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Hair Growth Promoting Effect of Urticadioica L
Khaliun Myagmar1, 2, Enkhsaikhan Lkhagvasuren2, Munkhbayar Semchin1
1Institute of Medical Sciences, Mongolian National University of Medical Sciences, Ulaanbaatar, Mongolia; 2Department of Microbiology and Immunology, School of
Biomedicine, Mongolian National University of Medical Sciences, Ulaanbaatar, Mongolia;
Objectives: We investigated the effect of Urticadioica L extracts on hair growth by using
in-vitro and ex vivo study methods. Methods: Human single hair follicle and dermal papilla
cells obtained from scalp skin samples of healthy volunteers. We evaluated the effect of
Urticadioica L on hDPCs and on ex vivo hair follicle organ culture. Hair follicle matrix cell’s
proliferation marker Ki-67 identied by immunourescence staining. Results: Urticadioica L
ethanol extracts promoted elongation of the hair shaft and reduced catagen transition of human
hair follicles in organ culture model. E.extract of Urticadioica L increased Ki-67 positive matrix
keratinocytes. Conclusions: Urticadioica L ethanol extract enhanced human hair growth in
ex vivo organ culture model. Future study is needed related to its mechanism of hair growth.
Keywords: Dermal Papilla Cells (hDPCs), Hair Follicle, Urdicadioica L, Ki-67
Submitted: August 30, 2018
Revised: September 10, 2018
Accepted: October 9, 2018
Corresponding Author
Munkhbayar Semchin MD, PhD
Institute of Medical Sciences,
Mongolian National University of
Medical Sciences, Ulaanbaatar
16081, Mongolia
Tel: +976-88028860
E-mail:
munkhbayar.ims@mnums.edu.mn
This is an Open Access article distributed
under the terms of the Creative Commons
Attribution Non-Commercial License (http://
creativecommons.org/licenses/bync/4.0/)
which permits unrestricted non-commercial
use, distribution, and reproduction in any
medium, provided the original work is
properly cited. Copyright© 2018 Mongolian
National University of Medical Sciences
Introduction
Stinging nettle (UrticadioicaL ) belongs to the family Urticaceae
[1]. About 50 species of Stinging nettle (Urticadioica) are grown
in the world. Three species of Urticaceae (Urtica cannabina,
Urtica angustifolia, Urtica dioica L) grow in Mongolia [2].
Urticadioica L is a herbaceous perennial owering plant that
native to Europe, Asia, northern Africa and North America. The
maximum typical height of this plant species ranges from 2 to
4 meters [3]. It produces pointed leaves and white to yellowish
owers [4]. The plant has many hollow stinging hairs called
trichomes on its leaves and stems, which act like hypodermic
needles, injecting histamine and other chemicals that produce
a stinging sensation when contacted by humans and other
animals [3]. Flavonoids, tanins, scopoletin, sterols, fatty acids,
polysaccharides, isolectins andsterols are phytochemicals which
are reported from this plant [5].
The whole plant is used in folk medicine to treat allergies,
kidney stones, burns, anemia, rashes, internal bleeding,
diabetes, etc. However only a few of these pharmacological
activities have been experimentally proved [6]. Stinging nettle
extracts has been different studies reported to have various
pharmacological antioxidant, anti-microbial, anti-inammatory,
anti-ulcer, anticolitis and analgesic [7, 8]. As well as this plant
Original Article
Cent Asian J Med Sci. 2018 Sep;4(3):187-193. https://doi.org/10.24079/CAJMS.2018.09.004
30 July, 2018
10 August, 2018
9 September, 2018
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Hair Growth Promoting Effect of Urticadioica L
has been also used in human nutrition such as food or tea and
also gathered commercially due to high content of chlorophyll,
which is used as green coloring agent (E140) in food and
medicines [5]. Despite the fact that science has not been proven
its effectiveness, Mongolians have been using extracts from this
plant to prevent hair loss and as a hair loss treatment.
Hair loss problem have approximately 2% of the world
population [9]. According French scientists, one out of every
ve people have hair loss [10]. Depending on the clinical form,
approximately 78% of men and 57% of women over the age of
80 are affected by androgenetic alopecia and 58% of men over
the age of 50 are affected [11]. In Mongolia, the prevalence of
hair loss has not yet been studied, but between in 2011-2015
a total of 1325 hair loss cases diagnosed in out-patient clinic of
National Dermatology Center of Mongolia.
The hair follicle (HF) is a very small-organ [12]. In normal
hair growth, the hair follicles cycle through successive phases:
anagen phase of growth, catagen phase of involution, and
telogen phase of rest [13]. The hair grown from the follicle will
pass through the 4 stages of hair cycle to repeat hair growth and
loss [12]. When generalized loss is present it is useful to assess
the ratio of anagen (growing) hair to that of the telogen hair. In
hair loss, there appears to be a dysfunction and disproportion
in hair cycling, resulting in a reduced length of anagen phase,
increased proportion of hair in catagen/telogen phase [14].
Many various factors including stress as social, psychological
and mental, eating habits, and living habits, drugs etc can affect
hair loss [15]. The most common form of human hair loss is
androgenic alopecia.
Alopecia has few physically harmful effects, but may
lead to psychological consequences, including high levels
of anxiety and depression [16]. However, only two drugs so
far have been approved for hair loss treatment by the Food
and Drug Administration (FDA), nasteride and minoxidil [11].
Researchers are still studying low-cost therapeutic methods that
are effectives against hair loss.
This study aimed to determine the effect of Urticadioica
L on human hair growth. To accomplish this, we investigated
the effect of Urticadioica L extracts on hair growth by using
in-vitro and ex- vivo study methods.
Material and Method
Ethical statements
Study protocols were approved by the institutional research
board of Mongolian National University of Medical Sciences [IRB
approval number], and written informed consent was obtained
from all subjects. All experimental procedures using human
tissues were performed according to the principles described in
the declaration of Helsinki.
Human hair follicle samples
A total of 479 human scalp HFs were obtained from 7 different
healthy male volunteers 20 to 40 years of age without current or
prior scalp diseases. The samples were obtained from 1.5x1.0 cm
of scalp from their occipital region yielding greater than 60 HFs
per subject. The samples were carefully dissected into single HFs
under a stereo microscope (Motic K Series). In this study, only
anagen HFs were used.
Preparation of Plant extract
The aerial part of the Urticadioica L was harvested according
to the standards of “A” Pharmaceutical Factory (Ulaanbaatar,
Mongolia). Urticadioica L extract was obtained by adding
water, ethanol, hexane as solvents to macerated method
Urticadioica L at the room temperature for 72 hours [17]. The
solvent and extraction ratio were same (1:10). After ltration
through lter paper, the ltrates were concentrated and dried by
vacuum evaporation and freeze drying method.
Cell culture
As previously described [18, 19], the hair dermal papilla cells
(hDPCs) were cultured at 37°C with 5% CO2 in Dulbecco’s
modied Eagle’s medium (DMEM; Welgene, Daegu, Korea),
supplemented with 10% fetal bovine serum (Welgene), 10 ng/ml
basic broblast growth factor (bFGF; R&D Systems, Minneapolis,
MN, USA), and antibiotic/antimycotic 1x solution (100 mg/ml
streptomycin and 100 U/ml penicillin).
Thiazolylblue (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazoliumbromide) (MTT) assay
Cell viability was measured by the MTT assay. Cells at 1x104
cellsper well were seeded into 96-well plates, serum-starved
for 24 hours, and then treated for 24 hours with the vehicle
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(ethanol at a diluted to 1:1,000 in serum-free DMEM) or with
Uricadioica L extracts (0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml
[nal concentration], diluted to 1:1,000 in serum-free DMEM).
Subsequently, 15 μl of MTT solution was added to each well, and
the plate was incubated for 4 hours at 37°C. The supernatant
containing the MTT was removed, and the formazan crystals in
the wells were dissolved by adding 100 μl of dimethyl sulfoxide.
The samples were nally incubated for 30 minutes at room
temperature before a plate reader enabled quantitative analysis
for viable cells by optical density measurement at 570 nm.
Human hair follicles organ culture and
immunouorescence staining
Isolated human scalp hair follicles were cultured as described
previously [20, 21]. Each HF was cut at the level of the
sebaceous duct, and then cultured for 12 days at 37°C (5%
CO2) in Williams’ E medium (Gibco-BRL laboratories, Grand
Island, NY, USA), supplemented with 10 ng/ml hydrocortisone,
10 μg/ml insulin, 2 mM L-glutamine, and antibiotic/antimycotic
1x solution (penicillin and streptomycin, Gibco-BRL laboratories).
Urticadioica L water, hexane and ethanol extracts was added
to culture medium at nal concentrations of 0.125 mg/ml. At
every third day, elongation of the hair shaft was measured, and
HFs in anagen phase were determined according to their growth.
Immunouorescence staining was performed to evaluate
proliferation of HF matrix keratinocytes. As previously described,
immunoreactivity for Ki-67 (DAKO, Carpinteria, CA, USA) was
used as an indicator of cell proliferation [22]. A DAPI mounting
media kit (Vector Laboratories, Burlingame, CA, USA) was used
to counterstain the nuclei.
Statistical analysis
Statistical signicance was determined using Student’s t-test.
Paired t-test was used to compare the HF culture results. All tests
Figure 1. The viability of cultured human dermal papilla cells (hDPC’s) on treatment of Urdicadioica L extracts. Cells were treated
with Urticadioica L extracts, or vehicle for 24 hours. (A) water extract (W.extract) of Urticadioica L, (B) ethanol extract (E.extract),
and (C) hexane extract (H.extract). The results are expressed as mean ± standard error.
Munkhbayar Semchin
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were two-tailed, and differences with a p-value of 0.05 were
considered statistically signicant. The statistical analyses were
done by using the STATA 13 software package.
Results
Effect of Urticadioica L extracts on DPC proliferation
We performed the MTT assay to evaluate the effect Urticadioica
L extracts on hDPCs’ survival. In this experiment, Urticadioica
L water, ethanol, hexane extracts (0.125 mg/ml) signicantly
enhanced viability of hDPCs compared to the vehicle-treated
controls. However, the hDPCs’ viability decreased at all extracts
concentrations that were greater than 0.250 mg/ml (Figure 1
A,B,C).
Effect of Urticadioica L extracts on matrix cell proliferation
and hair shaft elongation in cultured human hair follicles
We performed in vitro culture of the healthy human scalp
HFs to examine the effect of Urticadioica L extracts at the organ
level. Hair shaft elongation by treating culturing human HFs with
0.125 mg/ml concentration of Urticadioica L water, ethanol
and hexane extracts for 12 days was analyzed. HFs treated with
Urticadioica L ethanol extract signicantly enhanced hair shaft
elongation after 6, 9, and 12 days, compared to the vehicle-
treated controls. As well, Urticadioica L hexane extract grew
longer than HFs treated with vehicle (Figure 2A). Urticadioica
L ethanol extract reduced catagen transition compared to the
vehicle control and other extracts (Figure 2B).
To analyze the proliferation of human HFs matrix cells,
immunouorescence staining was performed for Ki-67 (a
proliferation marker) after HFs were cultured with 0.125 mg/
ml concentration of Urticadioica L water, ethanol and hexane
extracts for 3 days. The number of Ki-67+ matrix keratinocytes
were counted and normalized by using the number of DAPI
positive cells. We found that treatment with Urticadioica L
ethanol extract (0.125 mg/ml) signicantly increased Ki-67+
matrix keratinocytes than vehicle control (Figure 3A and B).
Discussion
The hair follicle (HF) is composed of epidermal (epithelial) and
dermal (mesenchymal) compartments and their interaction has
an important role in the morphogenesis of hair, growth of the
hair follicle, and the hair cycle [23]. The dermal papilla, composed
of specialized broblasts located in the bulb of the hair follicle,
contributes to the control of hair growth and the hair cycle [21].
Single follicle organ culture method, which is an ex vivo
model for the study of freshly isolated human scalp HFs under
serum-free conditions developed by Philpott et al, [22]. This
Figure 2. The effect of Urticadioica L extracts onhair shaft elongation and catagen transition in ex vivo hair follicle organ culture.
HF’s cultured with 0.125 mg/ml concentration of Urticadioica L water, ethanol and hexane extracts for 12 days (n = 7). (A) The
effect of E.extract of Urticadioica L on hair shaft elongation and (B) The effect of E.extract of Urticadioica L oncatagen transition as
compared to the vehicle control. The results were expressed as mean ± standard error. * p ≤ 0.05, vs. the control group. Ctl: Control,
W.extract: Water extract of Urticadioica L, E.extract: Ethanol extract of Urticadioica L, H.extract: Hexane extract of Urticadioica L.
Hair Growth Promoting Effect of Urticadioica L
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Figure 3. The proliferation of matrix keratinocytes in Urticadioica L ethanol extract treated HFs. (A) Human HFs were cultured with
the vehicle or water, ethanol and hexane extracts (0.125 mg/ml) for 3 days, and then subjected to immunouorescence staining, to
examine proliferation in the hair matrix keratinocytes, with Ki-67 (proliferation, green uorescence), and 4’,6-diamidino-2-phenylindole
(DAPI, blue uorescence) to counterstain the nuclei (X200). (B) For quantitative analysis, Ki67+ cells were counted and normalized to
DAPI-stained cells. E.extract of Urticadioica L increased proliferation in the hair matrix keratinocytes. The results were expressed as
mean ± standard error. * p ≤ 0.05, **p≤0.01, vs. the control group. Ctl: Control, W.extract: Water extract of Urticadioica L, E.extract:
Ethanol extract of Urticadioica L, H.extract: Hexane extract of Urticadioica L.
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method, not only compares the single hair shaft elongation
and hair cycle change within the study groups and also has the
advantage using immunohistochemical and immunouorescence
staining evaluate the desired markers related proliferation and
differentiation [24].
Ki-67 immunoreactivity (IR) is a useful tool to obtain
information on cell proliferation in situ, with the signicant
advantage that it can be used on both cryo and parafn
embedded sections [25]. The changes in Ki-67 IR during the hair
cycle transition are well demonstrated. During anagen VI, Ki-67
IR is most prominent in hair matrix keratinocytes and the onset
of catagen is marked by the reduction in the percentage of Ki-
67+ matrix keratinocytes, and no Ki-67+ keratinocytes present
in late catagen [26-28]. Therefore, in our study we also hair
shaft elongation and examined Ki-67 IR by immunouorescence
staining.
Urticadioica L elaborates different classes of organic
compounds of medicinal importance including phytosterols,
saponins, avanoids, tannins, sterols, fatty acids, carotenoids,
chlorophylls, proteins, amino acids, macro, micro-elements and
vitamins [5].
In our study, we conrmed that Urticadioica L ethanol
extracts enhanced hair shaft elongation, reduced catagen
transition and signicantly increased the Ki-67+ matrix
keratinocytes compared with the vehicle control and water and
hexane extracts.
Previous research has shown the Urticadioica L chemical
components which are mainly soluble in ethanol are avonoid,
polyphenol, lectins, sterol, lignans, beta carotene, keton, palmitic
and steric acid, oleic acid, etc [29]. Polyphenols and some
avonoids has been reported to hair growth promoting effect by
the proliferation of hDPCs with ROS scavenging and increasing
of growth factors IGF-1 and VEGF, also conrmed hair re-growth
promotion in mice [30-32].
Our study has some limitation. First, we used only whole
plants and single proliferation marker Ki67. Using additional
proliferation marker could make our study more effective.
Second, our sample size was small.
Further researches are required for nding the effective
molecules or compounds and related mechanism.
We conclude above mentioned components such as
polyphenols and avonoids which are soluble in ethanol of
Urticadioica L may promoteon hair growth.
Conict of Interest
The authors state no conict of interest.
Acknowledgement
This study was supported by Institute of Medical Sciences,
Core Laboratory and Mongolian National University of Health
Sciences.
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