Journal of Biomolecular Structure and Dynamics

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Understanding hypocholesterolemic activity of

soy isoflavones: Completing the puzzle through
computational simulations

Feri Eko Hermanto, Warsito Warsito, Muhaimin Rifa’i & Nashi Widodo

To cite this article: Feri Eko Hermanto, Warsito Warsito, Muhaimin Rifa’i & Nashi Widodo
(2022): Understanding hypocholesterolemic activity of soy isoflavones: Completing the puzzle
through computational simulations, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2022.2148752

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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2022.2148752

Understanding hypocholesterolemic activity of soy isoflavones: Completing the

puzzle through computational simulations
Feri Eko Hermantoa , Warsito Warsitob,c, Muhaimin Rifa’ia,d and Nashi Widodoa,d
a
Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Malang, Indonesia; bDepartment of Chemistry,
Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Malang, Indonesia; cEssential Oil Institute, Universitas Brawijaya,
Malang, Indonesia; dBiosystem Study Center, Universitas Brawijaya, Malang, Indonesia
Communicated by Ramaswamy H. Sarma

ABSTRACT ARTICLE HISTORY

The hypocholesterolemic activity of soy isoflavones has been studied, but the exact mechanism under- Received 14 March 2022
lying the activity remains unclear. This study reveals the proposed mechanism of the cholesterol-low- Accepted 12 November 2022
ering effect of soy isoflavones by computational simulations. Daidzin, Glycitin, Genistin, Daidzein,
KEYWORDS
Glycitein, Genistein, Glyceollin I, Glyceollin II, and Glyceollin III were selected to be analyzed their inter-
Genistein; Genistin; HMGCR
action with 3-Hydroxy-3-Methyl-Glutaryl-Coenzyme A Reductase (HMGCR) and Sterol Regulatory inhibitor; hypercholesterol-
Element-Binding Protein 2 (SREBP2) as key factors in cholesterol biosynthesis as well as Proprotein emia; isoflavones
Convertase Subtilisin/Kexin type 9 (PCSK9) as a common target for hypercholesterolemia. Protein-iso-
flavones interaction was analyzed using AutoDock. According to binding energy calculations, a total of
five out of those nine isoflavones, including Glycitin, Genistin, Genistein, Glyceollin II, and Glyceollin III,
were favored to be a HMGCR inhibitor but not with SREBP2 and PCSK9. Those isoflavones were then
compared with Simvastatin as known inhibitor of HMGCR. Isoflavone with binding energy lower than
Simvastatin then directed to molecular dynamics using YASARA and headed into toxicity estimations.
Almost all of those isoflavones could bind with HMGCR with better stability than Simvastatin accord-
ing to molecular dynamics simulations. Toxicity prediction filtered two out of the five isoflavones men-
tioned earlier as the proper candidate to be an HMGCR inhibitor. Those isoflavones were Genistin and
Genistein. In summary, the hypocholesterolemic activity of soy isoflavones may occur by blocking the
cholesterol biosynthesis pathway.

CONTACT Nashi Widodo widodo@ub.ac.id Biosystem Study Center, Universitas Brawijaya, Jl. Veteran, Malang, 65145, Indonesia
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2022.2148752.
ß 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.
0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in
any way.
2 F. E. HERMANTO ET AL.

1. Introduction Table 1. The binding energy of each isoflavone with HMGCR, PCSK9,
and SREBP2.
Hypercholesterolemia accounts for major public health prob- Binding Energy (kcal/mol)
lems in modern society (Taddei et al., 2020). People with Compound PubChem CID HMGCR PCSK9 SREBP2
hypercholesterolemia have a higher risk of developing car- Daidzin 107971
7.15
4.35
5.35
diovascular diseases (Soran et al., 2018; Zarate et al., 2016). Glycitin 187808
8.23
4.85
5.65
Besides conventional medicinal therapy, food intervention Genistin 5281377
8.93
4.68
6.08
Daidzein 5281708
7.06
3.94
5.24
also mitigates or accompanies medical treatment (Mannu Glycitein 5317750
6.64
5.60
4.91
et al., 2013). In particular, soybean consumption was discov- Genistein 5280961
7.69
3.87
4.56
ered to have hypocholesterolemic activity (Ramdath et al., Glyceollin I 162807
7.35
5.35
4.81
Glyceollin II 181883
7.85
5.74
4.86
2017; Sirtori et al., 1995; Wong et al., 1998), suggesting a Glyceollin III 11954193
9.04
4.49
5.68
promising aspect in hypercholesterolemia treatment Simvastatin 54454
7.54 – –
using food.
Beyond its high amino acid contents, soybeans also have 2004; Sung et al., 2004). In addition, Simvastatin was used as
high bioactive compounds, majorly from isoflavones (Wang a control molecule for HMGCR inhibitor, according to the
et al., 2013). Dietary isoflavones have been reported to have previous study (Marahatha et al., 2021). On the other hand,
a protective role in regulating plasma cholesterol levels dur- three dimensional (3 D) structures of isoflavones and
ing the clinical phase (Herwana et al., 2020; Taku et al., Simvastatin were retrieved from PubChem, while the protein
2007). This result is supported by the experimental evidence structure was downloaded from Protein Data Bank (PDB)
that isoflavones reduced LDL cholesterol in blood plasma with the following identity: HMGCR (PDB ID: 1DQ8), PCSK9
(Huang et al., 2013; Lee et al., 2007; Teixeira Damasceno (PDB ID: 5OCA), and SREBP2 (PDB ID: 1UKL).
et al., 2007). On the other hand, an animal model experiment
discovered that isoflavones treatment altered SREBP expres-
sion and several of its downstream genes (Mullen et al., 2.2. Binding energy calculation
2004). Further investigations also tried to uncover the lipid-
Binding energy was calculated using AutoDock 4.0 in PyRx
lowering properties of the isoflavones by altering PPAR, ATP-
8.0 software using Lamarckian Genetic Algorithm (Dallakyan
Binding Cassette super-family G member 5 (ABCG5), and
& Olson, 2015; Morris et al., 1998, 2009). The grid setting was
ABCG8 expressions (Huang et al., 2013; Mezei et al., 2003). determined according to key residues for each protein
Unfortunately, the authors still hypothesized the exact mech- (Gustafsen et al., 2017; Istvan et al., 2000; Lee et al., 2003).
anism of the hypolipidemic activity of the isoflavones. The grid center position was set as follows: x: 11.8747, y:
Besides, the actual compound from the isoflavones group 17.447, z:
2.9508 for HMGCR; x: 46.3085, y: 10.8373, z:
involved in cholesterol-lowering activity remains unknown. It 23.6289 for PCSK9; and x: 23.9692, y: 6.1774, z: 101.417 for
is still hypothesized that all isoflavones have a beneficial role SREBP2 with 0.375 Å spacing for all docking simulations.
in maintaining normal plasma cholesterol. Nevertheless, since
chemical structure influences its interaction and activity
towards biological macromolecules (Lewandowski et al., 2.3. Docking analysis and structure visualization
2020), the mechanism leaves a gap to be filled.
Binding energy, types of the chemistry of residue interaction,
Several pathways have been suggested to regulate
and hydrophobicity surface were considered as the parame-
plasma cholesterol levels, including blocking de novo choles-
ters of docking results. A complex with binding energy lower
terol biosynthesis through inhibition of HMGCR (Boucher &
than Simvastatin was considered for molecular dynamics
Vogel, 2016; Ma et al., 2019) or SREBP2 (Tang et al., 2011)
simulation. Finally, the complex structure of each protein-lig-
and preventing Low-Density Lipoprotein Receptor (LDLR)
and was visualized using Biovia Discovery Studio 2019.
degradation through inhibition of PCSK9 activity (Gustafsen
et al., 2017; Ma et al., 2019). Thus, targeting those proteins
may unveil the hypolipidemic activity of the isoflavones. 2.4. Molecular dynamic simulation and binding energy
Therefore, this study will explores the mechanism underlying calculations
hypocholesterolemic activity of soy-isoflavone as well as
YASARA version 19.12.14 software (Krieger & Vriend, 2015)
complements and comprehends aforementioned pro-
with AMBER14 force field (Maier et al., 2015) was employed
posed mechanism.
to perform molecular dynamics simulation using the follow-
ing condition: pH 7.4, 0.9% NaCl concentration, 310 K tem-
2. Materials and method perature, 0.997 water density, 1 atm pressure, and 100 ns
running time. The Root-Mean-Square Deviation (RMSD) and
2.1. Data mining of isoflavone and protein structures Root-Mean-Square Fluctuation (RMSF) outputs were used for
The list of nine isoflavones was selected according to the determining structural stability and rigidity. Also, number of
previously identified from elicited soybean (Atho’illah et al. hydrogen bond was analyzed. In addition, free binding
2019). HMGCR, SREBP2, and PCSK9 were selected as the tar- energy was calculated using YASARA macros with Boundary
get based on the previous studies related to cholesterol Fast method. The DEnergy was estimated according to the
metabolism regulation (Mannino et al., 2021; Mullen et al., following sum:

Figure 1. Amino acid interactions of daidzin (A), Glycitin (B), Genistin (C), Daidzein (D), Glycitein (E), Genistein (F), Glyceollin I (G), Glyceollin II (H), Glyceollin III (I),
and Simvastatin (J) with HMGCR.


DEbinding ¼ DEreceptor þ DEligand
DEcomplex
2.5. Toxicity prediction
ProTox II (Banerjee et al., 2018) was used to determine the
toxicity profile of selected isoflavones. Lethal dose 50 (LD50)
and toxicity class were measured, while probability to have
hepatotoxic, carcinogenic, immunotoxic, mutagen, and
JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 3
cytotoxic properties were estimated according to the prob-
ability score.
3. Results and discussion
3.1. Isoflavones interaction with target proteins
Molecular docking simulation revealed that all of the isofla-
vones interacted with HMGCR at lower binding energy than
other target proteins, i.e., PCSK9 and SREBP2. In addition,
4 F. E. HERMANTO ET AL.

Figure 2. The RMSD of the selected compounds describes the stability of interaction of HMGCR and Glycitin, Genistin, Genistein, and Simvastatin, respectively. The
RMSD of Protein Backbone (A) describes the stability of the protein, the RMSD of Ligand Conformation (B) describes the stability of ligand structure during its inter-
action with the protein, and the RMSD of Ligand Movement (C) describes the stability of the ligand during the interaction with the protein. Additionally, RMSF val-
ues of selected compounds specified on the active site residues (D) and all residues in chain A (E) and chain B (F) of HMGCR. Also, average number of hydrogen
bond (G) and its fluctuations during the simulation time (H) were described along with binding energy of selected isoflavones (I).

Glycitin, Genistin, Genistein, Glyceollin II, and Glyceollin III
have binding energy lower than Simvastatin as control mole-
cules for HMGCR inhibitor (Table 1). The isoflavones with
lower binding energy than Simvastatin, except Genistein,
also bounded in the hydrophobic area of the HMGCR.
Several catalytic residues of HMGCR (Istvan et al., 2000) also
interacted with these isoflavones, including ARG590, GLU559,
LEU853, ASP690, LYS691, ASN755, and ASP767 (Figure 1). On
the other hand, Genistein showed more hydrogen bonds
than the other four isoflavones (Supplementary Table 1).
These results agree with the previous studies describing the
potential of isoflavones as competitive inhibitors of the
HMGCR (Sung et al., 2004). Accordingly, Glycitin, Genistin,
Genistein, Glyceollin II, and Glyceollin III interactions with
HMGCR were further analyzed using molecular dynam-
ics simulation.
To understand the structural dynamics of the selected iso-
flavones with HMGCR, molecular dynamics were employed.
The RMSD of the protein backbone value showed that the
interactions of isoflavones and Simvastatin did not alter the
structural backbone of HMGCR as all of those ligands have a
value of less than 3 Å (Figure 2A). Meanwhile, the structural
dynamics of isoflavones were more stable compared to the
Simvastatin during the binding to HMGCR (Figure 2B).
However, the RMSD of ligand movement described that
Glyceollin III might be detached during simulation, indicated
by the dramatic escalation of the RMSD at around 50 ns. On
the other hand, other isoflavones, particularly Genistein and
Glyceollin II, have better interaction stability with the HMGCR
than the Simvastatin (Figure 2C).
Residual fluctuations represented by RMSF showed no dif-
ference among isoflavones and Simvastatin (Figure 2D-F).
Catalytic residues also stayed stable in the complex of
HMGCR with isoflavones and Simvastatin, suggesting no
alteration of residual fluctuation at the catalytic sites (Figure
2D). RMSF value represented that isoflavones’ binding to the
HMGCR did not influence the stability of each residue, par-
ticularly catalytic residues of HMGCR. Therefore, all of the iso-
flavones may bound with great affinity to the HMGCR to act
as its inhibitor.
Since hydrogen bond has a vital role in protein-ligand sta-
bility, the number of hydrogen bond will influence the bind-
ing of isoflavones to the HMGCR (Fu et al., 2018; Patil et al.,
2010). Genistin, Genistein and Glyceollin II have greater
 JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS 5

Table 2. Calculations on free binding energy of HMGCR with every selected ligands.
D Energy (kJ/mol)
Ligand Binding Potential Receptor Solvation Receptor Potential Ligand Solvation Ligand Potential Complex Solvation Complex
Simvastatin
20.158
43412.268
53826.624 53.461
77.618
43554.937
53687.954
±53.645 ±1023.807 ±1328.619 ±21.366 ±10.157 ±1025.471 ±1307.768
Glycitin
128.494
43161.864
54532.992
5.128
118.893
43373.147
54317.237
±56.788 ±908.797 ±1150.590 ±19.458 ±14.247 ±934.553 ±1176.149
Genistin 11.839
42178.139
53519.153
24.437
108.176
42372.696
53469.048
±35.886 ±874.601 ±1232.478 ±19.916 ±17.380 ±877.777 ±1229.739
Genistein
281.768
41574.401
54952.332 94.421
427.697
41804.823
54773.417
±59.381 ±888.422 ±1130.330 ±32.179 ±37.839 ±885.184 ±1129.452
Glyceollin II
70.18
41602.929
54606.344
65.247
150.833
41848.156
54507.017
±40.326 ±888.576 ±1192.082 ±17.687 ±12.381 ±890.733 ±1192.142
Glyceollin III
54.989
42107.701
53848.603 110.211
122.275
42113.457
53799.921
±65.678 ±895.644 ±1169.889 ±19.653 ±29.479 ±894.903 ±1171.589
Notes: Each value was presented in mean ± standard deviation.

Figure 3. Toxicity classification (A) and probability to induce several toxicity properties (B) of selected isoflavones.

number of formed hydrogen bond than other isoflavones
and Simvastatin (Figure 2G-H). This data also supported by
the RMSD of ligand movement where Genistein and
Glyceollin II remains stable and better than Simvastatin as
control inhibitor. However, binding energy calculations
described that Genistin was the most stable one even com-
pared to the Simvastatin. Among all of the complexes,
Genistein had lowest energy than other complex (Table 2,
Figure 2I).
Despite many experiments and clinical studies conducted
to evaluate the hypolipidemic activity of soy-isoflavones, the
exact mechanism is still poorly understood. This study
revealed that Genistin, Genistein, and Glyceollin II might per-
form as HMGCR inhibitors through their interaction with
HMGCR catalytic residues. The interaction remains stable
compared to Simvastatin, suggesting that those compounds
have excellent potential to block uncontrolled cholesterol
biosynthesis in hyperlipidemia. Isoflavones, mainly Genistein,
have been evaluated as an HMGCR inhibitor in vitro as Statin
(Sung et al., 2004). As a rate-limiting factor of cholesterol bio-
synthesis, HMGCR inhibition alleviated plasma cholesterol
levels (Lee et al., 2007). The HMGCR blocking activity was
also proven to have an atheroprotective role in vivo
(Mansouri et al., 2021). Advance shrinking of cholesterol lev-
els leads to upregulation of LDLR (Notarnicola et al., 2008)
through the SREBP2 pathway at the transcription scale (Lu
et al., 2019), thus allowing plasma cholesterol clearance for
cellular metabolic utilization (Goldstein & Brown, 2009).
Therefore, soy isoflavones may have hypolipidemic activity
through alterations of HMGCR expression and activity at the
transcriptomic and proteomic levels.
3.2. Toxicity profile of selected isoflavones
Toxicity prediction revealed that Glycitin, Genistin, and
Genistein were the less toxic isoflavones among selected iso-
flavones. Glyceollin III became the most toxic isoflavone with
LD50 25 mg/kg according to the ProTox II estimations
(Banerjee et al., 2018). Glyceollin II was classified as class 4
with slight toxicity profile based on LD50 calculation (Figure
3A). Further, Glycitin, Glyceollin II, and Glyceollin III showed
high probability to have immunotoxic effect (Figure 3B). In
view of that, Genistin and Genistein were suggested as the
best HMGCR inhibitors with the lowest toxicity profile.
4. Conclusion
Genistin and Genistein may have excellent properties as
HMGCR competitive inhibitors with the highest affinities and
low toxicity profiles. The cholesterol-regulating mechanism of
soy isoflavones may be achieved by inhibiting cholesterol
biosynthesis by blocking HMGCR activity.
6 F. E. HERMANTO ET AL.
Acknowledgement
The authors thank to Dr. Dinia R. Dwijayanti for her constructive com-
ments and suggestions during the manuscript arrangement.
Disclosure statement
The authors declare no competing interest in this work.
Funding
This research was funded by Ministry of Education, Culture, Research,
and Technology, Republic of Indonesia under PMDSU research scheme
funding (grant No. 438.31/UN10.C10/TU/2021 and 612.11/UN10.C10/
TU/2022).
ORCID
Feri Eko Hermanto http://orcid.org/0000-0002-6955-3688
Muhaimin Rifa’i http://orcid.org/0000-0001-5731-2951
Nashi Widodo http://orcid.org/0000-0002-1126-498X
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