L-Hypaphorine and D-hypaphorine: Specific antiacetylcholinesterase activity in rat brain tissue

Article abstract of DOI:10.1016/j.bmcl.2021.128206

Acetylcholinesterase (AChEis) inhibitors are used to treat neurodegenerative diseases like Alzheimer's disease (AD). L-Hypaphorine (L-HYP) is a natural indole alkaloid that has been shown to have effects on the central nervous system (CNS). The goal of this research was to synthesize L-HYP and D-HYP and test their anticholinesterasic properties in rat brain regions. L-HYP suppressed acetylcholinesterase (AChE) activity only in the cerebellum, whereas D-HYP inhibited AChE activity in all CNS regions studied. No cytotoxic effect on normal human cells (HaCaT) was observed in the case of L-HYP and D-HYP although an increase in cell proliferation. Molecular modeling studies revealed that D-HYP and L-HYP have significant differences in their binding mode positions and interact stereospecifically with AChE's amino acid residues.

Full text of DOI:10.1016/j.bmcl.2021.128206

Bioorg. Med. Chem. Lett. 47 (2021) 128206
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
L-Hypaphorine and D-hypaphorine: Specific antiacetylcholinesterase
activity in rat brain tissue
Murilo K.A. Yonekawaa, Bruna de B. Penteadoa, Amanda Dal’Ongaro Rodriguesa, Estela M.
b
c
d
b
d
´
G. Lourenço , Euzebio G. Barbosa , Silvia C. das Neves , Rodrigo J. de Oliveira ,
a
e
b
a
ˆ
Maria R. Marques , Denise B. Silva , Denis P. de Lima , Adilson Beatriz , Jean P. Oses ,
Jeandre A. dos S. Jaquesa, Edson dos A. dos Santosa
,
*
a
´
ˆ
Laboratorio de Bioquímica Geral e de Microrganismos, Instituto de Biociencias, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil
b
c
´
Laboratorio de Pesquisa 4, Instituto de Química, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil
´
ˆ
Departamento de Farmacia, Centro de Ciencias da Saúde, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil
d
e
´
´
´
Centro de Estudos e Celulas Tronco, Terapia Celular e Genetica Toxicologica, Universidade Federal de Mato Grosso do Sul, NHU, Campo Grande, MS, Brazil
´
ˆ
ˆ
˜
Laboratorio de Produtos Naturais e Espectrometria de Massas, Faculdade de Ciencias Farmaceuticas, Alimentos e Nutriçao, Universidade Federal do Mato Grosso do
Sul, Campo Grande, MS, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Acetylcholinesterase (AChEis) inhibitors are used to treat neurodegenerative diseases like Alzheimer’s disease
(AD). L-Hypaphorine (L-HYP) is a natural indole alkaloid that has been shown to have effects on the central
nervous system (CNS). The goal of this research was to synthesize L-HYP and D-HYP and test their anti-
cholinesterasic properties in rat brain regions. L-HYP suppressed acetylcholinesterase (AChE) activity only in the
cerebellum, whereas D-HYP inhibited AChE activity in all CNS regions studied. No cytotoxic effect on normal
human cells (HaCaT) was observed in the case of L-HYP and D-HYP although an increase in cell proliferation.
Molecular modeling studies revealed that D-HYP and L-HYP have significant differences in their binding mode
positions and interact stereospecifically with AChE’s amino acid residues.
Acetylcholinesterase
Indole
Neurodegenerative diseases
Betaine
Alzheimer’s disease (AD) causes a lack of neurotransmitters, which
are responsible for transmitting nerve stimulation from one neuron to
another. Acetylcholine (ACh) (Fig. 1) is the primary neurotransmitter
deficient in this disease.1 ACh is important in the modulation of many
functions throughout the central nervous system (CNS), including
excitatory effects, attention, and cognition. The choline acetyltransfer-
ase (ChAT) is produced with choline (Ch) and acetyl coenzyme A as
substrates. Acetylcholinesterase (AChE) terminates ACh neurotrans-
mission by rapidly hydrolyzing ACh, yielding Ch and acetic acid.2
Although there is no cure for AD, the use of Acetylcholinesterase in-
hibitors (AChEis) represents a significant advancement in the treatment
of this pathology.3 AChE remains one of the main well validated mo-
lecular targets for the treatment of neuromuscular disease and myas-
thenia gravis.4 The AChEis prevent the degradation of ACh by
neurotransmitters and consequently increases neurotransmission in
cholinergic structures and other cholinergic systems.1
example, are found in many medicinal plants and used as alternative
medicines in many countries.1,5 Several AChEis, such as galantamine,
rivastigmine, and donepezil, have been approved by the Food and Drug
Administration (FDA). Memantine, an N-methyl-D-aspartate (NMDA)
receptor antagonist, is another example. It is used in clinical procedures
to improve memory in some people with AD.6 The presence of L-hypa-
phorine (L-HYP) was found during phytochemical studies of Erythrina
mulungu (Erythrina verna) (Fig. 1). Different biological studies show that
Erythrina alkaloids act on the peripheral cholinergic system.7 L-HYP is
also present in large quantities in other species of Erythrina genus,5,8,9
and its extracts have shown sedative and anticonvulsant effects.7,10 L-
HYP is present in human nutrition through the ingestion of lentils,
peanuts, peanut butter, and chickpeas, and can be found in breast milk
at a concentration up to 1.24
μ
M.11 The alkaloid has many reported
biological activities, such as anti-inflammatory,12 antihyperglycemic
and,13 holds potential for treating obesity and insulin resistance.14
Moreover, L-HYP is linked to the bioenergy cascade of neurotransmitters
Several AChEis are based on natural products. The alkaloids, for
* Corresponding author.
E-mail address: edson.anjos@ufms.br (E.A. Santos).
https://doi.org/10.1016/j.bmcl.2021.128206
Received 29 March 2021; Received in revised form 7 June 2021; Accepted 13 June 2021
Available online 16 June 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

M.K.A. Yonekawa et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128206
column).
Even at basic (NaHCO3) and acid (2 M HCl) pHs, the HYPs were
obtained enantiomerically pure (Fig. 2), and no racemization reaction
occurred (Fig. 2, Figs. S15-17 Supplementary Data). The HYP products
were subjected to liquid enantioselective chromatography analyses, as
well as mixtures, to confirm the chromatography efficiency in separating
the enantiomers (Fig. 2). Because each product had only one chro-
matographic peak, it was easy to prove that they are enantiomerically
pure and that racemization did not occur throughout the reactions.
L-HYP and D-HYP were evaluated in the form of hydrochloride salts
(L-HYP⋅HCl and D-HYP⋅HCl), which are more soluble in water, to
determine their AChE inhibitory activity. In each structure, the inhibi-
tion of AChE activity was investigated separately (i.e., brain, cerebral
cortex, cerebellum, striatum and hippocampus). The AChE occurs
throughout the CNS and is found in these structures in different
Fig. 1. Structure of L-hypaphorine (L-HPY), acetylcholine (ACh) and neostig-
mine (NEO).
in the brain, according to the results of sleep induction in rats.8
Bel-Kassaoui and co-workers (2008) investigated the neurotoxic ac-
tion of L-HYP in goats.15 This study was based on the possibility that L-
HYP could be responsible for the neurotoxic action of Astragalus lusita-
nicus. The results showed no intoxication effect in the animals.15 How-
ever, there are no detailed studies of the effect of L-HYP on enzymes of
the cholinergic system, such as AChE. L-HYP and ACh have interesting
bioisosteric relationships. L-HYP is also similar to that of neostigmine
(NEO), a potent AChE inhibitor with a quaternary ammonium group
(Fig. 1).16
Fig. 2. Chromatograms at the wavelength 290 nm from D-HYP⋅HCl and L-
HYP⋅HCl on the Chirex® (S)-LEU/(S)-NEA column. (A) Mixture of enantiomers
D-HYP⋅HCl and L-HYP⋅HCl (2:1 w/w); (B) D-HYP⋅HCl; (C) L-HYP⋅HCl.
As a result, because these structures have different profiles of AChE
activity, this study proposed looking into the effect of L-HYP on AChE
activity in the cerebral cortex, cerebellum, striatum, and hippocam-
pus.17 In addition, this work also proposed the synthesis and evaluation
of the effect of D-HYP on AChE activities in view of the importance of the
analysis of enantiomers for evaluating biological effects.18,19
Table 1
IC50 of compounds on inhibition of AChE activity in different brain structures.
a
Compounds
IC50
(
μM ± S.E.M)
Cortex
Cerebellum
Striatum
Hippocampus
L-HPY and D-HPY were prepared from L-tryptophan (L-Trp) and D-
tryptophan (D-Trp), respectively, following the procedure depicted on
Scheme 1. The synthesis consisted of the N-methylation reaction of L-Trp
or D-Trp with CH3I in the presence of base at room temperature (rt) in
order to form a quaternary ammonium salt. The hypaphorines hydro-
chlorides (HYP⋅HCl) were obtained by adding a 2 M HCl solution under
constant agitation and cooled to 0 ◦C. All compounds were purified by
recrystallization with ethanol in high yields. The structures were
L-HYP⋅HCl
D-HYP⋅HCl
NEOBrb
>1000
18.63 ± 0.14A
19.12 ± 0.03A
37.18 ± 0.07B
>1000
>1000
0.24 ± 0.28A
0.65 ± 0.14B
34.60 ± 0.34A
3.17 ± 0.04B
0.11 ± 0.32A
0.06 ± 0.07B
a
Concentration of the compound (L-HYP⋅HCl, or D-HYP⋅HCl, or NEOBr) to
inhibit 50% of AChE activity.
b
Neostigmine bromide. The results are presented as IC50 ± standard error of
the mean (S.E.M). Different capital letters in the column indicate a significant
difference between the compounds (t-student, p < 0.001 or in the one-way
ANOVA test of repetitive measurements with post-test of Tukey, p < 0.05).
The concentration of substrate was saturating (1 mM) and the content of protein
(mean ± SD) in cerebral cortex, cerebellum, striatum and hippocampus was
23.6 ± 1.72 µg, 14.8 ± 1.72 µg, 18.4 ± 2.8 µg, and 10.4 ± 1.92 µg, respectively.
1
confirmed by H and 13C NMR, and ESI-HRMS analyses (Appendix A.
Supplementary data). The enantiomeric purities were documented
based on optical rotation data (Appendix A. Supplementary data) and
liquid enantioselective chromatography (Chirex® (S)-LEU/(S)-NEA
Scheme 1. Synthesis of L-HYP (isomer S), D-HYP (isomer R) and their hydrochlorides (HYP⋅HCl). Reaction yields are in parentheses.
2

M.K.A. Yonekawa et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128206
Fig. 3. Cell viability of normal human cells (HaCaT) treated with hypaphorine (HYP). (A) Effect of different concentrations L-HYP⋅HCl on HaCaT cells. (B) Com-
parison of the effects of L-HYP⋅HCl on cell viability in 24 h and 48 h. (C) Effect of different concentrations of D-HYP⋅HCl on HaCaT cells. (D) Comparison of the effects
of D-HYP⋅HCl on cell viability in 24 h and 48 h. DTIC: dacarbazine. Different letters denote a significant difference between the treatments (ANOVA/Tukey, p < 0.05).
amounts.20 The analysis of AChEis in different brain structures is crucial
as it may demonstrate selective effects of inhibition.17,21 Therefore, it is
important for the understanding of the role of drugs in the treatment of
neurodegenerative diseases that affect specific brain structures such as
AD,1 cerebellar degenerative disorders,22 Huntington’s disease,23 and
Parkinson’s disease.24 The concentration that inhibits 50% of AChE
activity hydrolysis was determined by selecting brain areas in which the
investigated compounds had an inhibitory effect on AChE activity. The
neostigmine bromide (NEOBr) IC50 has also been determined in the four
brain structures to compare the inhibitory potential of D-HYP⋅HCl and L-
HYP⋅HCl (Table 1).
D-HYP⋅HCl inhibited AChE activity in all brain regions tested
(Table 1). In the cortex and cerebellum, this enantiomer was more active
than NEOBr, with IC50 values of 0.24 ± 0.28 and 19.12 ± 0.03 µM,
respectively. The IC50 values of the enantiomers in the cerebellum did
not differ significantly, indicating that they have comparable activity.
However, unlike L-HYP⋅HCl, the inhibitory activity of D-HYP⋅HCl was
not restricted to a specific brain structure. On hippocampus AChE, D-
HYP⋅HCl showed the lowest IC50 (0.11 ± 0.32 M) when compared to
NEOBr. The D-enantiomer has a higher activity in the cortex and hip-
pocampus. The cholinergic system in these brain regions is the most
affected during the early stages of Alzheimer’s disease, causing memory
loss, speech problems, and social behavior issues as the disease pro-
gresses.29 Although D-HYP⋅HCl had lower inhibitory activity in the
striatum when compared to other brain structures, its effect, even at
higher concentrations, may contribute to cholinergic signaling, as the
striatum contains high levels of ACh and plays an important role in
motor control and learning.30
L-HYP⋅HCl promoted AChE inhibition had a selective effect with an
IC50 of 18.63 ± 0.14 μM only in cerebellum. This effect outperformed
the inhibitor NEOBr, which had an IC50 of 37.18 ± 0.07 µM. The cer-
ebellum has the lowest cholinergic activity of all the brain areas; yet, the
cholinergic system in this structure is vital for vestibulo-ocular reflexes,
as well as motor coordination and cardiovascular regulation.25
Cerebellar cholinergic disorders are among the neuropsychiatric
disorders that can cause cholinergic abnormalities, such as decreased
ACh levels, leading to pathological alterations.26 Autism spectrum dis-
order (ASD) is one such example.25,27 Although there is still only a small
understanding of the pathophysiology of ASD and cholinergic system,
lower levels of L-HYP in ASD and associated allergic diseases have been
observed.28 The relationship between ACh and L-HYP levels in the
bodies of ASD patients has not yet been reported. The result achieved in
our investigations however showed that there is a particular AChE in-
hibition activity in the brain that indicates the interaction of L-HYP to
ACh.
L-HYP, which is found in extracts of Vaccaria segetalis and E. mulungu,
has recently been shown to have anti-inflammatory properties.31,32 It is
worth noting that in AD, an inflammatory process plays a significant role
in the disease’s onset and progression.33 Moreover, evidence suggests
that centrally acting AChEis used in the treatment of AD can modulate
peripheral immune responses in order to activate the cholinergic anti-
inflammatory pathway.34 HYPs could act as an AChEi, increasing ACh
levels and promoting ACh rebalance. As a result, ACh could build up in
the synaptic cleft. Several studies on the effects of AChEis found that
they slow gray matter atrophy in the hippocampus, cortex, and basal
forebrain.35
3

M.K.A. Yonekawa et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128206
Fig. 4. Cell viability of normal human cells (HaCaT) treated with NEOBr. (A) Effect of different concentrations of NEOBr on HaCaT cells. (B) Comparison of the
effects of NEOBr on cell viability in 24 h and 48 h. DTIC: dacarbazine. Different letters denote a significant difference between the treatments (ANOVA/Tukey, p
< 0.05).
Previously, studies with racemic mixtures and pure enantiomers
were conducted to evaluate the inhibition of AChE activity, and the
results revealed that enantiomers may have different effects.36 Studies
show that AChE activities can vary in different brain structures due to
various AChE isoforms. A plausible biological mechanism by which the
same AChEi could act selectively on different brain structures remains
unanswered.17 The biological mechanism by which the same AChEi can
act selectively on various brain areas is currently unknown.17 The
striatum is the most AChE brain structure due to the dense intrinsic
cholinergic neurons20 that can be linked to several factors such as: age37
stresses,38 malnourishment,39 hypothyroidism,40 and ethanol intake.41
Consequently, ideal AChE inhibitors should be highly specific for the
various brain structures, have minimal effects on the peripheral
cholinergic system, and cause no toxicity in other organs. The use of
specific AChEis could lead to the development of more effective cogni-
tive stimulants.42,43 The survey of biological activity of pure enantio-
mers is important since studies show that the use of pure drugs has the
advantage that the total dose administered is reduced, the dos-
e–response ratio is simplified and the toxicity due to the inactive isomer
is lessened.44 This is also critical in drug development, in order to
enhance clinical benefit while minimizing pharmacological adverse
effects.19
keratinocytes.45,47 This explains in part the multiplication of cells
observed in our experiments.
The results of the cell viability experiment for NEOBr (Fig. 4) showed
that the compound exhibited cytotoxicity at concentrations of 10,000
µM (24 h), 5000 and 10,000 µM (48 h) (Fig. 4A). In the comparison
between the two time periods, an increase (p < 0.05) in cytotoxicity was
observed for the doses of 5000 and 10,000 µM (Fig. 4B).
When we compared the cytotoxic effects of L-HYP⋅HCl, D-HYP⋅HCl,
and NEOBr, we discovered that the enantiomers were not cytotoxic and
performed better than NEOBr. It is worth noting that NEO is one of the
most commonly used AChE inhibitor drugs in perioperative medicine,
especially after the administration of neuromuscular blockers.48 More
detailed studies of the increase in cell proliferation in response to AChE
inhibition in keratinocytes are needed to confirm the suggested hy-
pothesis comprising inhibitors such as NEOBr. The cytotoxicity, as well
as genotoxic and apoptotic effects of NEOBr exhibited on HaCaT were
also observed in human embryonic renal cells (HEK-293).49 These toxic
effects of NEO may account for the increased cell proliferation not
observed on HaCaT upon treatment with NEOBr.
The differences in potency and selectivity demonstrated by L-HYP
and D-HYP among the tested brain structures is an attractive result.
However, these results are not rare in literature. Although chiral
bioactive isomers, they have markedly different pharmacologic, toxi-
cologic, pharmacokinetic and, metabolic behavior. One enantiomer may
produce the aimed therapeutic effect and, the other may be inactive,
exhibit lower potency and, even toxic effects.50 The different biological
properties of each enantiomer may be due to distinct binding modes at
the active site of the target. Therefore, molecular modeling is an excel-
lent tool for better understanding our biological outcomes.
Cell viability assays in the presence of L-HYP⋅HCl, D-HYP⋅HCl (Fig. 3)
and, NEOBr (Fig. 4) were performed in normal human cells (HaCaT).45
Dacarbazine (DTIC) was used as a positive control and was cytotoxic to
29.74 and 44.46% of the cells treated for 24 and 48 h, respectively. L-
HYP⋅HCl and D-HYP⋅HCl showed no cytotoxic effect on HaCaT in the two
periods analyzed (24 and 48 h) and did not alter the cell viability rate
after 24 h of experiment (p < 0.05). The enantiomers promoted an in-
crease in cell number after 48 h of the experiment (Fig. 3A and C). The
comparison between the two periods demonstrated an increase (p <
0.05) in cell proliferation for the 19.5 and 5000 µM concentrations of L-
HYP⋅HCl after 48 h (Fig. 3B) and, 19.5–156.2 and 625 µM concentra-
tions of D-HYP⋅HCl after 48 h (Fig. 3D). The increased cell proliferation
in the experiments observed with L-HYP⋅HCl and D-HYP⋅HCl may be
associated with the concentration of ACh on HaCaT cells. HaCaT are
keratinocytes and have a functional nonneuronal cholinergic system,
including AChE, ChAT and, cholinergic receptors.46 The result led us to
assume L-HYP⋅HCl and D-HYP⋅HCl inhibit HaCaT’s AChE activity and
increase ACh concentration, which is related to increased cell viability,
proliferation and, migration by the presence of this neurotransmitter in
The position of the ligand at the active site of AChE is crucial for its
inhibitory capacity. The AChE has two main binding sites: one is a hy-
drophobic pocket with Ser, His and, Glu as the more significant amino
acid residues and, the other, composed of Tyr, Trp and, Asp, is known as
a peripheral anionic site of AChE.51,52 The literature vastly reports these
two pockets and, an array of binding modes are observed as a conse-
quence of the structural diversity of the studied inhibitors. The differ-
ences between the structural features make difficult the description of a
structure–activity relationship for the compounds. However, some
amino acids are considered a key to the AChE inhibition as His440,
Glu327, Ser200, Trp84, Trp279 and, Tyr121. Most of the inhibitors can
interact with more than one site.4 Due to the quantity of AChEis, the
4

M.K.A. Yonekawa et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128206
Fig. 7. Overlay of NEO (blue), L-HYP (pink) and D-HYP (orange).
showed many hydrophobic interactions with the amino acid Phe331,
Phe330, Trp84, His440 and, Gly117 and hydrogen bonds with Ser200,
Gly119 and, Gly118 (Fig. 5). Both are considered strong intermolecular
interactions and, the sum of the results in a very stable complex between
D-HYP and AChE. Such stability at the active site may explain the high
activity demonstrated by D-HYP in the AChE inhibitory assay.
Fig. 5. Intermolecular interactions between D-HYP and AChE (PDB ID: 1QTI).
The 2D diagram was provided by the program LigPlot+. Hydrogen bonds are
shown as green dotted lines.
In contrast, the output of molecular simulations of L-HYP (Fig. 6)
showed that the enantiomer binds to the active site through hydro-
phobic interactions and does not make hydrogen bonds as D-HYP
(Fig. 5). The main difference between the binding mode position of D-
HYP and L-HYP is the location of the carboxylic acid group. The absence
of hydrogen bonds may be responsible for the lower inhibition capacity
of L-HYP. The selective action of this enantiomer on the cerebellum is
unclear yet. The study of the intermolecular interaction alone between
the ligand and AChE seems insufficient since even ACh can have action
in different brain regions depending on the physiological situation.41
It is also important to emphasize that NEO, used in the biological
tests, has a different binding mode position when compared with D-HYP
and L-HYP (Fig. 7). However, the standard compound occupies the same
pocket at the active site of AChE. A direct comparison between the
biological activities of L-HYP, D-HYP and, NEO is feasible. It is possible to
verify a better overlap of the quaternary ammonium group of NEO and
D-HYP, corroborating the results of AChE inhibition observed for these
compounds concerning D-HYP.
L-HYP and D-HYP act differently to AChE inhibition in brain struc-
tures. L-HYP inhibited AChE only in the cerebellum, while D-HYP was
also active in the cortex, striatum and, hippocampus. In silico results
show that the location of the carboxylic acid group affects the binding
mode of D-HYP and L-HYP. The compounds were not cytotoxic to HaCaT
cells and, their role in increasing cell viability may be associated with
the availability of ACh. The function of HYPs in the nonneuronal
cholinergic system depends on additional research. L-HYP has many
biological properties and, its action seems to have an importance for
cognitive functionality, specialty in the cerebellum. In addition, D-HYP is
a potent AChEi and can be considered a prototype for the development
of drugs for neurodegenerative diseases.
Fig. 6. Intermolecular interactions between L-HYP and AChE (PDB ID: 1QTI).
The 2D diagram was provided by the program LigPlot+.
Declaration of Competing Interest
crystal of galantamine in complex with AChE (PDB ID: 1QTI)53 was used
in our molecular modeling studies since this compound is an alkaloid.
The analyses considered the high structural similarity between the
quaternary ammonium group D-HYP and L-HYP and ACh as well.
After the molecular docking simulations and geometry optimization,
L-HYP and D-HYP presented remarkable differences in their binding
mode positions interacting in a stereospecific way with the amino acid
residues of AChE. The analysis of D-HYP at the active site of AChE
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The authors owe a debt of gratitude to the following Brazilian
5

M.K.A. Yonekawa et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128206
24 Rizzi G, Tan KR. Front Neural Circuits. 2017;11:110. https://doi.org/10.3389/
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Supplementary data (synthetic procedures and analytical/spectral
data for compounds synthesized, procedures for in silico studies, AChE
inhibitory and cytotoxicity assays) to this article can be found online at
https://doi.org/10.1016/j.bmcl.2021.128206.
28 Yadama AP, Kelly RS, Lee-Sarwar K, et al. Brain Behav Immun 2020;83:293-297.
https://doi.org/10.1016/j.bbi.2019.10.008.
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