Mechanistic Studies on the Use of Polygonum multiflorum for the Treatment of Hair Graying

Abstract

Polygonum multiflorum
is a traditional Chinese medicine with a long history in hair growth promotion and hair blackening. The purpose of the study was to examine the effect and the mechanism of
Polygonum multiflorum
in hair blackening. C57BL/6 mice hair fade was induced with H
2
O
2
and used in this research. Hair pigmentogenesis promotion activities of Polygonum Multiflorum Radix (PMR, raw crude drug), Polygonum Multiflorum Radix Preparata (PMRP, processed crude drug), and their major chemical constituent TSG were investigated. The regulation effects of several cytokines and enzymes such as POMC,
α
-MSH, MC1R, ASIP, MITF, TYR, TRP-1, and TRP-2 were investigated. PMR group gave out the most outstanding black hair among all groups with the highest contents of total melanin,
α
-MSH, MC1R, and TYR. Promotion of hair pigmentogenesis was slightly decreased after processing in the PMRP group. TSG as the major constituent of PMR showed weaker hair color regulation effects than both PMR and PMRP. PMR, but not PMRP, should be used to blacken hair. The
α
-MSH, MC1R, and TYR were the major targets in the medicinal use of PMR in hair graying. Chemical constituents other than TSG may contribute to the hair color regulation activity of PMR.

Full text

Research Article
Mechanistic Studies on the Use of Polygonum multiflorum for
the Treatment of Hair Graying
Ming-Nuan Han, Jian-Mei Lu, Guang-Yuan Zhang, Jie Yu, and Rong-Hua Zhao
Yunnan University of Traditional Chinese Medicine, Kunming, Yunnan 650500, China
Correspondence should be addressed to Jie Yu; cz.yujie@gmail.com and Rong-Hua Zhao; kmzhaoronghua@hotmail.com
Received  September ; Revised  October ; Accepted  October 
Academic Editor: Rituraj Purohit
Copyright ©  Ming-Nuan Han et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Polygonum multiorum is a traditional Chinese medicine with a long history in hair growth promotion and hair blackening. e
purpose of the study was to examine the eect and the mechanism of Polygonum multiorum in hair blackening. CBL/ mice
hair fade was induced with H2O2and used in this research. Hair pigmentogenesis promotion activities of Polygonum Multiorum
Radix (PMR, raw crude drug), Polygonum Multiorum Radix Preparata (PMRP, processed crude drug), and their major chemical
constituent TSG were investigated. e regulation eects of several cytokines and enzymes such as POMC, 𝛼-MSH, MCR, ASIP,
MITF, TYR, TRP-, and TRP- were investigated. PMR group gave out the most outstanding black hair among all groups with
the highest contents of total melanin, 𝛼-MSH, MCR, and TYR. Promotion of hair pigmentogenesis was slightly decreased aer
processing in the PMRP group. TSG as the major constituent of PMR showed weaker hair color regulation eects than both PMR
and PMRP. PMR, but not PMRP, should be used to blacken hair. e 𝛼-MSH, MCR, and TYR were the major targets in the
medicinal use of PMR in hair graying. Chemical constituents other than TSG may contribute to the hair color regulation activity
of PMR.
1. Introduction
Hair color is instantly recognized during human interactions,
and hair graying is a sign of old age, ill health, and bodily
decline, especially in Asian countries where black is the
most common hair color. ere is a strong desire to be
young and vital, and thus premature graying has attracted
manyresearchers.Atthesametime,thepharmaceutical
or functional food industries are seeking targets for the
prevention and treatment of hair graying. Despite the variety
of products available with diering results and eciencies,
a completely satisfactory solution for hair graying remains
incomplete.
Conditions that aect hair color include aging, achro-
motrichia [], stress [], medical conditions [], and articial
factors []. Hair color is not only aected by genetic, age, and
environmental factors but also aected by many cytokines
and proteins. e generation of melanin is mainly regulated
by POMC (proopiomelanocortin), 𝛼-MSH (𝛼-melanocyte-
stimulating hormone), MCR (melanocortin  receptor),
ASIP (agouti signaling protein), MITF (microphthalmia-
associated transcription factor), TYR (tyrosinase), TRP-
(tyrosinase related protein ), and TRP- (tyrosinase related
protein ) (Figure ).
In traditional Chinese medicine, preparations from Poly-
gonum multiorum have a long history of use for hair
growth and blackening. Both oral and topical administration
of P. mu l t i  o r u m preparations were clinically used in the
treatment of hair graying sometimes simultaneously in tra-
ditional application methods. RPM (Polygonum Multiorum
Radix, raw crude drug) and RPMP (Polygonum Multiorum
Radix Preparata, processed crude drug) were thought to
be herbs that tonify the kidney and liver in traditional
Chinese medicine theory, so that they could be used in the
treatment of early graying of hair. Double blind, placebo-
controlled studies lasting over  months have demonstrated
marked benecial eects of PMR on hair quality for pre-
and postmenopausal women []. Aer taking extracts of P.
multiorum for  and  months, the surveys administered
to the active treatment revealed a signicant improvement
Hindawi Publishing Corporation
BioMed Research International
Volume 2015, Article ID 651048, 8 pages
http://dx.doi.org/10.1155/2015/651048

BioMed Research International
Cysteinyl dopa
Cysteinyl dopaquinone
Pheomelanin
POMC
MC1R
ASIP
MITF
TYR
TRP-1
Tyrosine Dopa Dopaquinone
Dopachrome
Dihydroxyindole carboxylic acid
Dioxoindoline acid
Eumelanin
Total melanin
Inhibitory eect on a particular biological process
Stimulatory eect on a particular biological process
Melano cytes
TRP-2
TRP-1
𝛼-MSH
F : Major proteins and factors involved in the hair pigmentation.
of hair loss ( in  participants, %) and perceived hair
appearance ( in  participants, %). Additionally, % of
womenintheP. m u l t i  o r u m group reported “thicker hair,”
which was rated as “signicant” and “dramatic” improvement
[]. In another study employing , –-year-old men
(𝑛=24) and women (𝑛=24) with diering origins
of hair loss (age-related, stress- and medication induced,
and postpartum) received a standardized extract of PMR
twice daily []. Aer  month of treatment, % of men and
% of women reported improvement. Additionally, none
of the study participants reported any side eects during
thetreatmentperiod.However,themechanismsbywhich
PMR and PMRP exhibit these benecial eects have yet to
be elucidated.
We have studied P. m u l t i  o r u m for decades. In our
previous research [], we found that both oral administration
of PMR and topical administration of PMRP promoted hair
growth. PMR was more suitable for oral administration,
while PMRP showed greater eects in external use. e
hair growth promotion eect of oral PMR was most likely
mediated by the expression of broblast growth factor-
(FGF-). In this research, CBL/ mice hair fade induced by
H2O2was used to investigate hair pigmentogenesis promo-
tion activity and a possible mechanism of PMR and PMRP.
eir extractions were administered orally and/or topically.
Hair pigmentogenesis promotion activities were investigated
by hair color and total melanin contents. e regulation
eects of several cytokines and enzymes such as POMC, 𝛼-
MSH, MCR, ASIP, MITF, tyrosinase, TRP-, and TRP- were
studied here.
2. Materials and Methods
2.1. Plants Materials and Chemicals. e root of Polygonum
multiorum unb.werecollectedinJunebytheauthors
in Luda Rode, Pingshan Town, Luquan County, Yunnan
Province and identied by Professor Ronghua Zhao of
Yunnan University who is a specialist in traditional Chinese
medicine (Specimen number: HMN ). Voucher

BioMed Research International 
O
HO
OH
HO
HO OH
OH
OH
F : Structure of TSG.
specimens were deposited in the Herbarium of Pharmacog-
nosy, Yunnan University of Traditional Chinese Medicine.
TSG (Figure ) was purchased from Nanjing Jingzhu Bio-
Technology Co., Ltd., China. e purity was over %
via a high performance liquid chromatography-diode array
detector.
2.2. Processingand Extraction of PMR and PMRP. PMRP was
steamed from PMR with black soybean decoction according
to the procedure recorded in Pharmacopoeia of the People’s
Republic of China ( edition) []. Black soybean decoc-
tion ( kg) was obtained from  kg black soybeans boiled
with water for two times ( h for the rst time and  h for the
second time).  kg Black soybean decoction was needed in
the steaming of  kg PRM.
e  g of nely ground PMR and PMRP (Figure )
powder was successively extracted with , ., and . L of
water. Extracts were then combined and lyophilized.
2.3. HPLC-DAD Analysis of PMR and PMRP. All experi-
ments were performed with a Dionex Ultimate  HPLC
system (Dionex Technologies, USA). Data were analyzed
with Chromeleon ..
e separations of RPM and RPMP extractions were
achieved on a Zorbax SB-C analytical column (. mm ×
 mm, ID,  𝜇m particle diameter from Agilent Technolo-
gies, USA).
e gradient elution used a mobile phase consisting of
(A) .% H3PO4and (B) methanol. e following gradient
program was used: % B (at the start), % ( min), %
( min), % ( min), % (– min), and then back
to % ( min). e detection wavelength was  nm.
e oven temperature was ∘C,andtheowratewas
. mL⋅min−1.
References st andards of TSG were accurately weighed and
dissolved in methanol. Extracts of PMR and PMRP were both
weighted accurately and dissolved in  mL of % methanol.
A𝜇L injection was used for all analyses.
2.4. Animals and Treatments. Fiy six-week-old CBL/
male mice (weighed 20 ± 2g) were provided by Beijing
HFK Bioscience Co., Ltd. ey were housed ten per cage
in stainless steel cages containing sterile paddy husk as
T:Animalgroupingandtreatments.
Groups Hair color
fading reagent Tre a tmen ts
Drug-delivery route
and dosage (g/kg)
Oral Topical
A—Physiological
saline ——
BH2O2Physiological
saline ——
CH2O2TSG . .
DH2O2PMR . .
EH2O2PMRP . .
bedding in ventilated animal rooms (temperature 22 ± 1∘C;
60 ± 10% humidity; and a  h/ h light/dark cycle) with
free access to water and a commercial laboratory complete
food. All animal experiments were performed in compliance
with the animal experimental ethics committee of Yunnan
University of Traditional Chinese Medicine (R-).
All reasonable eorts were made to minimize the animals’
suering.
e mice were randomly assigned to  groups (𝑛=
10) (Table ) aer adaptive feeding for three days. Group A
was the control group with physiological saline given orally.
Group B had nothing other than .% H2O2solution on
its back fur for six consecutive weeks. is served as the
modelgroup.e.%H
2O2solution was spread with
a cotton swab on back fur of all mice except for the control
group every morning. Six hours later, all mice were fasted for
 h. en, TSG (. g/mL in water), PMR (. g/mL
in water), and PMRP (.g/mL in water) were given by
gavage to mice in groups C, D, and E (. mL/ g). In the
meantime, TSG (. mg/mL in water), PMR (. mg/mL
in water), and PMRP (. mg/mL in water) were rubbed in
with a cotton swab on the back fur for topical given. e doses
of PMR and PMRP were conversed from the recommended
doses of RPM and RPMP in the Pharmacopoeia of the
People’s Republic of China,  edition []. e dose of
TSG was calculated from its concentration in PMR. No
appropriate positive drug was used because there is no
approved therapy for hair graying.
2.5. Investigation of Hair Growth Color Regulation Activities.
Hair samples of all mice were collected of the same group on
the last day of the experiment. All collected hair samples in
the group were immediately combined, vortexed, and washed
with  mL ddH2O twice to prevent color interference from
the extracts or compounds. en,  mg of hair were weighed
and placed in glass test tube and  mL water and  mL organic
solvent Soluene- (Soluene-, a strong organic solvent
that could dissolve a variety of tissues such as hairs) were
added to each test tube []. e mixtures were heated twice
for min, with a short cooling period in between. e
absorbance of the supernatants at  nm was measured for
three times with a UV-Vis spectrophotometer (UV-H,
Unico Instrument Co. Ltd., Shanghai).

BioMed Research International
(a) (b)
F : Photographs of raw (a) and processed (b) Polygoni Multiori Radix.
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
(mAU)
(min)
TSG
WVL: 254 nm
−15
(a)
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
TSG
WVL: 254 nm
−15
(min)
(mAU)
(b)
F : HLPC proles of PMR (a) and PMRP (b).
2.6. Assessment of Related Proteins and Growth Factors in
Skin. Mice skin was collected at the end on the last day
of the experiment. Skin samples were disinfected with %
ethanol and transferred to liquid nitrogen for storage aer
depositing immediately in −∘Cforhandthenskin
samples were weighed and washed with .% saline before
use.  mg skin samples in each group were cut into pieces
and homogenated on ice with  mL physiological saline.
e homogenates were centrifuged for  min at ∘C. e
POMC, 𝛼-MSH, MCR, MITF, ASIP, TYR, TRP-, and TRP-
 contents in the supernatant were measured by Elisa kits
(Cusabio Biotech Co., Ltd., and Bio-Swamp Life Science Lab).
2.7. Statistical Analysis. All data were expressed in the form
of 𝑋±SD.edatawereevaluatedbyone-wayanalysisof
variance (ANOVA) when multiple group comparisons were
performed. Relationships between variables were assessed
with Pearson’s correlation coecient.
3. Results
3.1. TSG Content in Tested Preparations. Figure  shows
HPLC-UV proles of water extracts of PMR and PMRP. e
peak eluting at . minutes was identied as TSG by com-
parison of the retention time and UV spectra with authentic
standards. Linear relationships between the injected amount
and the peak area were observed. e TSG concentration in
PMR extract was .. However, its concentration reduced
to . mg/g in PMRP aer processing. Because it was an
abundant constituent of both PMR and PMRP [], we tested
this compound individually.
3.2. Hair Color Regulation Eects. Obvious decolorization
wasobservedinmicehairaerH
2O2treatment (Figure (a)).
Mice hair in the model group was a red-brown color but
pure black in the control group mice. e total melanin
content was signicantly lower in the model group aer
fading with H2O2(Figure (b)).However, this fading could
be reversed by treatment with PMR and PMRP. e total
melanincontentswere.%and.%higherinthePMR
and PMRP group than in the model group.
Photomicrographs of mice hair in all groups demonstrate
their color alteration (Figure (c)). e red-brown mixed
with black could be observed only in model group, while all
other groups were darker.

BioMed Research International 
(a)
ABCDE
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Groups
Total melanin (A500)
(b)
(A) (B) (C) (D) (E)
(c)
F : Hair color (a), total melanin contents (b), and microscopic images (c) of mice hair at the end of the research. (a) Mice appearances
in control (le) and model (right) group. Obvious decolorization was observed in mice hair aer H2O2treatment. (b) Average total melanin
content in a group was measured with a UV-Vis spectrophotometer. (c) Photomicrographs of mice hair in all groups demonstrate their color
alteration. Mice were in control (A) or untreated (B), TSG (. g/kg and .g/kg, oral and topical) (C), PMR (. g/kg and . g/kg,
oral and topical) (D), and PMRP (. g/kg and . g/kg, oral and topical) (E) groups.
3.3. Hair Color Regulation Mechanisms. Concentrations of
hair color-related proteins and factors could be correlated
directly to the generation of melanin. erefore, we studied
major proteins and factors involved in hair pigmentation.
Figures (a)–(c) show that aer treatment with the color
fading reagent (.% H2O2solution) the expression of 𝛼-
MSH, MCR, and TYR decreased signicantly and that all
the treatments could reverse this eect. We found that the
eect of PMR was the most prominent. e expression of 𝛼-
MSH,MCR,andTYRinthePMRgroupincreasedby,,
and  times versus the model group, which were even higher
thanthecontrolgroup.ePMRPcouldalsoincreasethe
expression of 𝛼-MSH, MCR, and TYR; however, the eect
was slightly weaker than the PMR.
Expression of TRP- and TRP- proteins was not sig-
nicantly aected by .% H2O2solution (Figures (d)
and (e)). eir changes aer treatment were not obvious
except that the expression of TRP- increased slightly in
the PMRP group. No signicant dierences were observed
in POMC, MITF, and ASIP among all groups (Figure S in
Supplementary Material available online at http://dx.doi.org/
.//).

BioMed Research International
ABCDE
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
###
#
#
Groups
𝛼-MSH concentration (pg/mL)
∗∗
∗
∗
(a)
ABCDE
0
200
400
600
800
1000
1200
1400
###
###
###
MC1R concentration (pg/mL)
Groups
∗∗∗
∗∗
(b)
ABCDE
0
1000
2000
3000
4000
5000
6000
7000
8000
##
###
#
TYR concentration (pg/mL)
Groups
∗∗∗
∗∗∗
(c)
ABCDE
0
100
200
300
400
500
600
700
TRP-1 concentration (pg/mL)
Groups
(d)
ABCDE
0
50
100
150
200
250
300
350
400
450
##
TRP-2 concentration (pg/mL)
Groups
(e)
F : 𝛼-MSH (a), MCR (b), TYR (c), TRP- (d), and TRP- (e) concentrations in skins of dierent groups. (X ±SD, 𝑛=10). Mice were
in control (A) or untreated (B), TSG (. g/kg and .g/kg, oral and topical) (C), PMR (.g/kg and . g/kg, oral and topical) (D),
and PMRP (. g/kg and . g/kg, oral and topical) (E) groups. e 𝛼-MSH, MCR, TYR, TRP-, and TRP- contents in the skin tissue
were measured by Elisa kits. e  indicates a signicant dierence compared with untreated hair graying group. #𝑝< 0.05;##𝑝< 0.01;
###𝑝< 0.001.e∗indicates a signicant dierence compared with control group. ∗𝑝< 0.05;∗∗𝑝< 0.01;∗∗∗𝑝< 0.001.

BioMed Research International 
T : Pearson’s correlation coecients between total melanin
and related proteins and enzymes.
𝛼-MSH MCR TYR TRP- TRP-
Total
Melanin 𝑟 = 0.936∗𝑟 = 0.9155∗𝑟 = 0.989∗∗ ——
∗𝑝<.; ∗∗𝑝<..
3.4. Relationship Analysis between Total Melanin and Protein
Expression. e relationships between total melanin contents
and expressions of 𝛼-MSH, MCR, TYR, TRP-, and TRP-
 were presented in Table . As expected, the total melanin
contents were positively correlated with the expression of 𝛼-
MSH (𝑟 = 0.936,𝑝 < 0.05), MCR (𝑟 = 0.9155,𝑝 < 0.05),
and TYR (𝑟 = 0.989,𝑝 < 0.01).ePMRgrouphadthemost
outstanding black hair among all groups with the highest
total melanin content. At the same time, the expressions of 𝛼-
MSH, MCR, and TYR aer treatment with PMR increased
to levels even higher than before H2O2-induced fading.
4. Discussions
Human hair color is regulated by multiple factors (Figure ),
such as activation of the POMC gene [, ], expressions
of 𝛼-MSH [, ], binding activities of 𝛼-MSH to MCR
[], agonists of MITF, and antagonists of ASIP [, ].
Downregulation of TYR activity, which is the rate-limiting
enzymeforcontrollingtheproductionofmelanin[,],
has been proposed to be responsible for reduced melanin
production. In addition, expression or activity variations of
TRP- and TRP- are eventually aecting the production
of melanin [–]. However, underlying molecular and
biochemical mechanisms of graying remain under debate.
erefore, there is no positive control drug because there is
no approved therapy for hair graying.
Apart from various hair dyes of varying ecacy and
duration, fully satisfactory solutions for the graying problem
remain to be brought to market. erefore, searching for
eective and safe hair graying prevention and treatment
drugs from traditional Chinese medicine or natural products
has enormous social and economic benets.
Recent in vivo research indicated that human gray/white
scalp hair shas accumulate hydrogen peroxide (H2O2)in
millimolar concentrations. FT-Raman spectra showed in
vivo thepresenceof
−3 mol/L H2O2concentrations in
gray and completely white hair. e in vivo identication
of massive H2O2concentrations (determined by FT-Raman
spectroscopy) in the gray hair sha introduced a new step in
the understanding of human hair graying on the biochemical
and molecular biological level []. erefore, CBL/ mice
hair fade induced by H2O2wasusedinthisresearchinorder
to simulate human hair gray status.
In this research, our results provided justication for the
traditional use of P. m u l t i  o r u m against hair graying. We
found that treatment with PMR could completely reverse the
hair decolorization induced by H2O2.ePMRgrouphad
the most outstanding black hair among all groups with the
highest content of total melanin, 𝛼-MSH, MCR, and TYR.
e 𝛼-MSH, MCR, and TYR were the major targets for
the medicinal use of PMR in hair graying. 𝛼-MSH, MCR,
and TYR were all critical factors that aect the formation of
melanin from tyrosine via dopa (Figure ). However, these
eects were weaker in the PMRP group. erefore, PMR
shouldbeusedforhairblackening,butnotPMRP.
e doses of PMR and PMRP in this research were
conversed from the recommended doses of RPM and RPMP
in the Pharmacopoeia of the People’s Republic of China, 
edition. Fortunately, there was no adverse eect reports under
these recommended doses. erefore, the doses used in our
research were considered to be safe.
TSG showed hair color regulation eects that were weaker
than both PMR and PMRP. As we mentioned before, TSG is
major chemical constituent of P. m u l t i  o r u m before and aer
processing.WeconcludethatTSGmightpartlycontribute
to hair color regulation and that constituents other than
TSG are responsible for the hair color regulation activity of
PMR. e active chemical constituent(s) should be identied
in future work; this may be a promising area of future
research. Meanwhile, whether oral or topical application
ismoreimportantshouldalsobeclariedinthefuture
research.
5. Conclusion
Rationality of traditional use of P. m u l t i  o r u m against hair
grayingfromancienttimesisprovidedinthisresearch.
Extracts of P. mu l t i  o r u m ,especiallyfromrawcrudedrug,
could completely reverse the hair decolorization induced
by H2O2.Expressionsof𝛼-MSH, MCR, and TYR are
upregulated and then more melanin is produced as a conse-
quence.Inourresearch,PMR,butnotPMRP,showedbetter
eect on hair blackening. However, further clinical investi-
gations were still needed to provide more solid and scientic
evidences.
Conflict of Interests
e authors declare that there is no conict of interests. ey
declarethattheyhavenonancialandpersonalrelationships
withotherpeopleororganizationsthatcaninappropriately
inuence their work; there is no professional or other per-
sonal interest of any nature or kind in any product, service,
and/or company that could be construed as inuencing the
position presented in this paper.
Acknowledgments
is research was nancially supported by the National
Natural Science Foundation of China (Grant no. ,
Grant no. , and Grant no.  ), the Natural Sci-
ence Foundation of Yunnan Province (Grant no. FA
and Grant no. FD), and the Southern Medicine
Collaborative Innovation Center (Grant no. ).
e authors thank LetPub (http://www.letpub.com/) for its
linguistic assistance during the preparation of this paper.

BioMed Research International
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