pISSN 2466-1384 eISSN 2466-1392
大韓獸醫學會誌 (2016) 第 56 卷 第 2 號
Korean J Vet Res(2016) 56(2) : 97~101
http://dx.doi.org/10.14405/kjvr.2016.56.2.97
<Original Article>
Hair growth promoting effects of emodin in telogenic C57BL/6 mice
Jung-Min Yon1, Seul Gi Park1, Chunmei Lin2, Lee Wha Gwon1, Jong-Geol Lee1, In-Jeoung Baek3,
Beom Jun Lee1, Young Won Yun1, Sang-Yoon Nam1,*
1
College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University,
Cheongju 28644, Korea
2
College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 130-118, China
3
Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul 05505, Korea
(Received: May 2, 2016; Revised: May 24, 2016; Accepted: May 30, 2016)
Abstract : Emodin is an anthraquinone derivative from the roots of Rheum officinale Baill that possesses a variety
of biological activities, including inhibition of 5α-reductase and prostaglandin D2. In this study, we investigated whether
emodin promotes hair growth. After emodin was topically applied to the shaved dorsal skin of telogenic C57BL/6
N mice, the hair growth rate and morphological analysis were evaluated in dorsal skin for 15 days. After 13 days
of treatment, minoxidil or emodin (0.01% or 0.1%)-treated groups showed remarkable regrowth of hairs relative to
the vehicle control group. Scoring of the hair growth and rate of hair growth area for 15 days revealed that groups
treated with minoxidil and 0.1% emodin were significantly higher than the vehicle control group. Histological
examination revealed the emodin and minoxidil groups markedly recovered the number and morphology of hair follicles,
including the subcutis depth, relative to the vehicle group. These results suggest that emodin has an excellent promoting
effect in hair growth similar to that of minoxidil and might be useful for treatment of baldness or alopecia.
Keywords : C57BL/6 mice, alopecia, emodin, hair growth
dant in a bald scalp than a haired scalp, especially for
patients with androgenetic alopecia, and that it had the capacity to decrease hair lengthening [5, 11].
Emodin (from Rheum emodi, a Himalayan rhubarb), 6methyl-1,3,8-trihydroxyanthraquinone, which is the most
common natural anthraquinone, occurs in the roots and rhizomes of several higher plants, fungi, and lichens [19]. It has
been reported that emodin possesses various biological activities, including anti-inflammatory, anti-bacterial, and anticancer activities. Emodin also inhibits the 5α-reductase activity
and reduces serum PGD2 levels in a dose-dependent manner
[3, 9, 17, 20], suggesting that it would be a good candidate for
prevention of androgenetic alopecia.
The number of men and women who suffer hair loss and/
or hair thinning are increasing in accordance with changes in
lifestyle and nutritional balance. Therefore, it is important to
develop new materials to prevent hair loss and promote hair
growth. However, despite a variety of activities of emodin,
the hair growth promoting effects of emodin are currently
unknown. In this study, we examined the hair growth promoting effects of emodin in C57BL/6 mice that were synchronized in the telogen stage.
Introduction
Hair loss is a common and distressing symptom that
affects men and women of all ages. Hair loss, which has a
variety of causes including androgenetic alopecia, radiotherapy, chemotherapy, and nutrition, occurs when the normal
cycle of hair growth changes. Hair growth is controlled by a
unique repetitive cycle comprised of anagen, catagen and telogen phases. Therefore, hair follicles undergo cyclic changes
between phases of rapid growth (anagen), regression (catagen), and resting (telogen). Normally, hair follicles are contracted after the anagen phase and the hair shaft falls out
during the catagen or telogen phase. As the follicles begin a
new anagen phase, it grows back to its original size and produces new hair of normal thickness [13, 18].
Androgenetic alopecia is the most common cause of hair
loss affecting both men and women. The 5-α reductase related
to androgenetic alopecia is responsible for converting testosterone into dihydrotestosterone (DHT). Because DHT shortens
the growth phase of the follicles and size of the hair follicles, it is considered to be a potent agent at triggering hair
growth and/or hair loss [7, 8, 21]. Recent studies have reported
that prostaglandin D2 (PGD2) was abnormally more abun*Corresponding author
Tel: +82-43-261-2596, Fax: +82-43-271-3246
E-mail: [email protected]
97
98
Hair growth effect of emodin
Materials and Methods
Chemicals
Emodin was purchased from Sigma Chemical Co. (St,
Louis, MO, USA) and dissolved in 50% ethanol. We used
two concentrations of emodin (0.01% and 0.1%) in ethanol.
In addition, 5% minoxidil as the positive control was purchased from Hyundai Pharmaceutical (Korea).
Animal experiments
Male C57BL/6 mice (5 weeks old) were purchased from
KOATECH (Korea). The animals were housed in polysulfone cages in a well-ventilated room maintained at 21 ± 2oC
and 55 ± 10% relative humidity under a 12 hour light/dark
cycle. The animals were fed standard mouse chow (Samyang, Korea) and provided tap water ad libitum. All experiments were approved by the Chungbuk National University
Animal Care Committee and conducted according to the
Guide for Care and Use of Animals (Chungbuk National
University Animal Care Committee, CBNUA-469-13-02). To
produce new hairs, existing follicles undergo cycles of growth
(anagen), regression (catagen) and rest (telogen). The skin of
C57BL/6 mice in a resting telogen phase is a pale pink color
and skin color of an active hair growth anagen phase becomes
dark gray or black. Hair growth stops at catagen phase and
the skin transitions are back to the telogen phase, returning to
a pale pink color. During anagen phase, hair follicles produce an entire hair shaft from tip to root, but during catagen
and telogen phases, the follicles become to regress and lie
dormant in a resting phase [1, 10]. After confirming homogeneously-pink skin color in the back skin of C57BL/6 mice in
a telogen phase, the dorsal skin hair was shaved in this study.
The animals were randomly allotted to the experiment, and
then subdivided into 4 groups: 50% ethanol (vehicle control), minoxidil, 0.01% emodin, and 0.1% emodin. Starting
the following day (1 day), 200 µL of each sample was topically applied daily for 15 days.
Photographs of hair growth states
At 1, 4, 7, 10, 13, and 15 days after topical application,
Fig. 1. Hair growth promoting effects of emodin. Telogen-matched C57BL/6J mice (6 weeks old) were shaved and topically treated
with vehicle (50% ethanol), 5% minoxidil, or emodin (0.01% and 0.1%). The back skins were photographed at 1, 7, 10, 13, and 15
days after depilation under anesthesia with diethyl ether. After 13 days, hair growth in the minoxidil, 0.01% emodin, and 0.1% emodintreated groups was accelerated relative to the vehicle control group.
Jung-Min Yon, Seul Gi Park, Chunmei Lin, Lee Wha Gwon, Jong-Geol Lee, In-Jeoung Baek, Beom Jun Lee, Young Won Yun, Sang-Yoon Nam 99
photographs of each group of mice were taken while the animals were anesthetized under diethyl ether.
Hair growth scoring
Promotion of hair growth was evaluated by observing the
skin color, which is indicative of the telogen to anagen conversion [13]. The hair growth effect was morphometrically
calculated using the following hair growth score: score 0, no
hair growth; score 1, less than 30% growth; score 2, 30% to
less than 50% growth; score 3, 50% to less than 70% growth;
score 4, 70% to 100% growth [12]. The rate of hair growth
area to clipped area was (hair growth area/clipped area) ×100.
Histological examination
Mice were euthanized with diethyl ether, after which the
dorsal skin tissue was extracted. At 7 and 15 days, mice were
sacrificed to obtain dermal skin specimens. Skin samples
were removed, fixed in 10% neutral buffered formalin and
processed according to routine histological techniques. Paraplast-embedded tissues were sectioned to a thickness of 4 µm,
then stained with hematoxylin and eosin (H&E).
Statistical analysis
Statistical differences between groups were analyzed by
one-way analysis of variance (ANOVA) followed by Tukey’s
multiple comparison test. Statistical significance was established at p < 0.05. All data were expressed as the mean ± SE.
Results
Change in hair growth
Emodin application did not show any adverse effects in
dorsal dermal skin for the duration of the experiment. The
vehicle control group revealed a low recovery in hair growth.
However, the skin colors in groups treated with minoxidil,
0.01% emodin, and 0.1% emodin began to change to light
gray after 7 days, and on 10 days the skin color had changed
from pink to black in all groups of mice. After 13 days, hair
growth in the minoxidil, 0.01% emodin, and 0.1% emodintreated groups was accelerated relative to the vehicle control
group (Fig. 1).
Hair growth scores
As shown in Table 1, the dorsal skin of mice was hairless
on days 1 to 10 (hair growth score: 0) in all groups. However, at day 13, hair growth was significantly higher in the
minoxidil and 0.1% emodin groups than the vehicle group.
Hair growth scores in the minoxidil, 0.01% emodin, and
0.1% emodin groups were significantly elevated relative to
the vehicle group for 15 days (p < 0.05).
Rate of hair growth area to clipped area
At day 13, hair growth rates in the minoxidil (57 ± 8.5%)
and 0.1% emodin (53 ± 8.9%) groups were significantly higher
than in the vehicle group (34 ± 7.3%). At day 15, minoxidil
Fig. 2. Rate of hair growth area to denuded area in C57BL/6J
mice after topical application of emodin. At day 15, minoxidil,
0.01% emodin, and 0.1% emodin applications had significantly
enhanced hair growth relative to vehicle alone (p < 0.05). The
rate of hair growth area to clipped area was (hair growth area /
clipped area) ×100. The results are shown as the mean values ±
SE (*p < 0.05 vs. vehicle).
Table 1. Scores of hair growth in C57BL/6 mice after topical
application of emodin for 15 days
Vehicle
Day
Day
Day
Day
Day
Minoxidil 0.01% emodin 0.1% emodin
1
0
0
7
0
0
10
0
0
13 1.5 ± 0.33 2.7 ± 0.39*
15 2.8 ± 0.34 3.8 ± 0.17*
0
0
0
2.2 ± 0.59
3.3 ± 0.29*
0
0
0
2.4 ± 0.40*
3.6 ± 0.23*
Hair growth scoring index: no hair growth (0), < 25% hair growth
(1), 25–50% hair growth (2), 50–75% hair growth (3), and 75–
100% hair growth (4). The results shown are the mean values ± SE
(*p < 0.05 vs. vehicle).
(86 ± 3.9%), 0.01% emodin (74 ± 6.6%), and 0.1% emodin
(85 ± 5.3%) applications significantly enhanced hair growth
relative to the vehicle alone (60 ± 7.9%) group (p < 0.05; Fig. 2).
Histological examination
An increase in the number and size of hair follicles is considered an indicator of the transition of hair growth from the
telogen to the anagen phases [16]. An increase of the size
and number of hair follicles can be observed in the deep subcutis during the anagen phase [4]. To investigate the progression of hair follicles in the hair cycle, H&E staining was
conducted. At day 7, the hair follicles in the 0.1% emodin
group had progressed to the anagen stage, whereas those in
the other groups remained in the telogen stage. At day 15, the
emodin and minoxidil groups showed markedly increases in
the number and morphology of hair follicles, including the
subcutis depth, relative to the vehicle group (Fig. 3).
100
Hair growth effect of emodin
Fig. 3. Hair follicle growth in telogen-matched C57BL/6J mice treated with emodin. At day 15, the emodin and minoxidil groups
showed markedly increased number and morphology of hair follicles including the subcutis depth relative to the vehicle group. The
effects of 0.01% and 0.1% emodin on the hair follicles in mice were analyzed using hematoxylin-eosin staining. Longitudinal sections
of the back skins (7 and 15 days) were stained, and the image shown is a representative picture of mice. 100×.
Discussion
Hair loss is caused by many factors ranging from genetics
to the environment. Recently, the number of men and women
suffering from hair loss has increased dramatically. Although
hair loss disorder is not life-threatening, it is emotionally distressing to the patient and causes afflicted individuals to have
withering during daily social interactions. Therefore, it is of
great importance to develop new materials to prevent hair
loss and promote hair growth.
Minoxidil is widely used for the treatment of hair loss and
is available without a prescription. This compound effectively promotes hair growth by dilating blood vessels and
opening potassium channels in both males and females with
androgenic alopecia [6, 14]. It is generally well maintained,
but side effects including severe allergic reaction, hypertrichosis, and temporary hair loss have been reported [2, 15].
Recently, herbal medicines have received increased attention
owing to their hair promoting potential with no side effects.
In this study, we examined the hair growth promoting effects
of emodin in C57BL/6 mice.
Emodin showed no adverse side effects during 15 days of
dermal application to the mice. The color of dermal skin was
light gray in the emodin (0.01% and 0.1%) treated groups on
day 7 after depilation. At day 13, the emodin (0.01% and 0.1%)
groups surpassed vehicle group in hair growth activity, similar to the minoxidil group. Furthermore, the 0.1% emodin
group had significantly higher hair growth and rate of hair
growth area than the vehicle group after 13 days. In the representative longitudinal sections, increasing hair follicles and
a thickened epidermis were observed in the emodin treated
groups compared with the vehicle group. These findings
indicate that emodin has hair growth promoting activity similar to minoxidil in rodent models.
Jung-Min Yon, Seul Gi Park, Chunmei Lin, Lee Wha Gwon, Jong-Geol Lee, In-Jeoung Baek, Beom Jun Lee, Young Won Yun, Sang-Yoon Nam 101
Androgenetic alopecia, which is known to be the most
common cause of hair loss, is associated with increased DHT
levels. Five alpha-reductase, which is produced in many tissues of males and females, participates in steroid metabolism that leads to conversion of testosterone into DHT [3, 7,
21]. PGD2 also inhibits hair growth of patients with androgenetic alopecia and has the capacity to decrease hair lengthening [5, 11]. Emodin possesses antioxidative activities including
increased superoxide dismutase (SOD) activity, and SOD1,
SOD2, and cytosolic glutathione peroxidase mRNA as well
as anti-inflammatory activities. Also, emodin inhibited 5αreductase by the hydroxyl group and attenuated the PGD2
activities by blocking NF-κB, a major transcription factor for
COX-2 in a dose dependent manner [3, 9, 22]. Therefore,
based on the results of the present study, we can infer that
emodin may act via inhibitions of 5α-reductase and PGD2
activities, as well as through antioxidative and anti-inflammatory activities. In this study, we demonstrated morphologically that two doses of emodin (0.01% or 0.1%) promote the
hair growth in dorsal skin of the shaved telogenic mice.
In conclusion, emodin significantly promoted hair growth
with no side effects in a mouse model. It is possible that the
hair follicles were stimulated to enter into anagen by emodin
application, resulting in shortening of the time required for
full growth. Although emodin was found to have a definite
hair growth promoting effect in this study, further investigations are needed to clarify its molecular mechanism of action.
6.
7.
8.
9.
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Acknowledgments
15.
This work was supported by the Basic Science Research
Program (2013R1A2A2A03016519 and 2014R1A1A3052735)
and the Priority Research Centers Program (2015R1A6A1
A04020885) through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education, Science
and Technology and by a research grant from Chungbuk
National University in 2013.
16.
17.
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