 4.25 and 4.78 ppm which belonging to H-
1, H-30 , and H-600 , respectively. Two calculated aliphatic proton
groups H12eH14 and H50 -H15' were also determined by the
overlapped signals that range from 1.42 to 1.47 and 1.36e1.51 ppm,
respectively. However, due to shielding effect and particularly the
influence of electronegative oxygen atoms, the 13C NMR calculated
chemical shifts of C-1, C-3, C-10, C-20 , and C-100 appeared as upfiel-
ded at 63.0, 62.7, 162.6, 63.1 and 95.9 ppm, respectively [31].
3.2. UV spectroscopy
Although UVeVisible experimental spectra of cerebrosides were
available, the calculated procedure on this class of compound has
not yet been performed to date. Herein, the quantum chemical
analytical outcomes with the scope at the deepest comparison
between theoretical and experimental aspects were provided.
Based on using a reliable time dependent-density functional theory
(TD-DFT) method with B3LYP/6-31G (d,p) and B3LYP/6-311G (d,p)
levels to predict the UV spectrum for natural products [32], the
experimental (in methanol) and the predicted (in mediums gas,
methanol and water) electronic excitations (energy, wavelength,
absorbance, oscillator and transition assignment) of cerebroside (1)
were presented in Table 2 and Fig. 2. The results exhibited that the
experimental UVeVisible spectrum of cerebroside (1) shows a peak
at lmax 224 nm with absorbance log (ε) ¼ 3.559. In agreement with
this, the calculated band absorption reached only one peak in each
medium at both two studied levels. Especially, in term of 6-311G
(d,p) basis set, the gas values of lmax 224 nm (Evert ¼ 5.536 eV,
f ¼ 0.0887) and log (ε) ¼ 3.591 accurately coincided with experi-
ment, as compared to lmax 220 nm (Evert ¼ 5.643 eV, f ¼ 0.0992) and
5
log (ε) ¼ 3.660 in methanol were observed. As shown in Table 2, in
all studied mediums, the higher basis set gave the higher wave-
length absorption and absorbance, but the lower transition energy
and oscillator strength. The studied environment greatly affected
the outcomes, the UVeVisible spectrum of 1 in gas was due to
excitation transitions H-1/ L (ca. 30%) and H-1 / Lþ1 (ca. 67%),
while excitation transition H-1 / Lþ1 (more than 97%) might be
claimed responsible for UVeVisible spectrum of 1 in either meth-
anol or water. In addition, it depended upon the influence of sol-
vent, all band absorptions lmax were red-shift and the natural
electronic excitations which might have as arriving state p/p* on
the two double bonds of 1.
3.3. Geometric analysis
Numeric data reported that radicals scavenging powerful ca-
pacity, along with their three antioxidant pathways, are contingent
upon structural properties, especially in terms of the fluctuation of
hydroxyl groups [22]. As a consequence, the detailed analysis of
structural and electronic characteristic features seemed to be the
best way. In our present work, we provided the computational
outcomes on optimized structure 1 at DFT-B3LYP/6-31G (d,p) level.
As shown in Fig. 3 and Table 3, in the all mediums, the most striking
significance was observed from the polar solvents that the opti-
mized glucosylceramide (1) is able to form up to three intermo-
lecular hydrogen bonds between hydroxyl group of sugar and
carbonyl of amide [H(OeC200 )—O(C10 )/1.955e1.958 Å], and also
between hydroxyl groups in sugars [H(OeC400 )—O (C200 )/1.983 Å;
H(OeC300 )—O(C600 )/1.930e1.933 Å], but this phenomenon is absent
in gas. Local minimum energies had no significant distinction
among studied environments, averagely reached 2339.440 au
(Table 4). Taking the selective bond lengths into consideration, in
contrast NeC10 and C100 -O, almost bond lengths tended to slightly
increasing by 0.01e0.07 Å when transferred from gas to solvents. In
the same manner, the tendency of bond angle values q (C100 -O-C1),
q (C2eNeC10 ) and q (NeC10 -C20 ) regards to change medium from
gas to liquids in 1 was opposite with that of q (C1eC2eC3), q
(C3eC4eC5), and q (C4eC5eC6). Generally, our results described
bond lengths and bond angles are in accordance with X-ray data of
similar compound methylated b-D-glalactosyl-N-octadecanoyl-D-
sphingosine (MCER) [15]. However, as the effects of the around
groups, especially 20 -OH in 1 and CH3N in MCER, bond angle q
(C2eNeC1’) in 1 achieved approximately 133.8 and bigger than
those of MCER (118 ) [15]. In three mediums, the glycosyl plane
formulated L-shape with the long alkyl chain axis (from C-20 to C-
170 ). The serial torsional angles a1/a2/a3 also showed well agree-
ment with the status of sc/ac/ap in MCER [15]. The optimized
structure 1 was greatly affected by two double bonds and two hy-
droxyl groups 3-OH and 20 -OH. Herein, our results showed that two
sides of each double bond are twisted with establishing a4/a5/a6 as
sc/ap/ap. Meanwhile, because torsional angles a8/a9/a10/a11
possess sc/sp/ap/ap, it turned out that 20 -OH is more likely to be
the main reason that the planarity of plane C2(H)eNHeCOeC2’ has
lost as well as the bond C10 -C2’ did not belong to zigzag plane of
alkyl chain (C-20 to C-170 ).
3.4. Frontier molecular orbital theory and spin density
With regards to electron delocalization, it involved in the sta-
bilization of neutral and radicals after proton abstractions. The
frontier orbital theoretical calculation seemed to be an effective
model for determining the relation between the neutral and radical
forms, especially the electron delocalization [22]. At the theoretical
level of DFT-B3LYP/6-31G(d,p), the results exhibited no differences
in HOMO and LUMO neutral illustrations when changed from gas to
6 A.I. Elshamy et al. / Journal of Molecular Structure 1200 (2020) 127061

Table 2
Observed experimentally (MeOH) and calculated electronic transitions (gas, MeOH and water) of 1: Wavelength lmax (nm), absorbance log (ε), vertical transition energy E (eV),
oscillator strength f at TD-DFT/B3LYP/6-31G(d,p) level and TD-DFT/B3LYP/6-311G(d,p) level of theory.

Mediums Basis sets lmax (nm) Log (ε) Evert (eV) Oscillator strength f Excitation transition Transition type

Gas 6-31G(d,p) 220 3.641 5.623 0.1064 H-1/ L (30%) p-p*

H-1 / Lþ1 (67%)
6-311G(d,p) 224 3.591 5.536 0.0887 H-1 / L (31%)
H-1 / Lþ1 (64%)
Methanol 6-31G(d,p) 217 3.693 5.723 0.1131 H / Lþ1 (98%)
6-311G(d,p) 220 3.660 5.643 0.0992 H / Lþ1 (97%)
Water 6-31G(d,p) 216 3.696 5.730 0.1152 H / Lþ1 (98%)
6-311G(d,p) 219 3.664 5.650 0.1015 H / Lþ1 (98%)
Exp. 224 3.559

after H-abstraction from cerebroside (1) was presented in Fig. 5. It

should be noted that the more spin density delocalized in radicals,
the easier the radical formation, hence the BDE values are lower
[35]. As a general view, the calculated results reveal that strong spin
distributions of 3-OH radical of 1 remained on atoms around oxy-
gen atom radical. Among atoms contained the significant values of
spin density, C-2, C-4, C-5 and 3-O are centers of positive spin
density, the remaining carbon C-3 bears negative value. It sug-
gested that with regards to more delocalized spin, C2eC3eC4¼C5
fragment is a suitable site for radicals forming. The same result was
observed in the case of 2-OH radical, carbonyl group and 20 -O
charge positive except for carbon C-2' (0.034989).
As of a normal rule, in gas phase, the lower spin density values
will be equivalent to the lower BDE values. In 1, the gaseous spin
density values of O-atom radicals made an order as 3-O
(0.847895) > 20 -O (0.793353). It expected that BDE value of 20 -OH
radical is lower than that of 3-OH radical.

Fig. 2. UVeVis spectroscopy of 1 in studied mediums at TD-DFT/B3LYP at 6-31G(d,p)

and 6-311G(d,p) level of theory. 3.5. Natural bond orbital analysis (NBO)

To receive more insights into the delocalized phenomenon of

liquids (Fig. 4). This phenomenon happened similar to two radical the electron density from occupied Lewis-type (donor) to unoc-
cases. In detail, HOMO neutral image showed that the electrons cupied non-Lewis-type (acceptor), study of the natural bond orbital
delocalized over fragment from C4¼C5 to C20 (OH), and sugar unit. analysis (NBO) for studied 1 at the same theoretical level DFT-
Of particular interest, HOMO profiles of OH-radicals differently B3LYP/6-31G(d,p) was performed. The hypothetical suggestions of
distributed with that of neutral form. HOMO 3-OH radical visual NBOs were derived from both of conjugative and hyper-conjugative
image showed that the electrons highly concentrate on interactions and electron density transfer between filled donor and
C2eC3(OH)eC4¼C5 fragment. While, with regards to HOMO 20 -OH virtual empty acceptor NBOs. The result described the possible
radical, the high density of the electrons was found to focus on interactive E(2) energy using the second order perturbation theory
C20 (OH)eCONHeC2 unit. It is similar to LUMO neutral case, LUMO (Eq. (20)) [31].
3-OH radical was composed of charge density, which is absent in

2 .

sugar, but focuses on a fragment from C-2 to double bond C8¼C9. Eð2Þ ¼ Eij ¼  qi Fi j Ej eEi (20)
Meanwhile, from LUMO 20 -OH radical pictorial representation,
electronic distribution remained unchanged when compared with
where qi is orbital occupancy, Ei and Ej are diagonal elements, and Fi
HOMO 20 -OH radical. It may conclude that C2eC3(OH)eC4¼C5 and
j is off-diagonal NBO Fock matrix element.
C20 (OH)eCONHeC2 fragments facilitate antioxidant reaction, but
As observed, the higher the energy E(2) was, the more interac-
other parts of 1 failed to do so.
tion happened and the more donation from donors to acceptors
From Eqs. (9) and (10), the higher EHOMO (the lower ionization
have been occurred, thereby enhancing the stability of the mole-
potential Io) and the lower ELUMO (the higher electron affinity A),
cule. As can be seen from Table 5, related to resonance in 1, the most
were equal to the better capability of electrons donation and the
important interaction involved in electron donation from lone pair
better sensitivity of receiving electrons, respectively, whereas the
electron of nitrogen to anti-bonding p orbital [LP(1)N / p*(C1¼O),
easier electron transfer indicates the lower Egap ¼ ELUMO  EHOMO,
the interactive energy reached to maximum 49.02 kcal/mol]. By
and thus the better reactivity. From Table 4 and Fig. 4, the use of
this way, it would be helpful to stabilize amide unit. In the same
solvent should be a suitable tool to scale down the EHOMO, ELUMO
manner, the significant hyper-conjugative interactions LP(2)
values and their band gap energy in compound 1. These three pa-
O(C1' ¼ O) / s*(C1eN) (E(2) ¼ 20.72 kcal/mol) and LP(2)O(C1' ¼ O)
rameters were ruled following the visible order: Gas > meth-
/ s*(C1eC2) (E(2) ¼ 20.79 kcal/mol), as well as LP(2)O(C1eOeC100 )
anol > water. As expected, it clearly showed that the polar solvents
/ s*(C100 -O-C500 ) (E(2) ¼ 13.87 kcal/mol), LP(2)O(C100 -O-C500 ) /
will support ionization so that SET-PT and SPLET antioxidant
s*(C1eOeC100 ) (E(2) ¼ 16.26 kcal/mol) could be shown as the main
mechanisms can get more advantageous [33,34].
reason to explain the stability of amide unit, NHeC1eC2, and
Computing the atomic spin density distribution of two radicals
linkage between glycone and aglycone. It was also noticeable to be
 A.I. Elshamy et al. / Journal of Molecular Structure 1200 (2020) 127061
Fig. 3. The optimized forms of the studied compound 1 in mediums at B3LYP/6-31G(d,p) level of theory.
conclude that lone pair electrons of hetero atoms oxygens of amide
and sugar, and nitrogen of amide reveal the critical role to change
the stability of 1 and it's the contrast to the destabilization in the
radical reaction of hydroxyl groups 3-OH and 20 -OH.
3.6. Electronic properties
The global hardness h was used for identifying the resistance to
charge transfer [22]. Cerebroside (1) yielded the maximum chem-
ical hardness h value of 3.045 eV in gas phase, as compared with the
decrease h values in methanol (2.923 eV) and water (2.917 eV). In
polar solvents methanol and water, cerebroside (1) contained
larger polarizability than that in gas (Table 4). By means of theo-
retical HSAB discipline, it was concluded that environmental factors
are reasonable for inducing a transferable trend from “high
oxidation state and low polarizability” to “low oxidation state and
high polarizability” [36]. In a molecule, the dipole moment was a
useful tool to estimate the separation of positive (þ) and negative
() electrical charges. The high magnitudes of the dipole moment
accompanied with the high charge densities and high polarity in
bonds [37]. Moreover, Han and his partners figured out that mol-
ecules established the lower dipole moment which seemed to
7
associate with better biological evaluations [38]. In our present
work, methanol and water had directly affected to increase dipole
moment of 1 when compared with gas using. It expected that gas
environment should be suitable to promote antioxidant reaction.
The electronegativity c referred to as a trend of an atom to
attract electrons towards itself, together with the chemical poten-
tial m will be proportional to this parameter of a negative signal
[22]. Utilizing Sanderson's theory, a systematic compound revealed
the high electronegativity which might be reached quickly to
equalization and established establishes low reactivity [36].
Therefore, it was possible to pronounce the lower c is better for the
antioxidant reactions. Of notes there was no difference in the c
values (3.301 eV) in both two mediums methanol and water.
Nevertheless, cerebroside (1) had the lowest electronegativity c
value of 3.287 eV in gas phase.
Beside descriptors like the electron affinity, the ionization po-
tential, the global hardness, and the global electronegativity; the
increases of the global electrophilicity index u, u, and uþ values
had been investigated when gas converted into high polar solvents.
The u values of molecule 1 were approximately 7 times more than
those of uþ in all phases. This one was identical with the previous
researches on other natural products [22,39], in which cerebrosides
8 A.I. Elshamy et al. / Journal of Molecular Structure 1200 (2020) 127061

Table 3
Optimized bond distances, bond angles (q) and dihedral angles (a) of 1 in gas, methanol and water mediums at B3LYP/6-31G(d,p) level of theory.

Mediums Bond lengths

OeC1 C4¼C5 C8¼C9 NeH C2eN NeC10 10 (C]O) 3(OeH) 20 (OeH) C100 -O

Gas 1.425 1.337 1.343 1.013 1.474 1.363 1.237 0.967 0.966 1.426
Methanol 1.431 1.337 1.344 1.014 1.477 1.360 1.242 0.968 0.967 1.419
Water 1.432 1.337 1.344 1.014 1.477 1.360 1.242 0.968 0.967 1.419

Mediums Hydrogen bonds

H(OeC400 )—O(C200 ) H(OeC300 )—O(C600 ) H(OeC200 )—O(C10 )

Gas
Methanol 1.983 1.933 1.958
Water 1.983 1.930 1.955

Mediums Bond angles (q)

C1eC2eC3 C3eC4eC5 C4eC5eC6 C7eC8eC9 C8eC9eC10 C100 -O-C1 C2eNeC10 NeC10 -C20

Gas 112.3 124.3 125.5 128.8 120.1 115.7 133.5 119.3

Methanol 111.8 124.0 125.4 128.8 120.1 115.9 133.8 119.6
Water 111.7 124.0 125.4 128.8 120.1 115.9 133.8 119.6

Mediums Dihedral angles (a)

a1 a2 a3 a4 a5 a6 a7 a8 a9 a10 a11¼a12
Gas 88.8 100.9 179.6 65.5 175.8 178.7 48.9 72.6 30.0 165.0 177.8
Methanol 88.8 100.9 179.6 65.5 175.8 178.7 48.9 72.9 30.0 163.5 177.8
Water 90.4 98.3 179.5 65.5 175.7 179.1 48.9 72.9 27.6 163.4 177.9

a1 ¼ OeC100 -O-C1, a2 ¼ C100 -O-C1-C2, a3 ¼ OeC1eC2eC3, a4 ¼ C1eC2eC3eC4, a5 ¼ C5eC6eC7eC8, a6 ¼ C9eC10eC11eC12, a7 ¼ OeC1eC2eN, a8 ¼ C1eC2eNeC1’,
a9 ¼ C2eNeC10 -C2’, a10 ¼ NeC10 -C20 -C3’, a11 ¼ C10 -C20 -C30 -C4’, a11 ¼ C20 -C30 -C40 -C50 .

Table 4
Chemical reactivity indices obtained using DFT method in gas, MeOH and water mediums at B3LYP/6-31G(d,p) level of theory.

No Medium h (eV) c (eV) m (eV) Io (eV) A (eV) ɷ (eV) Dipole moment (Debye) Polarizability (au) Energy (au) EHOMO (eV) ELUMO (eV)

ɷ u uþ
1 Gas 3.045 3.287 3.287 6.332 0.241 1.774 3.798 0.511 9.466 494.88 2339.424 6.332 0.241
Methanol 2.923 3.301 3.301 6.224 0.378 1.864 3.880 0.579 12.357 610.57 2339.450 6.224 0.378
Water 2.917 3.301 3.301 6.218 0.384 1.868 3.883 0.582 12.476 615.81 2339.451 6.218 0.384

tended to donate electrons rather than capture.
Mulliken population analysis (MPA) had been resulted in net
charges of a chemical ring system also generate as an effective tool
to estimate a chemical reactivity. The Mulliken atomic charges
values using DFT method were shown in Table S1. In general,
charges have not been changed by poplar solvents using, but a
slight decrease was viewed when transferred from gas to liquids.
All hydrogen atoms in molecule 1 were composed of positive
charges (data not shown). The fact was that antioxidant activities of
molecules further depend on negative centers. Due to the signifi-
cant negative charges, the heteroatoms oxygen and nitrogen in
cerebroside (1) were always capable of donating their electrons. In
solvents methanol and water, the high negative charge was found
atoms oxygen of CO, and hydroxyls of sugar, thereby stabilizing
internal hydrogen bonds between sugar and amide units, and be-
tween several hydroxyls in sugar unit. But to further corroboration
with HOMO-LUMO and NBO analyses above, this research indicated
that the sugar and amide units did not show favorability for the
antioxidant reactive types. Resembling with various computational
results, glycone parts of natural products were not good sites to
facilitate radical scavenging reactions [22,40].
Apart from the frontier molecular orbital considerations, and
electronic structural analysis, Fukui indices were also one of the
most significant parameters and to be a quick solution to justify the
powerful reactive site of each atom. Fukui indices from Table 6 were
calculated in gaseous medium basing on theoretical HSAB princi-
ple. It was of interest to conclude that in cerebroside (1), carbons C-
1, C-5, C-6, C-7, C-10 to C-18, C-10, C-170, C-100, C-300 , C-400 , C-600 and
oxygens 20 -OH, 200 -OH, and 600 -OH, amide unit were nucleophilic
sites while electrophilic sites are remaining atoms. However,
because of the small values fþ k and fk, almost of carbons from two
long alkyl side chains were not favorably attractive nucleophilic
and electrophilic sites. It should be mentioned that the better fok
condensed Fukui values advocated for radical producing at atom
consideration. Even though the hydroxyl groups and amide unit,
saturated carbons, and several carbons around 2-OH and 30 -OH
were good sites for radical attacks, but from the view of HOMO-
LUMO, NBO and MPA analyses, especially spin density distribu-
tion, radical reactions might easily happen at 2-OH and 30 -OH.
3.7. Antioxidant mechanisms (antioxidant activity of 1)
Cerebroside (1) was further subjected to DPPH antioxidant ac-
tivity. The method of assay has been carried out as described in
experimental part [21]. The result was displayed by percent radical
scavenging activity (% RSA) and half inhibitory concentration IC50.
As shown in Fig. 6, RSA of 1 achieved the maximum percentage of
75.4% at the concentration of 100 mg/mL and the strong IC50 value
of 55.41 mg/mL when compared with the rate of standard com-
pound catechin (RSA ¼ 80.1%, IC50 ¼ 40.99 mg/mL). The mechanism
was discussed and established depending upon the DFT calculation
as below.
3.7.1. HAT mechanism
Apparently, HAT pathway occurred when a spontaneous H-
atom is extracted out of hydroxyl group and produced a free
 A.I. Elshamy et al. / Journal of Molecular Structure 1200 (2020) 127061 9

Fig. 4. HOMO and LUMO images together with Egap ¼ EL e EH of neutral and radical structures of 1 in studied mediums at B3LYP/6-31G(d,p) level of theory.

Fig. 5. Spin density distribution of structural radicals of 1 in gas medium at B3LYP/6-31G(d,p) level of theory.

Table 5 radical was only 0.12 kcal/mol. To the best of our knowledge, there
NBO analysis of charge delocalization of studied compound 1 in gas medium at no DFT study on antioxidant activity of cerebroside derivatives
B3LYP/6e31(d,p) level of theory.
were documented up to date. In comparison with the same DFT
Donor (i) Acceptor (j) E(2) (kcal mol1) researches for well-known classes of flavonoids and phenols, the
LP(1)N p*(C1¼O) 49.02 enthalpy BDE values of cerebroside (1) OH-radicals were quietly
LP(2)O(C1' ¼ O) s*(C1eN) 20.72 observed lesser [22,41]. Hence, cerebroside (1) and its analogous
s*(C1eC2) 20.79 could be considered as promising antioxidant agents.
LP(2)O(C1eOeC100 ) s*(C100 -O-C500 ) 13.87
LP(2)O(C100 -O-C500 ) s*(C1eOeC100 ) 16.26

3.7.2. SET-PT and SPLET mechanisms

cerebroside radical since the process of bond dissociation was ho-
molytic and essentially controlled by the physicochemical enthalpy
BDE. As shown in Table 7, in gas phase, 20 -OH radical included the
smallest enthalpy BDE value of 22.46 kcal/mol, while this param-
eter for 3-OH radical was more than 1.09 kcal/mol. This phenom-
enon showed an agreement with the findings of the spin density
result. Taking environment factor into account, the analogous ac-
tion had occurred: 20 -OH radical (22.79e22.81 kcal/mol) < 3-OH
radical (23.67 kcal/mol) in methanol and water. The gaseous
enthalpy BDE value of 20 -OH radical was found less than that of
methanol or water by 0.33e0.35 kcal/mol, while this thing for 3-OH
In polar solvents, it is popular knowledge that, SET-PT and SPLET
mechanisms might act as the key role in scavenging of free radicals
of natural products [22,34]. As discussed above, these two path-
ways have been undergone via two popular steps but favorably
decided by first steps [33]. Consequently, the calculated values of IP
appeared to determine for SET-PT way. From Table 7, it confirmed
that polarity of environment quietly affected to charged species
[34], methanolic enthalpy IP value of 33.18 kcal/mol is quietly less
than that of gas (40.49 kcal/mol) and water (44.62 kcal/mol).
Methanol facilitated to decrease the IP value as compared with gas,
but water failed to do. Therefore, it assumed that if SET-PT mech-
anism happened, methanol solvent should be the best choice for
10 A.I. Elshamy et al. / Journal of Molecular Structure 1200 (2020) 127061

Table 6
Condensed Fukui indices of studied compound 1 in gas medium at B3LYP/6-31G(d,p) level of theory.

No fþ
k fk e
fok Dfk No fþ
k fk e
fok Dfk
C-1 0.000 0.015 0.008 0.015 C-40 0.017 0.002 0.009 0.015
C-2 0.015 0.002 0.009 0.013 C-50 0.006 0.002 0.004 0.004
C-3 0.011 0.001 0.005 0.012 C-60 0.006 0.002 0.004 0.004
3-OH 0.004 0.009 0.007 0.005 C-70 0.003 0.001 0.002 0.002
C-4 0.027 0.006 0.011 0.033 C-80 0.003 0.001 0.002 0.002
C-5 0.018 0.003 0.011 0.015 C-90 0.002 0.001 0.002 0.001
C-6 0.010 0.014 0.012 0.004 C-100 0.001 0.001 0.001 0.000
C-7 0.003 0.018 0.011 0.015 C-110 0.001 0.001 0.001 0.000
C-8 0.006 0.047 0.021 0.053 C-120 0.001 0.001 0.001 0.000
C-9 0.005 0.014 0.009 0.009 C-130 0.001 0.000 0.001 0.001
C-10 0.004 0.018 0.010 0.016 C-140 0.001 0.000 0.001 0.001
C-11 0.004 0.010 0.007 0.006 C-150 0.001 0.000 0.001 0.001
C-12 0.002 0.004 0.003 0.002 C-160 0.001 0.000 0.001 0.001
C-13 0.000 0.005 0.003 0.005 C-170 0.000 0.001 0.001 0.001
C-14 0.001 0.004 0.003 0.003 10 -O-C-1 0.010 0.027 0.019 0.017
C-15 0.001 0.002 0.002 0.001 C-100 0.000 0.001 0.001 0.001
C-16 0.001 0.003 0.002 0.002 C-200 0.025 0.000 0.013 0.025
C-17 0.000 0.002 0.001 0.002 200 -OH 0.051 0.020 0.036 0.031
C-18 0.000 0.009 0.005 0.009 C-300 0.004 0.021 0.008 0.025
NH 0.026 0.000 0.013 0.026 300 -OH 0.009 0.040 0.025 0.031
CO 0.155 0.009 0.082 0.146 C-400 0.007 0.009 0.008 0.002
C-10 0.199 0.003 0.101 0.196 400 -OH 0.022 0.072 0.047 0.050
C-20 0.025 0.006 0.009 0.031 C-500 0.018 0.016 0.017 0.002
20 -OH 0.003 0.013 0.008 0.010 C-600 0.008 0.000 0.004 0.008
C-30 0.033 0.011 0.022 0.022 600 -OH 0.006 0.004 0.005 0.002

Fig. 6. Percent radical scavenging activity (% RSA) of new cerbroside 1 as compared with standard compound catechin.

Table 7
Studied phases reaction enthalpies (at 298 K) for radicals of the studied compound 1 at B3LYP/6-31G(d,p) level of theory (in kcal/mol).

Compounds BDE IP PDE

Gas Methanol Water Gas Methanol Water Gas Methanol Water

3-OH 23.55 23.67 23.67 40.49 33.18 44.62 58.19 41.42 29.55
20 -OH 22.46 22.79 22.81 57.10 40.53 28.66

Compounds PA ETE

Gas Methanol Water Gas Methanol Water

3-OH 84.83 52.50 51.43 13.86 22.55 22.74

20 -OH 83.06 50.90 50.26 14.52 22.81 23.05

antioxidant activity. The second action of SET-PT was understood water successfully decreased PDE values. In contrast to IP results,
deprotonation of radical cation and had normally been defined by the lowest PDE enthalpy of 28.66 kcal/mol was found in water and
the lowest PDE. Transferring from gas to liquids, both methanol and belongs to 20 -OH radical. 20 -OH radical also possesses lower PDE
 A.I. Elshamy et al. / Journal of Molecular Structure 1200 (2020) 127061
values than 3-OH radical in all three studied mediums. But it was
opposite with the trend of BDE enthalpies, in which PDE values of
each radical always make an arrangement as follows:
Water < methanol < gas.
Taking SPLET mechanism into consideration. In the first step of
this process, the PA value is quite sensitive to the changes in the
environment. It was clear to see that the deviation in enthalpy PA
values reaches up to 33.0 kcal/mol between gaseous medium and
the use of solvents in both two radical cases. The lowest PA value of
50.26 kcal/mol was occurred at 20 -OH radical in water phase, but
this one slightly differed from the result using methanol
(50.90 kcal/mol). Once again, 20 -OH radical associated with the
lower enthalpy PA parameters than those of 3-OH radical in all
mediums. It might have a conclusion that polar solvent such as
water and methanol seemed to be the best choice to SPLET action of
1 and 20 -OH radical is a good site for this mechanism. In the second
step, just like the enthalpy PA outcome, the electron transfer
enthalpy ETE values showed no more change in environmental
liquids but a dramatic observed decreased in gas. In contrast to BDE,
the lowest ETE values of 13.86 and 14.52 kcal/mol could able to
assign to 3-OH radical and 20 -OH radical. Furthermore, in contrast
to enthalpy BDE, PDE and PA values, ETE results of 3-OH radical
have been found lower than those of 20 -OH radical in all three
studied mediums.
3.7.3. Preferential mechanisms
The possible favorable mechanisms of antiradical of 1 might be
discussed through thermodynamically preferential enthalpies BDE
of HAT, IP of SET-PT, and PA of SPLET [22]. The minimum values in
enthalpies BDE, IP, PA were predicted, as well as that mechanism, to
happen easily. From Table 7, analyzing of the enthalpy reaction of 1,
values of BDE were always lower than those of IP and PA, and
establish the order BDE < IP < PA in both three mediums. Conse-
quently, thermodynamically preferable mechanisms HAT > SET-
PT > SPLET might simultaneously run for 1, and the performance to
produce 20 -OH radical is easier than 3-OH radical.
4. Conclusion
Collectively, phytochemical investigation of n-butanol extract of
O. longiissima led to isolation and determination of one new cere-
broside and one known steroid glycoside. Naturally occurring
cerebroside 1 have also shown to be associated with the strong
DPPH-free radical scavenging. DFT calculated method indicates that
the antioxidant activity of new compound 1 may due to OeH ho-
molytic bond dissociation in aglycone, and HAT mechanism is
suitable circumstance. This current result also proofs the effects of
structural conformation, especially the role of functional groups on
antioxidant activity of 1, in which 20 -OH seems to be a good site for
radical attack rather than 3-OH in all studied mediums gas, meth-
anol and water. This account provides necessary guidelines for
future research.
Acknowledgments
Dr. Elshamy wishes to gratefully acknowledge Takeda Science
Foundation; Tokushima Bunri University, Japan; National Research
Centre, Egypt and Vietnam Academy of Science and Technology,
Vietnam.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2019.127061.
11
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