Identification of Beta-Sitosterol and Stigmasterol as Possible Inhibitors of 5 Alpha-Reductase 1: An In-Silico Study

Abstract

Background: Dihydrotestosterone (DHT), the most critical pathogenic androgen in hair loss, is identified as an etiologic factor of androgenetic alopecia (AGA). The AGA is a genetically common disorder among men and is characterized by the progressive conversion of hair follicles into small vellus hair. Steroid 5 alpha-reductase type 1 (5AR1) is a crucial target responsible for this gradual replacement. The 5AR1 function is determined by converting testosterone to DHT. The inhibitors of 5AR1 play their role by blocking the DHT production pathway. Objectives: This study focused on the potent inhibitors of the 5AR1 enzyme to suggest effective synthetic drugs for restoring hair loss with fewer side effects. Methods: The three-dimensional structure of 5AR1 was created using homology modeling methods. Then, the inhibitory effects of some significant compounds from natural sources were examined on the 5AR1 protein using molecular docking approaches. Results: The obtained results suggest that two natural compounds isolated from Serenoa repens, including beta-sitosterol and stigmasterol, could inhibit the regular activity of 5AR1 and can be recommended as safe and novel AGA medicines for hair restoration.

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Precis Med Clin OMICS. 2021 December; 1(1):e121545.
Published online 2022 January 22.
doi: 10.5812/pmco.121545.
Research Article
Identification of Beta-Sitosterol and Stigmasterol as Possible
Inhibitors of 5 Alpha-Reductase 1: An In-Silico Study
Pardis Zamani 1, Omid Mokhtari1and Fariba Dehghanian 1, *
1Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran
*Corresponding author: Department of Celland Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran. Email:
fa.dehghanian@sci.ui.ac.ir
Received 2021 December 02; Revised 2022 January 04; Accepted 2022 January 05.
Abstract
Background: Dihydrotestosterone (DHT), the most critical pathogenic androgen in hair loss, is identified as an etiologic factor of
androgenetic alopecia (AGA). The AGA is a genetically common disorder among men and is characterized by the progressive con-
version of hair follicles into small vellus hair. Steroid 5 alpha-reductase type 1 (5AR1) is a crucial target responsible for this gradual
replacement. The 5AR1 function is determined by converting testosterone to DHT. The inhibitors of 5AR1 play their role by blocking
the DHT production pathway.
Objectives: This study focused on the potent inhibitors of the 5AR1 enzyme to suggest effective synthetic drugs for restoring hair
loss with fewer side effects.
Methods: The three-dimensional structure of 5AR1 was created using homology modeling methods. Then, the inhibitory effects of
some significant compounds from natural sources were examined on the 5AR1 protein using molecular docking approaches.
Results: The obtained results suggest that two natural compounds isolated from Serenoa repens, including beta-sitosterol and stig-
masterol, could inhibit the regular activity of 5AR1 and can be recommended as safe and novel AGA medicines for hair restoration.
Keywords: DHT, 5AR1, Homology Modeling, Molecular Docking
1. Background
Androgenetic alopecia (AGA), or male-pattern hair loss
(MPHL), is the most common form of hair loss occurring
in 80% of men aged 80 years or older (1). The AGA is an
androgen-dependent disorder, and the miniaturization of
scalp hair follicles results in decreased hair density, thin
fibers, and severe forms that cause baldness (2,3). Dihy-
drotestosterone (DHT) is the primary specific androgen
hormone involved in AGA. In the DHT biosynthesis path-
way, an integral membrane enzyme, 5 alpha-reductase
(5AR), plays a regulatory role by reducing testosterone to
DHT.
The 5AR family is related to androgen-dependent dis-
orders and consists of two isozymes, 5 alpha-reductase
type 1 (5AR1) and 5 alpha-reductase type 2 (5AR2) (4,5).
The 5AR1 and 5AR2 are nicotinamide adenine dinucleotide
phosphate-dependent enzymes and share a low sequence
identity of 47%. These two isozymes are encoded by SRD5A1
and SRD5A2 genes, located in different chromosomal loca-
tions. The 5AR1 is located on the 5p15.31 chromosome; how-
ever, 5AR2 is located on chromosome 2P23.1 in the human
genome (6).
Moreover, the tissue expression patterns of 5AR1 and
5AR2 are different. Type 1 is expressed in nongenital skin,
fetal scalp, and liver; nevertheless, type 2 is predominantly
expressed in male genital tissues, prostate, and seminal
vesicles. The 5AR2 is responsible for virilizing the external
male genitalia during puberty, benign prostatic hyperpla-
sia (BPH), and prostate adenocarcinoma tissues (7-9). Since
it has been reported that DHT-related disorders include
AGA, BPH, and prostate cancer,which can be treated by low-
ering DHT levels, 5AR is identified as an efficient drug tar-
get (10).
The 5AR inhibitors are applied in restoring hair loss
and regenerating miniaturized hair. Hair restoration is
defined by the change in terminal hair count and width.
Nonsurgical hair restoration options ranging from drug
therapy to laser treatments are cheaper and do not carry
risks associated with surgical hairline restoration, includ-
ing hair transplantation (11). In this regard, two potent syn-
thetic drugs, including finasteride and dutasteride, have
been established to inhibit 5AR1 and 5AR2. Finasteride, a
synthetic azo-steroid, is a more potent 5AR2 inhibitor than
5AR1, and dutasteride, widely used as AGA or BPH drug, in-
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Zamani P et al.
hibits both 5AR1 and 5AR2 (12).
According to the research conducted on the above-
mentioned two drugs, it has been detected that finasteride
can reduce the DHT level in serum by 70%, and the dutas-
teride inhibition effect showed a reducing amount of DHT
level up to 90% in serum (13). Although finasteride and du-
tasteride performed well as efficient 5AR1 inhibitors and
have been approved by the United States Food and Drug Ad-
ministration (FDA), there have been more concerns about
these two drugs’ long-lasting side effects occurring in ther-
apy within the last few years. Recent research has shown
that finasteride and dutasteride have several sexual ad-
verse effects, including libido reduction, dysfunction of
erectile, ejaculation disorders, and gynecomastia (14,15).
Due to the safety concerns of these drugs, the detection of
new, safe, and more effective 5AR inhibitors has become a
new medication approach in the drug industry. As it is ev-
idenced that 5AR1 is primarily expressed in the scalp, the
inhibition of 5AR1 is the primary approach for AGA medi-
cation (16).
2. Objectives
Multiple traditional herbals are suggested as potent
drugs to prevent this enzyme from working normally. The
current study provided a library of herbal components as
natural inhibitors and evaluated 5AR1 through homology
modeling and molecular docking techniques. The current
study library includes Cedrus and the henna tree (Lawso-
nia inermis), widely used in home remedy medications as
topical therapy on the scalp. In various studies, other com-
pounds mentioned as effective 5AR1 inhibitors that sug-
gested stimulating hair regrowth and strengthening hair
follicles are saw palmetto (Serenoa repens) and green tea
(Camellia sinensis). Another highly recommended com-
pound is vitamin B2, also known as riboflavin. Moreover,
two approved drugs, finasteride, and dutasteride, were
chosen as positive controls to be compared with the afore-
mentioned natural compounds (17-21).
3. Materials and Methods
3.1. Molecular Modeling
Since no three-dimensional (3D) structure of 5AR1 is
currently available in the Protein Data Bank, homology
modeling was utilized to generate the structure of SRD5A.
Modeling approaches are the most reliable methods of
predicting the 3D structure of an unknown protein based
on known homologous reference protein with high iden-
tity to the novel protein as a template. The FASTA format
of the 5AR1 enzyme protein sequence was retrieved from
the UniProt database (UniProtKB) with the accession num-
ber P18405. Based on best sequence alignment, the 3D
structure of the protein was modeled using I-TASSER on-
line server. The model was validated using various tools,
such as ERRAT (22) and PROCHECK (23), to generate the Ra-
machandran plot.
3.2. Dataset Collection
The data of this study contained several medicinal
plants widely used in home remedy medications. The
present study identified the main components of these
selected herbs. Cedrol, Cedrene, and Thujopsene are de-
tected in Cedar (Cedrus). Cedarwood essential oil is used
as a skincare agent and hair growth medication (24). 1,4-
naphthoquinone and Lawsone are active compounds iso-
lated from Lawsonia inermis, also known as the henna tree.
Studies evaluated that Lawsonia inermis compounds can
slow hair loss and help nourish the scalp (25). Epicatechin
gallate (ECG) and Epigallactocatechin gallate (EGCG) are
extracted from Camellia sinensis leaves (26-28). Other com-
pounds are β-sitosterol and stigmasterol isolated from
Serenoa repens. It is a popular herbal remedy that boosts
hair growth and helps maintain scalp health (29,30). Ri-
boflavin (vitamin B2) is a known antioxidant present in var-
ious plants. Research showed that riboflavin deficiency is
associated with hair loss (31). Finasteride and dutasteride,
used orally as 5AR1 inhibitors, were selected as positive con-
trols. Figure 1 depicts the two-dimensional (2D) structure
of all compounds.
3.3. Protein and Herbal Compounds Preparation
AutoDock Vina tools 1.5.6 were used to prepare 5AR1
and all compounds. The modeled structure of 5AR1 was
obtained from I-TASSER online server and imported to the
AutoDock Vina tools program. All water molecules were
merged, polar hydrogens were added, and Kollman charge
(32) and AD4 atom type were assigned. All the above-
mentioned 3D structures of compounds were retrieved
from PubChem data bank. Then, nonpolar hydrogens were
eliminated; finally, Gasteiger charge was assigned to calcu-
late the partial charge of all inhibitors.
3.4. Docking Studies
A molecular docking experiment was employed to de-
termine the binding modes of selected components with
5AR1 active site residues. This study used AutoDock Vina to
perform docking procedures to predict all inhibitors’ best
configuration within 5AR1 protein. The grid box parame-
ter values were adjusted with the size of 25*25*25 in spe-
cific coordinates of x, y, and z-axis (x = 64.899, y = 62.246,
and z = 66.772) and the space value of 1 Å to confirm the
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Zamani P et al.
Figure 1. Chemical structure of 5 alpha-reductase type 1 inhibitors from natural sources
best binding affinity of the inhibitor-protein complex. The
Lamarckian genetic algorithms (33) were selected to per-
form docking. The best confirmation with the lowest bind-
ing energy estimated by Autodock Vina was visualized with
PyMOL (34) and LigPlot plus (35) to provide a 2D diagram of
the compound’s interaction against 5AR1.
4. Results
4.1. Constructed Model of 5AR1 and Validation
Figure 2A illustrates the constructed homology model
of 5AR obtained from I-TASSER. PROCHECK and ERRAT
were used to evaluate the model structure. ERRAT results
showed an overall quality factor of 92.0319. The Ramachan-
dran plot of the model in Figure 2B shows that 86.97% of
residues are in the most favored regions (red), 9.8% in addi-
tional allowed regions, 1.8% in generously allowed regions,
and 1.8% in disallowed regions. These statistics indicated
that the modeled structure was suitable for docking simu-
lation.
4.2. Docking Studies and Visualization
AutoDock Vina results revealed the orientation and in-
teraction of all natural compounds against 5AR1. The most
active compounds of the present study data collection are
listed in Table 1. In addition, the binding energy, H bonds,
and hydrophobic interactions are also shown. The docking
results demonstrated that two compounds, β-sitosterol,
and stigmasterol from Serenoa repens, had the strongest
docking affinity of -10.7 and -10.1 kcal/mol, respectively,
compared to the rest of the collection. In the second place,
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Zamani P et al.
Figure 2. (A) Three-dimensional modeled structure of 5 alpha-reductase type 1 (5AR1) enzyme using I-TASSER online server; (B) Ramachandran plot of 5AR1 model built via
PROCHECK
ECG and EGCG isolated from Camellia sinensis leaves had
the binding energy values of -9.9 and -9.6. kcal/mol, respec-
tively. Riboflavin also showed high affinity (-9.1 kcal/mol)
but weaker than the last two groups and equal to or greater
than finasteride and dutasteride. Cedrus’s three main com-
pounds (ie, Cedrol, Cedrene, and Thujopsene) displayed
well but were weaker than the positive controls with the
binding energy of -8.2, -8.3, and -7.9 kcal/mol, respectively.
Lawsone and 1,4-naphthoquinone isolated from Lawsonia
inermis exhibited the weakest affinity among other com-
pounds with the active site residues of 5AR1. Screening the
interaction details of the 5AR1-inhibitor complex from Lig-
Plot+ showed that almost every compound interacts with
Ser36, Tyr38, Trp56, Glu60, Tyr95, Ala116, Met119, Ala120,
Phe123, Phe224, and Phe228. It could be inferred that these
residues are critical in the binding site and can be consid-
ered important residues for inhibitors to bind within the
active pocket of 5AR1. The most vital interaction regions
of 5AR1 are shown in brown parts in Figure 3A. The 5AR1
residues involved in the interaction with stigmasterol and
β-sitosterol are represented in 3D and 2D structures in Fig-
ures 3B-3C and 4A-4B, respectively.
5. Discussion
This study investigated the possible interactions be-
tween herbal inhibitors and 5AR1 protein using bioinfor-
matics tools for AGA medication. Previously, Lin et al.
worked on 5AR1 inhibitors to treat BPH; however, contrary
to the present work, they used AutoDock for the docking
procedure (36). In comparison to AutoDock 4, AutoDock
Vina significantly increases the average accuracy of the
binding mode predictions and is incredibly faster. It has
always been among the six first choices for docking analy-
sis globally. In the present study, this software could define
the interactions properly.
The current study findings revealed that all inhibitors
have possible interactions with 5AR1. Moreover, among
the selected inhibitors, the highest binding (-10.7 kcal/mol)
belongs to stigmasterol. The present analyses indicated
that stigmasterol binds to 5AR1 through only hydropho-
bic forces, and 13 amino acid residues were detected in
the interaction site (Figure 3B). Therefore, it can be con-
cluded that stigmasterol might be the best inhibitor. This
inhibitor is suggested as the new choice for further inves-
tigations in this field. In the second rank, β-sitosterol was
placed, with -10.1 kcal/mol. In the interaction site of β-
sitosterol, 13 amino acid residues weredetected (Figure 3C).
The highenergy value of thisinhibitor showed thepossible
potential of this component to inhibit 5AR1.
Similar to the present study results, previous stud-
ies confirmed the possible ability of stigmasterol and β-
sitosterol to inhibit 5AR. Prager et al. established the
effectiveness of β-sitosterol to control AGA. For the first
time, Prager et al. used a placebo-controlled double-blind
method to examine the benefit of this botanical substance
(37). In addition, Cabeza et al. reported the effect of β-
sitosterol to inhibit 5AR1 in the hamster prostate (38). They
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Zamani P et al.
Table1. List of Most Active Compounds
Compounds Binding Energy
(kcal/mol)
H-bond Hydrophobic Interaction
Stigmasterol -10.7 - Ser36, Val37, Tyr38,Trp56, GLN59, Glu60, Tyr95, Ala116, Met119, Ala120, Glu202,
Phe224, and Phe228
Beta-sitosterol -10.1 Tyr95 and Glu202 Arg30, Ser36, Tyr38, His46, Glu60, Leu115, Ala116, Met119, Ala120, Phe224, and
Phe228
Epigallactocatechin
gallate
-9.9 Tyr56, Arg176, Tyr183,
Arg232, and Tyr240
Tyr38, Gly39, Ala52, Tyr102, Met106, Gly108, Gly109, Met112, Leu172, Tyr199, and
His236
Epicatechin gallate -9.4 Arg98, Asn165, Asp169,
and Asn198
Tyr38, Trp56, Glu60, Met119, Ala120, Phe123, Glu202, Phe221, Phe224, Phe228,
and Leu229
Riboflavin -9.1 Arg98 and Glu202 Tyr38, Trp56,Gln59, Glu60, Tyr95, Ala116, Met119, Ala120, Phe123, Phe224,
Phe228, and Leu229
Dutasteride -9.1 Arg30, Asn31, and Glu60 Ser36, Val37, Tyr38,His46, Gln59, Trp56, Leo115, Ala116, Met119, Ala120, Phe224,
and Phe228
Finasteride -8.5 Tyr95 and Arg98 Arg30, Ser36, Val37, Glu60, Ala116, Met119, Ala120, Phe123, Phe224, and Phe228
Cedrene -8.3 - Trp56, GLN59, Glu60, TYR95, Ala120, Phe123, Glu202, Phe221, Phe224, Thr225,
and Phe228
Cedrol -8.2 Glu60 Gln59, Tyr95, Ala120, Phe123, Phe221, Phe224, and Phe228
Thujopsene -7.9 - Glu60, Tyr95, Met119, Ala120, Phe123, Phe221, Phe224, THR225, and Phe228
Lawsone -7.4 Arg30 and Ser36 Val27, Tyr38, Ala116, Met119, Ala120, Phe224, and Phe228
1,4-naphthoquinone -7.1 Arg30 and Ser36 Tyr38, Ala116, Met119, Ala120, Phe224, and Phe228
Figure 3. Most Critical interaction sites (brown regions) of 5 alpha-reductase type 1 (5AR1) protein (A); Active site residues of 5AR1 binding within stigmasterol (B) and beta-
sitosterol (C)
showed β-sitosterol could decrease the prostate weight,
and this effect was not related to the binding of β-sitosterol
to the androgen receptor but the inhibition of 5AR. How-
ever, both targets were present in the prostate.
Furthermore, Upadhyay et al. investigated the effect
of β-sitosterol phyto-vesicles to treat AGA. The aforemen-
tioned study confirmed the ability of β-sitosterol in the
control of alopecia, and phyto-vesicles could increase this
compound’s water and lipid solubility (39). Finally, Chen et
al. showed the potential of stigmasterol and β-sitosterol to
target 5AR and inflammatory pathways, which confirmed
the efficacy of these components to treat AGA and BPH.
In Chen et al.’s study, in vitro assays were used. The re-
sults of the aforementioned study determined the down-
regulation of messenger ribonucleic acid expression pro-
file characteristics of both disease processes in AGA (hair
follicle dermal papilla cells) and BPH (LNCaP prostate
cells) cell lines treated with stigmasterol or β-sitosterol.
The aforementioned study proved that stigmasterol and
β-sitosterol targeting 5AR and inflammatory mediators
might represent a rational approach in treating AGA and
BPH (40). Currently, available drugs for AGA are finasteride
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Zamani P et al.
Figure 4. Two-dimensional diagram of most potent inhibitors, namely stigmasterol (A) and beta-sitosterol (B), interactions within 5 alpha-reductase type 1
and dutasteride, which are synthetic. However, these two
FDA-approved drugs have good inhibitory activity. Recent
studies have shown that they can lead to severe adverse
effects, including ejaculation disfunction, reduction of li-
bido, gynecomastia, and erectile disorder (41).
Docking analysis showed that dutasteride had a
stronger binding affinity (-9.1 kcal/mol) than finasteride.
In both inhibitors’ interaction sites, H-bonds and hy-
drophobic forces get involved. In line with the present
study results, some previous studies showed the in-
hibitory activity of these inhibitors. Rabasseda et al. and
Olsen et al. indicated that dutasteride increased scalp
hair growth in men with MPHL and suggested that type 1
and type 2 5AR might be important in the pathogenesis
and treatment of MPHL (42,43). In addition, Gubelin
Harcha et al. determined that dutasteride increased hair
growth and restoration in men with AGA. Gubelin Harcha
et al. showed that dutasteride 0.5 mg could remarkably
increase hair width and hair terminal count more than
finasteride 1 mg, suggesting that dutasteride is more effec-
tive than finasteride in hair loss restoration (44). Eun et al.
cited in Dhurat and Shanshanwal showed the potential of
dutasteride to inhibit 5AR (type 1 and type 2), converting
testosterone to DHT (45). Moreover, Inadomi et al., Shapiro
et al., and Yanagisawa et al. conducted different pilot stud-
ies, and their results showed that finasteride is effective
in the treatment of AGA patients (46-48). Finally, Arif et al.
and Jung et al. introduced dutasteride as a treatment of
choice for AGA (49,50).
Liao et al. showed that EGCG and ECG extracted from
green tea could inhibit 5AR in the sex gland in the rat. Fur-
thermore, Hiipakka et al. determined that EGCG can affect
5AR in cell-free but not whole-cell assays. They suggested
that EGCG with long-chain fatty acids is active in both cell-
free and whole-cell assay systems (26,51). Koseki et al. sug-
gested the potential of ECG to inhibit 5AR, which results
in inhibiting the androgen-related pathogenesis of acne,
testosterone conversion, and sebum synthesis. Therefore,
it was proposed that it can be a helpful agent in the thera-
peutic strategy of acne (52). The present study confirmed
the possible interactions between EGCG and ECG with 5AR
by computational methods, similar to the aforementioned
studies. Both inhibitors’ energy values were about -9
kcal/mol.
The docking results of the present study estimated the
strong interaction with -9 kcal/mol binding energy. More-
over, Nakayama et al. introduced riboflavin as a 5AR in-
hibitor. It was confirmed by Cho et al. that riboflavin has
stronger potent inhibitory activity than other compounds,
such as emodin and alizarin (53,54). Although the bind-
ing energy of Cedrol (-8 kcal/mol) is not close to the high-
est ones, it was predicted to be a strong inhibitor. Zhang
et al. showed the beneficial effect of Cedrol on hair loss
and confirmed that it has a strong hair growth promo-
tion effect (55). In addition, Deng et al. suggested that
Cedrol nanoemulsion significantly improved pharmacoki-
netic properties and hair growth (56).
1,4-naphthoquinone was extracted from a natural
source for the first time by Ishiguro et al. that showed
the significant testosterone 5AR inhibitory activity (57). Al-
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Zamani P et al.
though Ishiguro et al. examined the inhibitory activity
of 1,4-naphthoquinone, the current study findings showed
that it has the lowest binding energy (-7.1 kcal/mol), com-
pared to other inhibitors. Therefore, it can be concluded
that this inhibitor has the weakest interaction with 5AR1.
Although Lawsone, Thujopsene, and Cedrene were listed as
5AR1 inhibitors in some reviews, any experimental studies
related to these components were detected. By consider-
ing the docking results of the present study, it can be con-
cluded that among these inhibitors, Cedrene is more capa-
ble of inhibiting 5AR1 due to the highest binding affinity
(-8.3 kcal/mol), compared to Thujopsene and Lawsone with
binding energy values of -7.9 and -7.4 kcal/mol, respectively.
Docking analysis showed that some regions were criti-
cal in the interaction between selected inhibitors and 5AR1,
shown in Figure 3A as brown regions. Among amino acid
residues involved in all interactions, 11 amino acids (ie,
Ser36, Tyr38, Trp56, Glu60, Tyr95, Ala116, Met119, Ala120,
Phe123, Phe224, and Phe228) were detected in the majority
of inhibitors and 5AR1 interaction sites. Therefore, it can be
suggested that the aforementioned residues are essential
to inhibit this protein and might be helpful in designing
new targets for the next generation of 5AR1 inhibitors.
5.1. Conclusions
The present study aimed to examine the potential ac-
tivity of herbal components as 5AR1 inhibitors using in-
silico analysis, which can be helpful for AGA treatments
and restoration of hair follicles. In conclusion, the present
predictions determined two inhibitors (ie, β-sitosterol and
stigmasterol) with the most robust interactions as the sug-
gested compounds to be used in further investigations as
the next generation of drugs to control AGA.
Footnotes
Authors’ Contribution: Pardis Zamani and Omid
Mokhtari performed in-silico analyses and wrote the
manuscript. Fariba Dehghanian helped in editing the
manuscript.
Conflict of Interests: The authors declare that they have
no conflict of interest.
Funding/Support: This study was performed at the Uni-
versity of Isfahan, Iran, and supported by the Graduate
Studies Office at this university.
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