Grape skin and seed extracts as a potential natural solution for hair loss: A bioactivity evaluation

Abstract

Scalp yeast infections and androgenic effects can lead to hair loss respectively by disrupting hair follicle function and by causing miniaturization. Herbal extracts, including grape skin and seed extracts, show promise for hair growth improvement and hair loss prevention due to their safety and diverse biological activities. The aim of this study was to evaluate the antifungal and 5α-reductase inhibition activities of grape skin and seed extracts. The results revealed that grape skin and grape seed extracts exhibited excellent antifungal activity against T. mentagrophytes, M. gypseum, C. albicans, P. ovale, and M. canis in descending order. Furthermore, ethanolic grape skin and seed extracts demonstrated promising anti-hair loss activity through an increase in the proliferation of human hair follicle dermal papilla cells (HFDPC) and 5α-reductase inhibition analyzed in vitro on HFDPC. Therefore, the results of this study imply that grape skin and seed extracts may be utilized in the development of multifunctional hair products with anti-hair loss and antifungal actions.

Full text

*Corresponding author
Email address: liudmila.ya@buu.ac.th
Songklanakarin J. Sci. Technol.
45 (5), 578–584, Sep. – Oct. 2023
Original Article
Grape skin and seed extracts as a potential natural solution for hair loss:
A bioactivity evaluation
Pathomthat Srisuk1, Watcharee Khunkitti1, Chontira Khawee1, and Liudmila Yarovaya2*
1 Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences,
Khon Kaen University, Mueang, Khon Kaen, 40002 Thailand
2 Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences,
Burapha University, Mueang, Chon Buri, 20131 Thailand
Received: 29 June 2023; Revised: 26 September 2023; Accepted: 27 September 2023
Abstract
Scalp yeast infections and androgenic effects can lead to hair loss respectively by disrupting hair follicle function and
by causing miniaturization. Herbal extracts, including grape skin and seed extracts, show promise for hair growth improvement
and hair loss prevention due to their safety and diverse biological activities. The aim of this study was to evaluate the antifungal
and 5α-reductase inhibition activities of grape skin and seed extracts. The results revealed that grape skin and grape seed extracts
exhibited excellent antifungal activity against T. mentagrophytes, M. gypseum, C. albicans, P. ovale, and M. canis in descending
order. Furthermore, ethanolic grape skin and seed extracts demonstrated promising anti-hair loss activity through an increase in
the proliferation of human hair follicle dermal papilla cells (HFDPC) and 5α-reductase inhibition analyzed in vitro on HFDPC.
Therefore, the results of this study imply that grape skin and seed extracts may be utilized in the development of multifunctional
hair products with anti-hair loss and antifungal actions.
Keywords: grape extract, antifungal activity, 5α-reductase, anti-hair loss
1. Introduction
Among hair loss disorders, androgenetic alopecia
(AGA) is the most prevalent form characterized by
progressive hair thinning, shortening, and loss of
pigmentation, affecting both genders. It is suggested that AGA
is primarily associated with a genetic predisposition to
sensitivity to dihydrotestosterone (DHT) (Sawaya & Price,
1997). The mechanism behind AGA involves the enzyme 5α-
reductase that converts testosterone into a potent androgen
DHT. Androgen receptors mainly expressed in the dermal
papilla cells of the hair follicle are a primary target for DHT,
and this binding leads to an inhibition of hair growth
(Chandrashekar, Nandhini, Vasanth, Sriram, & Navale, 2015).
Moreover, maintaining a balance among the
numerous microorganisms inhabiting the skin and hair of an
individual becomes vital for their health condition. While
research on the microbiome of the skin and hair follicles is
limited, it has been established that the invasion of fungi into
the hair follicle bulge can result in permanent hair loss
(Nematian, Ravaghi, Gholamrezanezhad, & Nematian, 2006;
Sohnle, Collins-Lech, & Hahn, 1986).
The two drugs approved by the regulatory
authorities of various countries and widely used for the
treatment of alopecia are minoxidil and finasteride. The
mechanism of action of finasteride, as a type II 5α-reductase
inhibitor, is directly related to AGA, as it reduces the
conversion of testosterone to DHT, while for minoxidil,
although it has been used for the treatment of AGA for several
decades, the mechanism of action is still not fully understood
(Madaan, Verma, Singh, & Jaggi, 2018). One of the main
assumptions revolves around the notion that, given
minoxidil’s vasodilatory properties, its mechanism of action

P. Srisuk et al. / Songklanakarin J. Sci. Technol. 45 (5), 578-584, 2023 579
involves increasing blood flow to the follicles. However, a
recent study revealed another possible mechanism of action of
minoxidil based on its antiandrogenic effect through
significant downregulation of the expression of the 5α-R2
gene in HaCaT cells (Pekmezci & Türkoğlu, 2017). Despite
different anti-hair loss mechanisms, both drugs cause
undesirable side effects that have prompted the search for
alternative treatments (Madaan et al., 2018).
The fact that the hair follicles provide a favorable
route for the delivery of topical preparations containing
compounds that penetrate these channels more efficiently than
the stratum corneum has brought to the fore the potential for
the topical application of herbal products. Among them are
grape skin and grape seeds (Vitis vinifera L.), a rich resource
of polyphenols with numerous documented biological benefits
for skin and hair (Obreque-Slier et al., 2010; Yarovaya,
Waranuch, Wisuitiprot, & Khunkitti, 2022). Regarding the
latter, the antioxidant and anti-inflammatory properties of the
grape extract are believed to help protect hair follicles from
damage and stimulate hair growth (Ferri et al., 2017; Kwon et
al., 2007; Takahashi, Kamiya, & Yokoo, 1998). Increases in
the number of hair follicles and in hair growth length have
been reported in a study on rats topically treated with grape
sap (Esmaeilzadeh et al., 2021). The antifungal and
antibacterial efficacy of rich polymeric flavan-3-ols extracts
from grape seed extract has also been reported (Khawee,
Aromdee, Monthakantirat, & Khunkitti, 2015; Simonetti et
al., 2017). Furthermore, grape skin and seeds as wine-making
by-products are an example of a natural resource that meets
the sustainability criteria of production, which further justifies
their use in cosmetics.
In our previous study, it was found that grape skin
and seed extracts contain high quantities of polyphenols along
with significant antioxidant and antibacterial properties
(Khawee et al., 2015). However, it was of interest to extend
the evaluation of their antifungal activity. Moreover, many
studies on grape properties have validated a remarkable ability
to promote cell growth in hair follicles and regulate
physiological cycles of hair growth. However, there is still a
lack of studies demonstrating the potential of grape extracts to
inhibit 5α‐reductase. Therefore, the hair loss prevention
potential of grape extracts was also analyzed in vitro on
human dermal papilla cells.
2. Material and Methods
2.1 Materials
Kojic acid, testosterone, minoxidil, and finasteride
were obtained from Sigma-Aldrich (Taufkirchen, Germany).
Tween 80 was ordered from Namsiang (Bangkok, Thailand).
Sabouraud dextrose agar, sabouraud dextrose broth, and yeast
extract were purchased from Difco (Detroit, MI, USA).
Human hair follicle dermal papilla cells (HFDPC), ready-to-
use HFDPC growth medium, HEPES Buffered Saline
Solution (HBSS), trypsin ethylene diamine tetra acetic acid
(Trypsin/EDTA), and trypsin inhibitor solution were ordered
from Promo Cell (Heidelberg, Germany). PrestoBlueTM Cell
Viability Reagent was obtained from Invitrogen (Waltham,
MA, USA). Testosterone (ab174569) Human Elisa Kit was
purchased from Abcam (Cambridge, UK). Dimethyl sulfoxide
(DMSO) was obtained from Gibco (Gaithersburg, MD, USA).
Grape skin and grape seeds (Vitis vinifera L.) were provided
by Village Farm & Winery (Nakhon Ratchasima, Thailand).
All solvents used were of analytical grade.
2.2 Preparation of grape skin and grape seed
extracts
The studied grape cultivars of Vitis vinifera were
ripe and had a dark purple color. The waste from grapes was
separated into grape skin and seeds and dried in an oven at 55
°C for 24 hr to obtain a moisture content of 8-10%. The
material was then ground to pass through a 60-mesh sieve
(Endecotts, London, United Kingdom). The sifted grape skin
and grape seeds were stored at –20 °C until the extractions
were carried out. One gram sample of either grape skin or
grape seeds was extracted with 3 ml of the following
alternative solvents: deionized water adjusted to pH 3 with
citric acid, ethanol, or acetone. The mixtures were left in the
dark at room temperature (RT) for 1 week and centrifuged at
1,200 rpm for 15 min in a centrifuge (mPW-350, MPW Med
Instruments, Warsaw, Poland). The supernatants were filtered
using Whatman No. 4 filter paper. The filtrates were collected
and concentrated using a Buchi-3 rotary vacuum evaporator
(Flawil, Switzerland) at 50 °C under reduced pressure.
2.3 Antifungal activity
2.3.1 Fungal strains and growth conditions
Candida albicans TISTR 5779, Trichophyton
mentagrophytes DMST 19735, Microsporum gypseum DMST
21146, Microsporum canis DMST 11875 were grown in
sabouraud dextrose broth (SDB), and incubated at 37 °C for
48 hr. Pityrosporum ovale ATCC 64061 was grown on
sabouraud dextrose agar (SDA) supplemented with 1% w/v of
yeast extract and 1% w/v Tween 80 at 25 °C for 72 hr.
2.3.2 Antifungal assay using agar diffusion method
All tested fungi, except P. ovale, were adjusted to an
optical density (OD) of 0.1 at 600 nm and seeded into 20 ml
of SDA. A sterilized spreader was used to spread the
microorganisms evenly over the agar surface. For P. ovale,
100 µL of the tested yeast and 100 µL of sterile olive oil were
simultaneously seeded on SDA supplemented with 1% w/v
yeast extract and 1% w/v Tween 80. Using a cork borer with a
diameter of 6 mm, wells were made on the SDA plate, and 45
µl of the grape skin or grape seed extract samples at a
concentration of 3% w/v in 95% ethanol were added to each
well. Selenium sulfide (0.25% w/v) and ketoconazole (0.2%
w/v) were used as positive controls. The plates with C.
albicans were incubated at 25 ºC for 72 hr and all other tested
fungi were incubated at 37 °C for 48 hr. The diameter (mm)
of the inhibition zone was measured (Punyoyai, Sirilun,
Chantawannakul, & Chaiyana, 2018).
2.3.3 Determination of the minimum inhibitory
concentrations (MICs) and minimum
fungicidal concentrations (MFCs)
A broth microdilution method was used to
determine the minimum inhibitory concentrations (MICs) and

580 P. Srisuk et al. / Songklanakarin J. Sci. Technol. 45 (5), 578-584, 2023
minimum fungicidal concentrations (MFCs) of grape seed and
grape skin extracts. Briefly, 50 µl of two-fold serial dilutions
of grape seed and grape skin extracts in SDB were prepared in
a 96-well plate. Then, 50 µl of tested fungal culture was added
into each well to make a final concentration of approximately
106 CFU/ml. In each test, all tested fungi in SDB and P. ovale
in SDB supplemented with 1% w/v of yeast extract and 1%
w/v Tween 80 were used as the positive controls, and broth
alone was the negative growth control. The plates were
incubated at 37 °C for 72 hr. The MICs and MFCs were then
determined. The MFC value was determined by removing 10
µl of the broth from each well and spotting onto the
corresponding agar, then incubating for 72 hr at 25°C for C.
albicans and at 37 °C for the other fungi
(Taweechaisupapong, Ngaonee, Patsuk, Pitiphat, & Khunkitti,
2012).
2.4 In vitro anti-hair loss activity
2.4.1 Human hair follicle dermal papilla cell
(HFDPC) culture
The HFDPCs were purchased from Promo Cell
(Heidelberg, Germany) and cultured in Follicle Dermal
Papilla Cell Growth Medium Kit (Promo Cell) supplemented
with 1% (v/v) penicillin/streptomycin (PS, Gibco BRL). The
growth medium contained growth factors of fetal calf serum
0.04 ml/ml, bovine pituitary extract 0.004 ml/ml, basic
fibroblast growth factor (recombinant human) 1 ng/ml, and
insulin (recombinant human) 5 μg/ml. Dermal papilla cells
were incubated in the appropriate cell culture conditions of
95% relative humidity (RH), 5% CO2, and 37°C. The cells
were sub-cultured until they grew to about 80% confluence
using HEPES buffered saline solution (HBSS), trypsin/EDTA
solution, and trypsin inhibitor solution (Reiter, Pfaffi,
Schönfelder & Meyer, 2009).
2.4.2 Human hair follicle dermal papilla cell
(HFDPC) viability assay
The cytotoxicity of grape skin and grape seed
extracts against human dermal papilla cells was examined
using the PrestoBlueTM cell viability protocol. Briefly, dermal
papilla cells (104 cells/well) were seeded into each well of a
96-well microliter plate and incubated for 24 hr. For this
assay, the stock solutions of 3% w/w grape extracts were
dissolved in HFDPC medium, and 1% minoxidil and 1%
finasteride were dissolved in 30% ethanol. The cells were
treated with 50 µl of samples of grape skin and grape seed
extracts (0.012–0.2 mg/ml) in HFDPC medium, minoxidil
(0.05-0.2 mg/ml), and finasteride (0.05-0.2 mg/ml) in HFDPC
medium (positive control), and with a blank control
containing HFDPC medium only. After 24-hr incubation, 10
µl of PrestoBlueTM reagent in HFDPC medium was added to
each well and incubated at 37°C for 2 hr. The absorbance was
determined at 570 nm using a Multiskan MS microplate
reader (Thermo Electron Corp., Waltham, MA, USA) at 0, 30,
60, 90, and 120 min. The % cell viability was calculated as
follows:
% cell viability =
Abssample
x 100,
Absblank
where Absblank is the absorbance of cells in the medium, and
Abssample is the absorbance of cells treated with the sample in
the medium.
2.4.3 Determination of remaining testosterone in
human hair follicle dermal papilla cell
(HFDPC)
The 5α-reductase activity was determined by means
of remaining testosterone using the method modified from
McCoy and Ziering (2012). Dermal papilla cells (104
cells/well) were placed into 96-well plates and incubated for
24 hr at 95% RH, 5% CO2, and 37°C. In accordance with the
cell viability assay, the cell treatments (50 µl) were divided
into the following groups: grape skin and grape seed extracts
(0.012–0.2 mg/ml) in HFDPC medium, minoxidil (0.05-0.2
mg/ml) and finasteride (0.05-0.2 mg/ml) in HFDPC medium
(positive control), and blank control containing HFDPC
medium only. Testosterone was dissolved in dimethyl
sulfoxide (DMSO) to a concentration of 40 ng/ml and a 100 µl
aliquot was added to each well except for the blank control.
After 24 hr of incubation, the supernatant was collected. The
Testosterone Human Elisa Kit Assay was carried out
following the Abcam protocol. The absorbance was measured
at 450 nm using a microplate reader.
3. Results and Discussion
The antimicrobial assay of grape extracts conducted
by agar diffusion method against five fungal strains is shown
in Table 1. Overall, the agar diffusion assay revealed
comparable zones of inhibition for 3% w/w ethanolic grape
extracts and 0.2% ketoconazole and 0.25% selenium sulfide
against all fungal strains. The ethanolic grape seed extract was
able to inhibit all fungal strains with an inhibition zone in the
range of 22.17 ± 0.94 mm to 23.76 ± 0.01 mm and appeared
closely similar to 0.2% ketoconazole (22.13 ± 0.47 – 23.67 ±
1.01 mm) and 0.25% selenium sulfide (23.40 ± 0.13 – 24.76 ±
1.03 mm) except against P. ovale that showed a significantly
smaller inhibition zone (20.60 ± 0.22 mm) compared to both
reference standards (22.73 ± 0.01 mm and 23.12 ± 0.85 mm,
respectively). Moreover, a significantly higher antifungal
activity of ethanolic grape seed extract compared to ethanolic
grape skin extract was found against C. albicans (22.17 ± 0.94
mm and 18.18 ± 0.34 mm, respectively), and M. canis (23.08
± 0.32 mm and 19.84 ± 0.03 mm, respectively) (p<0.05).
Other grape extracts demonstrated significantly smaller
inhibition zones (p<0.05).
The antifungal activities (MICs and MFCs) of 3%
w/w grape skin and 3% w/w grape seed extracts compared to
0.2% ketoconazole and 0.25% selenium sulfide are shown in
Table 2. In accordance with the results of the agar diffusion
assay, the ethanolic extracts of grape skin and seed showed
greater antifungal activity compared to aqueous and acetone
extracts against all fungal strains, with MIC/MFCs in the
range of 0.25 – 1 μl/ml and mg/ml, repectively. Ethanolic
extract of grape skin exhibited the lowest MIC of 0.25 μl/ml
and MFCs in the range of 0.5 – 0.75 mg/ml against T.
mentagrophytes, and M. gypseum, while ethanolic extract of
grape seed performed similarly against C. albicans, T.
mentagrophytes, M. gypseum, and P. ovale. The MICs against

P. Srisuk et al. / Songklanakarin J. Sci. Technol. 45 (5), 578-584, 2023 581
Table 1. Antifungal efficacy of 3% w/w grape skin extract and 3% w/w grape seed extract by agar diffusion method
Grape part
Extraction solvent
Inhibition zone (mm)
C. albicans
T. mentagrophytes
M. gypseum
M. canis
P. ovale
Skin
Water pH 3
16.48 ± 0.12a
16.50 ± 0.63a
16.44 ± 0.27 a
17.88 ± 0.61a
18.04 ± 1.00ab
Ethanol
18.18 ± 0.34b
22.22 ± 0.54bc
22.16 ± 0.65bc
19.84 ± 0.03b
19.72 ± 0.24ac
Acetone
16.40 ± 0.55a
16.10 ± 0.42 a
17.56 ± 1.00 a
16.30 ± 0.55a
16.28 ± 0.01b
Seed
Water pH 3
19.73 ± 1.00b
20.12 ± 1.20b
20.04 ± 1.21b
19.80 ± 0.66b
18.18 ± 1.11ab
Ethanol
22.17 ± 0.94c
23.72 ± 1.00c
23.76 ± 0.01c
23.08 ± 0.32c
20.60 ± 0.22c
Acetone
16.05 ± 0.01a
16.12 ± 0.10a
18.40 ± 0.54a
18.54 ± 1.00b
18.48 ± 1.01a
Ketoconazole 0.2%
22.13 ± 0.47c
22.41 ± 0.22c
23.67 ± 1.01c
22.82 ± 0.21c
22.73 ± 0.01d
Selenium sulfide 0.25%
23.40 ± 0.13c
24.72 ± 0.54c
24.76 ± 1.03c
24.08 ± 1.12c
23.12 ± 0.85d
Different superscripts in the same column indicate significant differences (p < 0.05) between grape skin and grape seed extracts and between each
reference shampoo product using one-way ANOVA with Tukey’s multiple comparisons. Reported values are mean ± SD of triplicate assay for
each sample.
Table 2. MIC and MFC of 3% w/w grape skin and 3% w/w grape seed extracts
Grape
part
Extraction
solvent
C. albicans
T. mentagrophytes
M. gypseum
M. canis
P. ovale
MIC*
(μl/ml)
MFC**
(mg/ml)
MIC
(μl/ml)
MFC
(mg/ml)
MIC
(μl/ml)
MFC
(mg/ml)
MIC
(μl/ml)
MFC
(mg/ml)
MIC
(μl/ml)
MFC
(mg/ml)
Skin
Water pH 3
0.5
1.0
0.75
1.0
0.75
1.0
1.0
1.0
0.75
1.0
Ethanol
0.5
1.0
0.25
0.5
0.25
0.75
0.75
0.75
0.5
0.75
Acetone
0.5
1.0
0.5
0.5
0.5
0.75
0.75
1.0
0.5
0.75
Seed
Water pH 3
0.75
0.75
0.5
1.0
0.5
1.0
0.5
1.0
0.75
1.0
Ethanol
0.25
0.75
0.25
0.5
0.25
0.75
0.5
0.5
0.25
0.5
Acetone
0.25
0.75
0.5
0.75
0.25
0.75
0.5
0.75
0.25
0.75
Ketoconazole 0.2%
0.125
0.25
0.25
0.25
0.125
0.25
0.25
0.25
0.25
0.25
Selenium sulfide 0.25%
0.125
0.25
0.25
0.5
0.125
0.25
0.125
0.25
0.125
0.25
* MIC = Minimum Inhibitory Concentration
** MFC = Minimum Fungicidal Concentration
T. mentagrophytes (0.25 μl//ml) of both ethanolic grape
extracts were the same as those of 0.2% ketoconazole and
0.25% selenium sulfide, while the MFCs was 0.25% for
selenium sulfide only (0.5 mg/ml). Overall, both grape
extracts showed antifungal activities comparable to those of
the reference standards with ethanolic grape seed extract being
slightly superior to grape skin extract. The effectiveness of
antifungal activity for both grape extracts ranked in
descending order was as follows: T. mentagrophytes, M.
gypseum, C. albicans, P. ovale, and M. canis.
This is in accordance with other studies on the
antifungal activity of Vitis vinifera. A study evaluating the
antifungal activity of ethanol/water grape seed extract (7:3
v/v) showed significant inhibition of C. albicans in an
experimental murine model of vaginal candidiasis, which was
correlated with the high content of polyphenols, particularly
flavan-3-ols (Simonetti et al., 2014). In other studies, the
antifungal activities of grape seed extracts against
dermatophytes of T. mentagrophytes, M. gypseum, and
Malassezia furfur were reported and found to be greater with
ethanol/water grape seed extract compared to ethanol or
methanol alone (Simonetti et al., 2017). In a recent study,
Vitis vinifera juice extract loaded on chitosan nanoparticles
showed an antifungal effect against C. albicans and
Aspergillus niger and demonstrated complete wound-healing
after 7 days on experimental rats with wounded skin fungal
infection (Elshaer, Elwakil, Eskandrani, Elshewemi, &
Olama, 2022). In addition, in our previous study, it was found
that both grape skin and seed extracts showed antibacterial
activity against Staphylococcus aureus, Staphylococcus
epidermidis, Bacillus subtilis, and Escherichia coli (Khawee
et al., 2015). These results indicate that grape extracts, as a
high polyphenol resource, are effective antimicrobial agents
that can find applications in cosmetic and pharmaceutical hair
products. Since ethanol was the most efficient solvent for
achieving the highest yield of polyphenols, as well as
antioxidant and antimicrobial activities in our previous and
current studies, the ethanolic extracts were selected for further
studies on human follicle dermal papilla cells (Khawee et al.,
2015). The cytotoxicity of ethanolic grape skin and grape seed
extracts on HFDPC at various concentrations (0.012 – 0.6
mg/ml) was assessed in comparison to minoxidil and
finasteride reference standards (Figure 1). Cell proliferation of
HFDPC treated with both grape extracts increased in a dose-
depended manner and exceeded 100% at all concentrations
compared to untreated control cells. Cell proliferation
increased by up to 128% and 138% after treatment with
concentrations as low as 0.012 mg/ml of grape seed and grape
skin extracts, respectively. Treatment of HFDPC with 0.3
mg/ml of grape skin extract and 0.06 mg/ml and 0.3 mg/ml of
grape seed extract significantly increased cell proliferation

582 P. Srisuk et al. / Songklanakarin J. Sci. Technol. 45 (5), 578-584, 2023
Figure 1. Cell proliferation assessment of grape skin and grape seed
extracts compared to minoxidil and finasteride as reference
standards. The reported values are expressed as a
percentage of control and presented as mean ± SD of the
triplicate assay for each sample. Data with different letters
have a significant difference (p<0.05) between different
concentrations within the sample. Data with different
symbols have a significant difference (p<0.05) between
samples and reference standards.
(167%, 187%, and 204%, respectively) compared to other
concentrations within the type of extract (p<0.05). This result
indicates that both grape extracts not only preserved the
viability of the cells but also elevated dermal papilla cell
proliferation.
In an earlier study, grape seeds’ proanthocyanidins
increased the proliferation of mouse hair follicle cells and
converted the hair cycle in mice from the telogen phase to the
anagen phase, potentially through effects on signal
transduction pathways (Takahashi et al., 1998). Moreover,
epigallocatechin-gallate (EGCG), commonly found in flavan-
3-ol, in green tea and grape seeds improved hair growth in
hair follicles ex vivo and increased the proliferation of HDPCs
in vitro (Kwon et al., 2007).
In accordance with other studies, minoxidil
increased cell proliferation of HFDPC in a dose-dependent
manner at all concentrations (Chaksupa, Sookvanichsilp,
Soonthornchareonnon, Moongkarndi, & Gerdprasert, 2022).
However, for finasteride, the results were inconsistent with the
recent study, where HFDPC cell viability was exceeding 80%
after treatment with finasteride in the concentration range of
0.01-100 M (Rattanachitthawat, Pinkhien, Opanasopit,
Ngawhirunpat, & Chanvorachote, 2019). In contrast, in this
study, treatment with concentrations of 0.05 – 0.1%
finasteride reduced HFDPC cell viability to 61% and 68%,
respectively, while 0.2% finasteride maintained cell viability
in a non-cytotoxic range above 80% compared to untreated
cells. Although there was no explanation for this phenomenon
in other studies, we do not rule out that the discrepancy in
results could be due to the different methodologies, reagents,
and cell resources used in studies.
In androgenetic alopecia, miniaturization of the
dermal papilla is associated either with the direct action of
testosterone or with conversion by the enzyme 5α-reductase to
the more potent 5α-DHT. The action of these androgens on
human hair follicles causes inhibition of dermal papilla cell
proliferation and induction of programmed cell death by
androgens (Sawaya & Price, 1997). Therefore, the experiment
using the Testosterone ELISA kit was used as an indirect
method to identify the conversion of testosterone to DHT by
5α-reductase produced by dermal papilla cells (Figure 2).
Testosterone was added to the tested grape extracts and
reference standards at an initial concentration of 40 ng/ml, and
the remaining testosterone content was measured after its
conversion to DHT as a factor responsible for hair loss. The
effectiveness of the sample in inhibiting the activity of 5α-
reductase can be assessed by the amount of testosterone
remaining. It was found that both grape skin and seed extracts
at all concentrations inhibited 5α-reductase to a similar extent
and were comparable to finasteride. Moreover, both grape
extracts at concentrations of 0.06 mg/ml and 0.3 mg/ml
demonstrated significantly higher effectiveness in inhibition
of 5α-reductase compared to minoxidil (p<0.05). These results
can be explained by the difference in the mechanisms of
action of the two drugs. While finasteride selectively inhibits
type II 5α-reductase, minoxidil dilates the blood vessels of the
scalp, promoting blood flow to the hair follicles without
affecting hair follicle enzymes, which makes the effectiveness
in this context limited (Chandrashekar et al., 2015; Mata et
al., 2020).
Figure 2. Testosterone remaining in hair follicle dermal papilla cells after treatment with ethanolic grape seed and skin extracts in comparison to
minoxidil and finasteride. The results indirectly represent inhibition of 5α-reductase activity and conversion of testosterone to
dehydrotestostetone (DHT). Data with different letters have a significant difference (p<0.05) between different concentrations within
the sample. Data with different symbols have a significant difference (p<0.05) between samples and reference standards.

P. Srisuk et al. / Songklanakarin J. Sci. Technol. 45 (5), 578-584, 2023 583
Although minoxidil and finasteride are still at the
forefront of conventional therapeutic agents used to treat
alopecia, their side effects have prompted the search for
effective alternative therapies with limited side effects among
plant-based resources. To the best of our knowledge, this is
the first study to show the inhibitory activity of grape extracts
on 5α-reductase tested in HFDPC. However, there were
several studies reporting the potent inhibiting capacity of
EGCG and other flavonoids containing catechol moieties
against 5α-reductase (Hiipakka, Zhang, Dai, Dai, & Liao,
2002; Kwon et al., 2007). The proposed mechanism of
competitive 5α-reductase inhibition might be explained by the
interaction of catechol groups in flavonoids with amino acid
residues in the carboxyl-terminal portion of the protein
important for binding of nicotinamide adenine dinucleotide
phosphate (NADPH) by 5α-reductase. Other mechanisms of
inhibition associated with their ability to form complexes with
certain metal ions and proteins, as well as their antioxidant
and pro-oxidant activities are unlikely (Hiipakka et al., 2002).
These findings suggest that grape extracts have the potential
to be applied to cosmetic products for hair loss.
4. Conclusions
In this study, ethanol was found to be superior to
water and acetone solvents in terms of the bioactivities of
grape skin and seed extracts. Moreover, both ethanolic grape
extracts exhibited excellent antifungal activity against T.
mentagrophytes, M. gypseum, C. albicans, P. ovale, and M.
canis, ranked in descending order. In addition, a promising
anti-hair loss activity through an increase in the proliferation
of HFDPC and indirectly assessed 5α-reductase inhibition
were discovered for grape skin and seed extracts showing
their potential as alternatives to conventional hair loss
medications. The results of this study imply that grape skin
and seed extracts may be useful in the development of hair
products with multifaceted benefits including anti-hair loss
and antifungal properties. However, human studies are
required to validate their effectiveness.
Acknowledgements
The authors are grateful to the Faculty of
Pharmaceutical Sciences, Khon Kaen University, Thailand for
technical support.
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