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Hair regenerative activities of flavonoid-rich extract of Equisetum hyemale L. (Equisetaceae) in chemically-induced alopecia in Sprague Dawley rats

Authors:

Abstract

Context: The prevalence of alopecia around the world is high and awareness continuously increased due to social stigma. There are limited commercially-available medications for the management of different forms of baldness, most of which are prohibitively expensive and presents with various adverse effects. Aims: To evaluate the flavonoid-rich ethyl acetate extract of Equisetum hyemale stem for its hair-regenerative properties in chemically-induced alopecia in Sprague Dawley rats. Methods: Various concentrations of the flavonoid-rich extract of E. hyemale were applied in depilated areas in dorsal rat skin after chemical induction of alopecia. Evidence of hair growth was observed for 28 days in a weekly basis. Histopathological analysis of rat skin was performed to demonstrate evidence of follicular hair growth. Results: The abundance of flavonoids in the ethyl acetate extract was established by total flavonoid contents and LC-MS analyses. The 2.5% E. hyemale extract exhibited hair-regenerative properties with high tensile strengths, combined masses and follicular growth, which was comparable to the positive control, 5% minoxidil (p>0.05). The highest rate of hair follicular development was observed in the telogen phase in rat skin after 28 days of treatment. Conclusions: The flavonoid-rich ethyl acetate extract of E. hyemale exhibits in situ hair-regenerative properties in chemically-induced alopecia in rats at 2.5% w/v concentration.
© 2019 Journal of Pharmacy & Pharmacognosy Research, 7 (5), 323-330, 2019
ISSN 0719-4250
http://jppres.com/jppres
Original Article | Artículo Original
_____________________________________
Hair regenerative activities of flavonoid-rich extract of Equisetum
hyemale L. (Equisetaceae) in chemically-induced alopecia in Sprague
Dawley rats
[Actividad regeneradora del cabello de extracto rico en flavonoides de Equisetum hyemale L. (Equisetaceae) en
alopecia inducida químicamente en ratas Sprague Dawley]
Penuel P. David1,2*, Cecilia D. Santiago1,3, Anges L. Castillo4, Gerard Q. De Guzman1,3,5,6,7
1Graduate School, Centro Escolar University, Manila, Philippines.
2Department of Pharmacy, Centro Escolar University, Malolos, Philippines.
3School of Pharmacy, Centro Escolar University, Manila, Philippines.
4Faculty of Pharmacy and the Graduate School, University of Santo Tomas, Manila, Philippines.
5College of Pharmacy and Graduate School, Adamson University, Manila, Philippines.
6Francisco Q. Duque Medical Foundation, College of Medicine, Lyceum Northwestern University, Dagupan, Pangasinan, Philippines .
7The Graduate School, Virgen Milagrosa University Foundation, San Carlos, Pangasinan, Philippines.
*E-mail: pdavid@ceu.edu.ph
Abstract
Resumen
Context: The prevalence of alopecia around the world is high and
awareness continuously increased due to social stigma. There are
limited commercially-available medications for the management of
different forms of baldness, most of which are prohibitively expensive
and presents with various adverse effects.
Aims: To evaluate the flavonoid-rich ethyl acetate extract of Equisetum
hyemale stem for its hair-regenerative properties in chemically-induced
alopecia in Sprague Dawley rats.
Methods: Various concentrations of the flavonoid-rich extract of E.
hyemale were applied in depilated areas in dorsal rat skin after chemical
induction of alopecia. Evidence of hair growth was observed for 28 days
in a weekly basis. Histopathological analysis of rat skin was performed
to demonstrate evidence of follicular hair growth.
Results: The abundance of flavonoids in the ethyl acetate extract was
established by total flavonoid contents and LC-MS analyses. The 2.5% E.
hyemale extract exhibited hair-regenerative properties with high tensile
strengths, combined masses and follicular growth, which was
comparable to the positive control, 5% minoxidil (p>0.05). The highest
rate of hair follicular development was observed in the telogen phase in
rat skin after 28 days of treatment.
Conclusions: The flavonoid-rich ethyl acetate extract of E. hyemale
exhibits in situ hair-regenerative properties in chemically-induced
alopecia in rats at 2.5% w/v concentration.
Contexto: La prevalencia de alopecia en todo el mundo es alta y la
conciencia aumenta continuamente debido al estigma social. Existen
medicamentos disponibles pero limitados para el tratamiento de
diferentes formas de calvicie, la mayoría de ellos son muy caros y
presentan diversos efectos adversos.
Objetivos: Evaluar el extracto de acetato de etilo, rico en flavonoides, del
tallo de Equisetum hyemale por sus propiedades regenerativas del cabello
en la alopecia inducida químicamente en ratas Sprague Dawley.
Métodos: Se aplicaron diversas concentraciones del extracto rico en
flavonoides de E. hyemale en áreas depiladas en la piel dorsal de rata
después de la inducción de la alopecia. La evidencia de crecimiento del
pelo se observó semanalmente durante 28 días. El análisis
histopatológico de la piel de rata se realizó para demostrar la evidencia
de crecimiento folicular del pelo.
Resultados: La abundancia de flavonoides en el extracto de acetato de
etilo se estableció mediante el contenido total de estos y el análisis LC-
MS. El extracto de E. hyemale al 2,5% mostró propiedades de
regeneración del cabello con altas resistencias a la tracción, masas
combinadas y crecimiento folicular, que fue comparable al control
positivo, 5% de minoxidil (p> 0.05). La mayor tasa de desarrollo folicular
del cabello se observó en la fase telógena en la piel de rata después de 28
días de tratamiento.
Conclusiones: El extracto de acetato de etilo, rico en flavonoides, de E.
hyemale exhibe propiedades regenerativas del cabello in situ en la
alopecia inducida en ratas a una concentración de 2,5% p/v.
Keywords: alopecia; Equisetum hyemale; flavonoids; hair-regenerative;
horsetail.
Palabras Clave: alopecia; cola de caballo; Equisetum hyemale; flavonoides;
regenerador del cabello.
ARTICLE INFO
Accepted: June 29, 2019.
Available Online: July 8, 2019.
David et al.
Philippines Equisetum hyemale as potential hair grower
http://jppres.com/jppres
J Pharm Pharmacogn Res (2019) 7(5): 324
INTRODUCTION
Alopecia is one of the most frequent dermato-
logical disorders with universal prevalence, affect-
ing more males than females at a ratio of 2:1
(Fricke and Miteva, 2015). The only FDA-approved
medication available for the management of hair
loss includes finasteride, dutasteride and minox-
idil which are costly and presents with numerous
adverse effects (Yamada et al., 2013). There was,
therefore, a need to outsource naturally-derived
drugs from plants which exhibit hair-regenerative
properties. Equisetum is a genus of ferns known as
horsetails, which consist of 15 species (Purcell,
2018). Several species were proven to have hair
grower activities, e.g., Equisetum arvense (Pingale et
al., 2016) whilst the Philippine horsetail plant, E.
hyemale, is gaining attention to the hair-growth
stimulatory properties of its flavonoid-rich extracts
(Al-Snafi, 2010; Jiang et al., 2014). This research
aimed to screen the abundance of flavonoids in
ethyl acetate stem extract of E. hyemale and to
screen if this extract demonstrates hair-
regenerative properties in animal model of alope-
cia.
MATERIAL AND METHODS
Chemicals
Hexane, ethyl acetate, methanol and tween 80
were purchased from Belman Laboratories Que-
zon City, Philippines. Minoxidil 5% solution (Re-
grow, Derm Pharma Inc, Pasig City, Philippines)
and Veet hair removal cream were procured at
Mercury Drugstore Malolos City, Philippines.
Quercetin, NaNO2, AlCl3, NaOH, formic acid and
acetonitrile were provided by the University of the
Philippines (UP), Manila.
Plant material and extraction
Whole plant of E. hyemale was randomly col-
lected from Bulacan, Philippines (GPS 14.887058,
120.785283). A voucher specimen of the plant was
verified by Mr. John Rey C. Collado, a botanist at
the Botany Division of the Philippine National
Museum with a control number of 15-07-124. The
stems were air-dried after removal of unwanted
particles by hand picking, cut into small pieces
and grounded to course powder using a blender
(Magic Bullet 400W, Homeland Housewares,
USA). About 250 g portions of the dried material
were separately macerated with exhaustion with
hexane, ethyl acetate, methanol and distilled wa-
ter. Combined hexane, ethyl acetate and methanol
extracts were concentrated in vacuo (Heodolph,
Schwabach, Germany) and then air-dried while
combine aqueous extracts were lyophilized (KD
Freeze Dryer, Zhengzhou Keda Machinery, Main-
land, China) (Kumar et al., 2011; Hossain et al.,
2013).
Flavonoids evaluation
The presence and abundance of flavonoids in
the ethyl acetate extract of E. hyemale was estab-
lished and termed flavonoid-rich extract of E. hye-
male (FREEh) by the quantification of total flavo-
noids and liquid chromatographic mass spectral
(LC-MS) analysis (Asha and Kumar, 2015; Khati-
wora et al., 2010).
Analysis for total flavonoid was performed at
the Institute of Pharmaceutical Sciences National
Institute of Health UP Manila, Philippines with
control number of IPSNP 18-139. This assay facili-
tates standardization of the FREEh for its total fla-
vonoid contents in terms of the standard querce-
tin, based on the oxidative coupling and condensa-
tion of phenolic groups of individual flavonoid
moieties (i.e., flavanols, proanthocyanin, anthocy-
anins, flavones, flavonones, chalcones and their
glycosides) by trivalent aluminum. Briefly, exactly
one mL of the FREEh, prepared as one mg/mL in
methanol, and each of the standard solutions of
quercetin in methanol (0 - 100 μg/mL), were dilut-
ed with four mL of distilled water in a 10-mL vol-
umetric flask, followed by the addition of 0.3 mL
of 5% NaNO2; after five minutes, 0.3 mL of 10%
AlCl3 was added. After six minutes, two mL of 1
M NaOH was added and then water was finally
added to volume and then mixed. Absorbance
(Beckman Coulter DU 730 UV/Vis Spectropho-
tometer, China ) were read at 510 nm and the total
flavonoids were measured as quercetin in mg/g,
David et al.
Philippines Equisetum hyemale as potential hair grower
http://jppres.com/jppres
J Pharm Pharmacogn Res (2019) 7(5): 325
thus: total flavonoids = C × 10, where C was the
concentration of quercetin obtained by linear re-
gression analysis of the calibration curve that plots
quercetin concentrations and absorbance values
while 10 was a dilution factor (Khatiwora et al.,
2010).
The FREEh was submitted and analyzed at the
Biochemistry Department, College of Medicine,
UP Manila, Philippines for LC-MS (Waters Xevo
G2-XS QToF, Massachusetts, USA) analysis using
an ACQUITY HSS T3 C18, 1.8 µm, 2.1 x 100 mm
and a mobile phase consisting of phase A: water +
0.1% formic acid and phase B: acetonitrile + 0.1%
formic acid at a flow rate of 0.4 mL/min, a volt-
age: 40 V Source at 120°C at a gas flow of 40 L/h;
desolvation temperature was set at 550°C at a gas
flow of 950 L/h and a scan range of 50 - 1,200 m/z;
scan time was approximately 0.150 seconds at a
collision energy of 15 to 50 eV. Leucine enkephalin
was used as an internal reference compound for
mass correction; data were processed using a Wa-
ters UNIFI Scientific Information System v1.8.1.073
Library and Waters Traditional Chinese Medicine
Library, which allows for the putative identifica-
tion of separated molecules based on their reten-
tion times and molar masses (Annex 1), which are
monitored by a library fingerprint (Meng et al.,
2018).
Evaluation of hair-regenerative properties
The protocol for screening of hair regenerative
properties in rats was approved by the Institution-
al Animal Care and Use Committee (IACUC) with
reference number of IACUC 2017-18/071. Healthy
male Sprague Dawley rats, weighing at least 200 g,
were purchased from the Department of Science
and Technology (DOST) in Bicutan, Taguig City
Philippines. Rats were acclimatized at the animal
holding facilities of Centro Escolar University
(CEU) Malolos City Bulacan Philippines for seven
days at 22 ± 3°C and 55-75% RH with 10 - 15 cycles
of air and an alternating 12-hour light and dark
cycle. Rats were housed in rectangular transparent
plastic cage with an adequate space and holes to
breathe in and to allow ad libitum feeding with
standard pellets and water. Soft wood shavings
(Chipsi, Rosenberg, Germany) were used to pro-
vide beddings, which were changed daily as the
need arises.
The in situ screening method used in the study
was a modification of the methods used by Ad-
hirajan et al. (2003), Kurup et al. (2013), Allayle et
al. (2012) and Lanzafame et al. (2013). Sprague
Dawley rats were randomly distributed into five
groups (n=5 rats per group); the first three groups
were treated with 2.5%, 5% and 10% concentration
of the predetermined flavonoid-rich extract in
0.5% Tween 80. Minoxidil 5% solution and 0.5%
Tween 80 were used as positive and negative con-
trols, respectively, and were administered to the
4th and 5th groups, respectively.
A commercially-available hair remover (Veet,
Reckitt Benckiser, Maharashtra, India) was topical-
ly applied into the dorsal skin of the rats, covering
an area of four x four cm2 where template was
used to ensure uniformity of size of the bald areas.
Exactly 0.5 mL portions of the samples were ap-
plied once daily, for 28 days, using an applicator.
Caution was observed to ensure evenness of appli-
cation in the bald areas. The growth of hair in each
bald region was evaluated by visual observations
and recorded using photographs (Vivo Y53, Ma-
nila, Philippines) at weekly basis after daily topical
application of the samples (Young Oh et al., 2014).
On the seventh, 14th and 28th day, ten strands of
hair were plucked randomly from each one cm2
bald area for measurement of hair length using a
digital micrometer (Mitutoyo, Illinois, USA)
whereas the combined mass were weighed accu-
rately using analytical balance (Denver Instrument
SI-234 Colorado, USA). For tensile strength analy-
sis, ten strands of hair per treatment group were
randomly plucked after 28 days of treatment and
mounted manually by supporting the ends of each
hair with mechanical weights, which are incre-
mentally added up to the maximum weight that
can be held. The evaluation of hair regenerating
effects was based on the scoring index used by
Young Oh et al. (2014), as follows: one = 0 - 19%
growth; two = 20 - 39% growth; three = 40 - 59%
growth; four = 60 - 79% growth and five = 80 -
100% growth.
David et al.
Philippines Equisetum hyemale as potential hair grower
http://jppres.com/jppres
J Pharm Pharmacogn Res (2019) 7(5): 326
The percentage growth was calculated as fol-
lowed:
[1]
Histopathological study
After 28 days, the rats were sacrificed and the
dermal skin samples were fixed in 10% buffered
formalin, followed by paraffin wax embedding
and stained with hematoxylin-eosin (Belman La-
boratories Quezon City, Philippines) and observed
under digital microscope with LCD screen (Stein-
dorff, New York, USA) at 400x magnification. The
number, elongation and depth of hair follicular
growth, graded according to the scale by Young
Oh et al. (2014), thus: five = presence of fully de-
veloped hair with shafts, four is characterized by
developing hairs in dermal, epidermal and slightly
above epidermal layer; three noticed the presence
of developing hair in both dermal and epidermal
layers; two indicated presence of developing hair
in the dermal layer, while one indicated absence of
any hair.
Statistical analysis
Data are compared by the one-way analysis of
variance (ANOVA), the two-tailed t-test and the
90% confidence interval using SPSS software (Sta-
tistical Package for the Social Sciences, version
20.0, SPSS Inc, Chicago, IL). Replicate analyses are
expressed as means ± standard error of the mean.
P<0.05 was considered significant level.
RESULTS AND DISCUSSION
From among the hexane, ethyl acetate, metha-
nol and aqueous extracts of E. hyemale stem, the
ethyl acetate extract was designated as the flavo-
noid rich extract of E. hyemale (FREEh) based on
the quantification of total flavonoids registered
quercetin equivalent of 57.14 ± 2.51 mg/g (n = 5), a
quantity that is comparatively high based on the
quercetin equivalent, at 17.47 ± 0.25 mg/g, of the
ethanolic extract of E. arvense, another equisetum
species with known hair grower properties (Mim-
ica-Dukic et al., 2008).
LC-MS separation of the FREEh afforded the
identification of 27 substances (Table 1). Most of
these substances are flavonoids, mostly flavones
and chalcones, as well as their derivatives that
include two glycosides.
Chemically-induced alopecia in Sprague Daw-
ley rats were used to screen the FREEh for its in
situ hair regenerative properties after 28 days of
treatment (Figs. 1 - 4). There were converse dose-
response relationship responses when various
concentrations of the FREEh were tested in situ in
chemically-induced alopecia in rats based on mean
tensile strengths (i.e., r = -0.0567) and hair mass
(i.e., r = - 0.9588). In this case, 2.5% concentration
was significantly more effective than higher con-
centrations. This is due to the phenomenon called
ceiling effect, where increasing dose or concentra-
tions of a drug produces progressively small in-
cremental therapeutic effects (Richardson and
Raymond, 2018). This is possible when both ago-
nists and antagonists are present in the sample.
This mixed agonist-antagonist may affect -
reductase inhibitory properties or any phases of
the hair growth cycle in chemically-induced alope-
cia in rats (Bhasin et al., 2012). These can be due to
saturation of receptors, which include the -
reductase enzymes found within hair follicles in
bald areas during alopecia, by bioactive secondary
metabolites present in the sample, such as flavo-
noids (Hiipakka et al., 2002). The advantage of
these findings is based on achieving optimum
therapeutic effects at lower concentrations without
posing risks of adverse effects associated with
higher concentrations. Usually, these adverse ef-
fects occur long after the desired outcome from
therapeutic ceiling concentrations are achieved
(Gal, 2009). The popular tradition in the manage-
ment of hair loss is by using botanical products as
it lowers incidence of side effects as observed in
synthetic products, recent studies showed that
herbal and synthetic hair growers were combined
producing synergistic effects (Keaney et al., 2016).
CONCLUSIONS
This study demonstrated the in situ efficacy of
2.5% of the flavonoid-rich ethyl acetate stem ex-
David et al.
Philippines Equisetum hyemale as potential hair grower
http://jppres.com/jppres
J Pharm Pharmacogn Res (2019) 7(5): 327
tract of E. hyemale as a hair-regenerative agent in
chemically-induced alopecia in rats as observed in
a weekly basis for 28 days, which showed evi-
dences of higher hair tensile strengths, combined
masses and follicular growth.
In the present study, ethnomedical knowledge
on the use of E. hyemale as hair grower has been
experimentally validated. Furthermore, the au-
thors recommend a thorough study on the mecha-
nism of action of FREEh on its hair growth activi-
ties.
Figure 1. Mean hair tensile strength after 28 days of treatment
with flavonoid-rich extract of E. hyemale in Sprague Dawley
rats.
Data represent mean ± SEM (n=5). Significant differences (**p<0.05)
were detected with respect to 5% FREEh, 10% FREEh and the positive
control 5% minoxidil where as a *p>0.05 were detected with respect
to 2.5% FREEh and 5% minoxidil.
Figure 2. Mean hair weight in mg within 1 cm2 after 28 days
of treatment with flavonoid-rich extract of E. hyemale in
Sprague Dawley rats.
Data represent mean ± SEM (n=5). Significant differences (**p<0.05)
were detected with respect to 5% FREEh, 10% FREEh and the posi-
tive control 5% minoxidil where as a *p>0.05 were detected with
respect to 2.5% FREEh and 5% minoxidil.
Figure 3. Mean hair tensile strength after 28 days of treatment
with flavonoid-rich extract of E. hyemale in Sprague Dawley
rats.
Data represent mean ± SEM (n=5). Significant differences (**p<0.05)
were detected with respect to 5% FREEh, 10% FREEh and the positive
control 5% minoxidil where as a *p>0.05 were detected with respect
to 2.5% FREEh and 5% minoxidil.
David et al.
Philippines Equisetum hyemale as potential hair grower
http://jppres.com/jppres
J Pharm Pharmacogn Res (2019) 7(5): 328
2.5% FREEh
5% FREEh
10% FREEh
0.5% Tween 80
5% Minoxidil
Figure 4. Representative histological
photomicrographs of depilated skin within 28
days of treatment with flavonoid-rich extract
of E. hyemale in Sprague-Dawley rats.
The blue arrows are evidence of hair growth where the hair follicles and shaft are visible. Scale by Young Oh et al. (2014): 5 = presence of fully
developed hair with shafts (e.g., 5% minoxidil or positive control and 2.5% FREEh); 4 is characterized by developing hairs in dermal, epidermal
and slightly above epidermal layer (e.g., 2.5% FREEh); 3 notices the presence of developing hair in both dermal and epidermal layers(e.g., 5%
FREEh) ; 2 indicates presence of developing hair in the dermal layer (e.g., 10% FREEh); while 1 indicates absence of any hair (e.g., 0.5% Tween
80 or negative control). Magnification 400x.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGMENTS
This research work did not receive any grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Authors wish to thank their families and school, Centro Esco-
lar University, in their continued support and understanding
in order for this study be materialized.
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AUTHOR CONTRIBUTION:
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Santiago CD
Castillo AL
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Citation Format: David PP, Santiago CD, Castillo AL, De Guzman GQ (2019) Hair regenerative activities of flavonoid-rich extract of Equisetum
hyemale L. (Equisetaceae) in chemically-induced alopecia in Sprague Dawley rats. J Pharm Pharmacogn Res 7(5): 323330.
David et al.
Philippines Equisetum hyemale as potential hair grower
http://jppres.com/jppres
J Pharm Pharmacogn Res (2019) 7(5): 330
Annex 1. LC-MS chromatographic spectra of flavonoid-rich extract of E. hyemale.
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