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44 Vol. 37, No. 1Biol. Pharm. Bull. 37(1) 44–53 (2014)
© 2014 The Pharmaceutical Society of Japan
Regular Article
In Vivo Hair Growth-Promoting Effect of Rice Bran Extract Prepared
by Supercritical Carbon Dioxide Fluid
Jae-Suk Choi,a Min-Hee Jeon,b Woi-Sook Moon,b Jin-Nam Moon,b Eun Jin Cheon,c
Jo o -Wa n K im,d Sung Kyu Jung,e Yi-Hwa Ji,e Sang Wook Son,*,e and Mi-Ryung Kim*,c
a RIS Center, IACF, Silla University; c Department of Bio-Food Materials, Silla University; Sasang-gu, Busan
617–736, Republic of Korea: b Department of R&D, ECOMINE Co., Ltd.; Nam-gu, Busan 608–736, Republic of
Korea: d Department of Veterinar y Medicine, Kyungpook National University; Buk-gu, Daegu 702–701, Republic of
Korea: and e Department of Dermatology, Korea University Ansan Hospital; Ansan 425–701, Republic of Korea.
Received July 2, 2013; accepted September 30, 2013
The potential hair growth-promoting activity of rice bran supercritical CO2 extract (R B-SCE) and
major components of RB-SCE, linoleic acid, policosanol, γ- oryzanol, and γ-tocotrienol, were evaluated with
the histological morphology and mR NA expression levels of cell growth factors using real-time reverse
transcriptase-polymerase chain reaction (PCR) in C57BL/6 mice. RB-SCE showed hair growth-promoting
potential to a similar extent as 3% minoxidil, showing that the hair follicles were induced to be in the anagen
stage. The numbers of the hair follicles were significantly increased. In addition, mRNA expression levels of
vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and keratinocyte growth
factor (KGF) were also significantly increased and that of transforming growth factor-β (TGF-β) decreased
in RB -SCE-treated groups. Among the major components of RB-SCE, linoleic acid and γ- oryzanol induced
the formation of hair follicles according to examination of histological morphology and mRNA expression
levels of cell growth factors. In conclusion, our results demonstrate that RB-SCE, particularly linoleic acid
and γ-oryzanol, promotes hair growth and suggests RB- SCE can be applied as hair loss treatment.
Key words rice bran supercritical CO2 extract; hair growth-promoting activity; in vivo
Although hair loss is not a mortal disorder, it has a great
impact on a person’s self-respect, mental health, and overall
quality of life. Approximately 50–60 hairs are normally lost
per day, which does not have a noticeable effect on appear-
ance; however, excess loss (>100) results in baldness. Andro-
genetic alopecia is the most common type of hair loss, affect-
ing millions of both men and women.1) There are many causes
of hair loss in men and women, including diseases, nutritional
deciency, aging, hormone imbalance, and stress. Androge-
netic alopecia may occur as early as the teenage years, but
typically begin in the later decades of life.2, 3) Hair loss affects
at least half of all men by the age of 50 and up to 70% of
70-year- old men.4)
Topical minoxidil and oral nasteride approved by the
Food and Drug Administration (U.S.A.) are typically used to
treat androgenetic alopecia. Topical minoxidil, an adenosine
triphosphate-sensitive potassium channel opener, was shown
to be effective in 54% of treated patients as opposed to 34%
in placebo control groups. However, there are signicant
adverse dermatological effects associated with minoxidil,
including pruritis, dryness, scaling, local irritation, and der-
matitis.5–8) Oral nasteride, a competitive inhibitor of type-2
5-α-reductase, is known to increase hair growth in patients
with male pattern baldness (androgenetic alopecia). It was re-
ported that 48% of hair regrowth was observed in nasteride
recipients in one year. Finasteride is generally well tolerated
by patients, but some patients withdrew from treatment due to
drug-related sexual disorders.9,10)
Therefore, there remains a demand for highly effective
pharmacotherapy for treating androgenetic alopecia with an
excellent safety and efcacy prole. In the past several years,
there have been numerous attempts to develop new agents
capable of preventing and/or treating pattern baldness.3,11,12)
Currently, natural extracts from several plants have been used
for hair growth promotion, including Asiasari radix,13) Eclipta
alba,14) essential oil of Chamaecyparis obtuse,15) Zizyphus
jujube,16) and Sophora flavescens.12)
Rice (Oryza sativa) is one of the most important crops
worldwide. It is a staple food for over half of the world’s
population with approximately 95% of rice produced in Asia
and about 600 million tons of rice produced annually world-
wide.17,18) Rice bran is the major by-product of rice milling
process and accounts for nearly 8% of milled rice. Between
20–30% of produced rice bran is used for oil production,
while the remaining rice bran is discarded or used as livestock
feed and fertilizer.19) It has recently been reported that rice
bran extract has various health benecial effects, including an-
tioxidant,19) anticancer,2 0) and anti-hyperlipidemia activities.2 1)
In addition, rice bran extract shows 5-α-reductase inhibitory
activities in vitro cell lines.2 2) However, it is unknown whether
rice-bran extract is effective for treating hair loss in vivo.
Typically, rice bran oil is extracted using organic solvents,
commonly hexane. Hexane is relatively simple and excellent
for extracting nonpolar lipids.23) However, it is highly volatile
and is considered toxic to animal and humans at relatively
low concentrations. In addition, removing residual hexane is
expensive and time consuming.23,2 4) Supercritical uid extrac-
tion has been introduced as an alter native one-step method
conducted at low temperature for oil extraction. Extraction of
oil at the critical point minimizes the thermal degradation of
proteins, antioxidants, and other nutritionally valuable compo-
nents. Supercritical carbon dioxide (SC-CO2) has been used as
a substitute for organic solvents during oil extraction, with ad-
vantages including that it is environmental friendly, non-toxic,
nonammable, and inexpensive. In addition, it can be easily
removed from the nal products.25)
* To whom correspondence should be addressed. e-mail: skin4u@korea.ac.kr; haha7kmr@silla.ac.kr
The authors declare no conict of interest.
January 2014 45
In the present study, we evaluated the potential hair
growth-promoting activity of rice bran by comparing histo-
logical results and expression levels of cell growth factors
from the skin of C57BL/6 mice treated with crude rice bran-
supercritical carbon dioxide uid extracts (RB-SCE) and its
major components.
MATERIALS AND METHODS
Oryza sativa Bran Preparation and Materials Rice
(Oryza sativa LINN. var. japonica; the Korean cultivars,
Dongjin; Gramineae) used in this study were harvested in
Gijang, Busan during the fall of 2011 and the rice bran was
milled and provided by PN RICE Co., Ltd. (Kimhae, Gyeon-
sanggnamdo, Korea) in March of 2012. Linoleic acid (LA) and
authentic fatty acids for quantitative analysis were purchased
from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). Gamma-
oryzanol (OZ) and policosanol (PS) were supplied from Oryza
Oil and Fat Chemical Co. (Ichinomiya City, Japan) and Sino-
chem Qindao. Co. (Qingdao, China), respectively. Gamma-
tocotrienol (TT) was obtained from Cayman Chemical Co.
(Ann Arbor, MI, U.S.A.). All chemicals and solvents used
were of analytical grade.
Preparation of RB-SCE Supercritical CO2 extractions
of rice bran were performed in a semi-continuous ow-type
apparatus with a 3-L extractor.26) Carbon dioxide was pumped
into the extractor by a positive displacement controlled-
volume metering-pump. A ow rate of 135-g CO2/min was
used for extraction. Pressure in the extractor was controlled
using a back-pressure regulator. The extraction vessel was
loosely packed with glass wool, and 1 kg of rice bran sample
was added and distributed throughout the packing. A small
plug of glass wool was then placed in the outlet end of the
tube before closure to reduce entrainment. The extract was
collected in a separator and chilled with ice by expanding
the loaded solvent to ambient pressure. Extractions were per-
formed at 32°C and 270 bar for 240 min. RB-SCE was stored
at −80°C until use.
Analysis of Fatty Acids Fatty acid methyl ester mixtures
(FAME) were prepared by esterication with alcoholic sulfu-
ric acid reagent according to the International Union of Pure
and Applied Chemistry (IUPAC) procedure.27) A GC-2010
series (Shimadzu Co., Ltd., Kyoto, Japan) equipped with a
ame ionization detector (FID) was used for gas chroma-
tography (GC) analysis of methyl esters. Methyl esters were
analyzed on an SPTM-2560 (Fused Silica Capillary Column,
100 m×0.25 mm×0.2 µm, Supelco, Bellefonte, PA, U.S.A.).
The injection and detector temperatures were maintained
at 225°C and 285°C, respectively. The ow rate of the car-
rier gas (helium) was 0.75 mL/min. The oven temperature was
programmed to increase from 100°C to 240°C at the rate of
3°C/min after maintaining the temperature at 100°C for 4 min.
FAME was identied using authentic standards, and peaks
were quantied using digital integration according to the
American Oil Chemists’ Society ofcial method Ce 1–62.2 8)
Analysis of OZ, Tocols, Squalene, Policosanol, and Py-
tosterols The content of OZ was determined using spectro-
photometry at 315 nm according to the method of Kim and
Kim.29) To determine the tocols, phytosterols, policosanol,
and squalene contents, 30 mL of ethanol was added into 0.5 g
of RB-SCE with 5-mL 5% pyrogallol solution while heat-
ing with a reux condenser. The solution was saponied
with 1 mL aqueous 50% KOH solution for 5 min and mixed
with 20 mL water and 30 mL diethyl ether. The mixture was
extracted twice with 30 mL diethyl ether in a separator fun-
nel. The pooled diethyl ether layer was washed 3 times with
20 mL distilled water, ltrated through anhydrous sodium
sulfate, and evaporated at 30°C. After diluting with 10 mL
chloroform and ltering through 0.45-µm FH membrane
(Millipore, Billerica, MA, U.S.A.), the ltrate was analyzed
for tocols using high-performance liquid chromatography
(HPLC), while phytosterols, squalene, and policosanol were
analyzed by GC. The HPLC apparatus (PU-1580; JASCO,
Tokyo, Japan) was equipped with a Lichrospher Si-60 col-
um n (250×4.6 mm id; Merck Co., Darmstadt, Germany) and
a uorescence detector (FP-1520, JASCO) with excitation set
at 298 nm and emission set at 325 nm. The isocratic mobile
phase contained 1% 2-propanol in n-hexane. The ow rate
was 1.0 mL/min. Tocopherol and tocotrienol peaks were evalu-
ated by comparison external standards in the linear measur-
ing ranges of 0.5– 40 µg/mL. The GC (Varian 3800, Varian
Inc., Walnut Creek, CA, U.S.A.) consisted of an SAC-5 fused
silica capillar y column (30 m×0.32 mm i.d.; Supelco) and
ame-ionization detector. The column was held at 270°C for
1 min and programmed to 290°C for 20 min at a rate of 10°C/
min. The carrier gas was helium, and the total gas ow rate
was 20 mL/min. The injector and detector temperatures were
300°C and 320°C, respectively. Squalene, policosanol, and
phytosterol peaks were identied by comparing retention
times (RT) of each peak to those of pure standards.
Animals All animal procedures were approved by the
Institutional Animal Care and Use Committee of Korea Uni-
versity. Five-week-old C57BL/6 mice (SLC, Shizuoka, Japan)
were treated after acclimatizing to laboratory conditions for 1
week. Animals were allocated at 5 per polycarbonate cage in a
temperature (20°C) and humidity (40–45%)-controlled room.
The light : dark cycle was 12 : 12 h, and food (Samyang, Wonju,
Korea) and water were supplied ad libitum.
To conrm the hair growth-promoting activity of RB-
SCE, 6-week-old C57BL/6 mice were randomly divided into
3 groups as follows: negative control (NC; 10% ethanol as a
vehicle), positive control (PC; 3% Minoxidil), and RB-SCE
(3% in 10% ethanol) groups. To examine the hair growth-
promoting activity of major components of RB-SCE, 6
groups were examined, including the NC group and groups
treated with PC, LA (11.1 mg/mL), PS (0.03 mg/mL), OZ
(0.22 mg/mL), and TT (0.0093 mg/mL). The concentrations of
each component were corresponding amounts of LA, PS, OZ,
and TT in 3% RB-SCE. Each 5 female mice per group were
tested.
Determination of Hair Growth-Promoting Activity Hair
growth-promoting activity of the RB-SCE was examined
using the method reported by Roh et al. 12) with some modi-
cations. Briey, mouse hair was removed from a 2 cm×3 cm
dorsal area of mice by carefully shaving with an electric clip-
per. Test materials (100 µL) were applied topically on the back
skin of the mice once a day for 4 weeks. The hair growth-
promoting activity of the substances was evaluated as darken-
ing of the dorsal skin, indicating that the hair follicles were
in the anagen phase. Hair growth scoring was performed by
2 independent dermatologists who were unaware of treatment
regimen. The average of the each 2 scores was used as the
46 Vol. 37, No. 1
hair growth index. Hair growth was measured once per week
for 4 weeks by assigning a hair growth score as follows: score
0= no growth observed; 1= up to 20% growth; 2= 20–40%
growth; 3= 40–60% growth; 4= 60–80% growth; and 5= 80%
to full growth observed. Digital images of total hair growth
on 4 weeks were obtained using Nikon Cool Pix P100 (Tokyo,
Jap an).
RNA Extraction and Real-Time Polymerase Chain Re-
action (PCR) Total RNA was extracted with Trizol reagent
(Life Technologies, Gaithersburg, MD, U.S.A.) and the cDNA
was synthesized by a reverse transcription reaction using the
RNA PCR kit (Applied Biosystems, Roche Inc., Foster City,
CA, U.S.A.) in a 20 µL mixture containing 1 µg RNA, 50 mM
KCl, 10 mM Tris–HCl, 5 mM MgCl2, 1 mM of each dNTPs,
oligo(dT) primers, 20 U of RNase inhibitor, and 50 U of MuLV
reverse transcriptase. Nucleotide sequences of the primers
for vascular endothelial growth factor (VEGF), insulin-like
growth factor-1 (IGF-1), keratinocyte growth factor (KGF),
transforming growth factor-β (TGF-β), and glyceraldehyde
3-phosphate dehydrogenase (GAPDH) are shown in Table 1.
The reaction mixture was incubated for 60 min at 42°C, and
then heated at 90°C for 7 min in a thermocycler (GeneAmp
PCR system 9600, PerkinElmer, Inc., Roche Molecular Sys-
tem, Waltham, MA, U.S.A.). Real-time PCR was performed
using a Lightcycler instrument using FastStart DNA Master
SYBR Green І PCR kit (Roche, Basel, Switzerland). Quanti-
cation of VEGF, IGF-1, KGF, and TGF-β mRNA expression
was corrected by GAPDH.
Histological Analysis of Hair Follicles The test materi-
als were applied topically on the lower side of dorsal skin of
the mice once per day for 4 weeks. After week 4, all mice
were sacriced. Their dorsal skins were removed and xed
in 4% formaldehyde solution and embedded in parafn. The
fragments were sliced into transverse sections for determina-
tion of hair follicle count. In order to minimize the variation
of histopathologic evaluation, the biopsy sites were set as the
center of treated areas. The 3-µm sections were stained with
hematoxylin-eosin and toluidine blue and examined under a
light microscope (Olympus, Tokyo, Japan).
Statistical Analysis Analysis of variance (ANOVA) and
Tukey HSD post-hoc tests were performed for statistical anal-
ysis of data using SPSS (version 12.00, SPSS Inc., Chicago,
IL, U.S.A.). A value of p<0.05 was considered statistically
signicant.
RESULTS
Hair Growth-Promoting Effect of RB -SCE To evaluate
the hair growth-promoting activity of rice bran extract, 3%
RB-SCE was applied on dorsal skin of C57BL/6 mice once
per day for 4 weeks. As a negative control (NC) and positive
control (PC), 10% ethanol and 3% minoxidil were topically
applied, respectively. Figure 1 demonstrated the hair growth-
promoting effects at 4 weeks for RB-SCE on C57BL/6 mice.
In NC group, most mice showed only faint hair appearance
after treatment for 4 weeks. In PC and RB-SCE treatment
groups, mature hair was mostly occupied on the back of mice.
The hair growth index of the RB-SCE group was compared
with those of NC and PC groups as shown in Fig. 2. The hair
growth index of RB-SCE group showed signicantly higher
values than that of the NC group, but similar values to the PC
group after treatment for 4 weeks ( p=0.002), indicating the
hair growth-promoting activity of RB-SCE.
Anagen Induction and Hair Restoration by RB-SCE on
C57BL/6 Mice To evaluate the morphological structure of
skin tissue, the histology of hair skin slices for each treatment
group was tested. The results of histopathological examina-
tion at 4 weeks treatment showed no signs of irritation on
treated area such as epidermal thickening or inammatory
cell inltrations and so on (data not shown). The dorsal skin
fragments of sacriced mice after treatment for 4 weeks were
stained with hematoxylin–eosin and toluidine blue (Fig. 3).
The hair follicle formation of NC group was rarely observed.
In contrast, the formation of hair follicles was observed in
PC and RB-SCE treatment groups than that of the NC group.
Particularly, in the skin of PC and R B-SCE groups, most hair
follicles were fully induced and the hair root reached the deep
subcutis, distinctly revealing growth of the inner and outer
Table 1. Nucleotides Sequences of the Primers Used for PCR Amplica-
tion in This Study
Growth factor Primer sequence
VEGF Forward ACS CGG TGG TGG AAG AAG AG
Reverse CAA GTC TCC TGG GGA CAG AA
IGF-1 Forward TCA TGT CGT CTT CAC ACC TCT
TCT
Reverse CCA CAC ACG AAC TGA AGA
GCA T
KGF Forward ACG AGG CAA AGT GAA AGG GA
Reverse TGC CAC AAT TCC AAC TGC CA
TGF-βForward GCG GCA GCT GTA CAT TGA CT
Reverse ACT GTG TGT CCA GGC TCC AA
GAPDH Forward CAA TGA ATA CGG CTA CAG
CAA C
Reverse AGG GAG ATG CTC AGT GTT GG
Fig. 1. Macroscopic Evalu ation on Hair Growth Prompt ing Effects of
RB-SCE on C57BL/6 Mice
(a): NC (negative co ntrol; 10% eth anol as a veh icle), (b): PC (positive c ontrol; 3%
minoxid il), (c): 3% RB-SCE (3% rice bran ScCO2 e xt ra ct) .
January 2014 47
root sheaths of hair according to toluidine blue staining.
To conrm changes in the hair growth cycle by applying
the RB-SCE, the hair follicle formation in each group were
compared (Fig. 4). The number of hair follicle in the PC and
RB-SCE groups were 24 and 18 count/mm2, respectively,
which were signicantly higher than 4 count/mm2 in the NC
group ( p=0.000, Fig. 4).
Effect of RB -SCE on mRNA Levels of Growth Factors
To investigate the ability of RB-SCE to restore or inhibit
hair loss, mRNA expression levels of VEGF, IGF-I, KGF,
and TGF-β, hair growth related cytokines on the dorsal skin
tissue of sacriced mice after treatment for 4 weeks were
determined using real-time PCR (Fig. 5). Expression levels of
VEGF and IGF-1 of the mouse skin tissue treated with PC and
RB-SCE were signicantly higher than that of NC (p=0.004
for VEGF and p=0.000 for IGF-1). The expression level of
KGF in the RB-SCE group was also signicantly higher than
that in the NC group. In addition, expression was signicantly
higher than that of the PC group (p=0.000). However, the
level of TGF-β on the PC and RB-SCE groups was signi-
cantly lower than that of the NC group ( p=0.001).
Based on these results, RB-SCE treatment on the dorsal
skin of mice appeared to induce changes in the expression
levels of growth factors, including VEGF, KGF, IGF-I, and
Fig. 3. Hematoxylin–Eosin (Magnication ×40: a, b, c. Magn ication ×100: d, e, f) and Toluidine Blue (Magnication ×40: g, h, i. Magnication
×100: j, k, l) Stai ning of the Skin Sections
The test mater ials were applied topically on th e back ski n of the mice once per d ay for 4 weeks. Neg ative cont rol (10% ethanol as a vehicle; a, d, g, j), positive control
(3% minoxidi l; b, e, h, k), 3% RB- SCE (rice br an scCO2 ex trac t; c, f, i, l).
Fig. 2. Hair-Growth Index of C57BL/6 Mice after Topical Application of Rice Br an Extracts during 4 Weeks
Group 1: NC (negative control; 10% ethanol as a veh icle), group 2: PC (positive control; 3% mi noxidil), g roup 3: 3% RB- SCE (3% rice bra n scCO2 ext ra ct).
48 Vol. 37, No. 1
TGF-β genes, and the differentiation and proliferation of hair
follicles, showing the ability of RB-SCE to promote hair
growth.
Composition of R B- SCE To examine the hair growth-
promoting activity of RB-SCE, the major composition of
RB-SCE was analyzed (Table 2). RB-SCE was primar-
ily composed of lipids. The amount of total triglyceride (TG)
was 83.9 g/100 g RB-SCE, which was the major component.
The TG of RB-SCE was mainly composed of 40.63% oleic
acid, 38.42% linoleic acid, and 16.49% palmitic acid. Tocols,
policosanol, phytosterols, and squalene contents were deter-
mined to be 46.88, 92.33, 655.68, and 141.03 mg/100 g RB-
SCE, respectively. Particularly, γ-tocotrienol was 66.76% of
total tocols. The amount of γ-oryzanol, which is known to
be an anti-oxidant, was 0.7 mg/100 g RB-SCE. Based on their
known biological activities, LA, PS, TT, and OZ were selected
as candidate materials for hair growth-promoting activity.
Hair Growth-Promoting Effects of Major Components
of RB-SCE To compare the hair growth-promoting ac-
tivities of the major components of RB-SCE, LA, PS, OZ, and
TT were applied to the dorsal skin of C57BL/6 mice at cor-
responding concentrations included in 3% RB-SCE once per
day for 4 weeks. The hair growth indices for each component
group were compared with those of NC and PC groups as
shown in Fig. 6. LA- and OZ-treated groups exhibited out-
standing hair growth-promoting potential, showing similar re-
sults with the PC group at treatment for 4 weeks (p=0.0004).
However, PS- and TT-treated groups did not exhibit hair
growth-promoting potential, showing similar results to the NC
group.
Anagen Induction and Hair Restoration on Male
C57BL/6 Mice by Major Components of RB -SCE The
morphological structures of the tissue, obtained by examining
transverse sections of the dorsal skin of each major compo-
nent-treated group, are shown in Fig. 7. Dorsal skin fragments
of sacriced mice were stained with hematoxylin–eosin and
toluidine blue. In the skin of the NC group, the formation of
hair follicles was rarely observed and the follicles were cited
in around boundary of dermis and adipose layer. In the PC,
Fig. 4. Compar ison of Follicle Number in C57BL/6 Mice af ter Topical
Application of Experimental Mater ials
Group 1: NC (negative control; 10% et hanol as a veh icle), group 2: PC (positive
control; 3% m inoxidil), group 3: 3% RB -SCE (3% rice bran scCO2 extra ct) for 4
weeks. The aste risk indic ates a statist ically sign icant dif ferenc e compared with
negative co ntrol (*** p<0. 0 01).
Fig. 5. mR NA Expression Levels of Growth Factors in the Skin of C57BL/6 Mice after Topical Application of Experimental Materials for 4 Weeks
(a) VEGF, (b) IGF-1, (c) KGF, and (d) TGF-β; group 1: NC (negative con trol; 10% eth anol as a vehicle), group 2: PC (positive cont rol; 3% minoxidil), group 3: 3% RB-
SCE (3% rice bra n scCO2 extract) for 4 weeks. The aster isk indicates a st atist ically signicant difference compared with contr ol (** p<0.01).
January 2014 49
LA, and OZ groups, most hair follicles were fully induced and
the hair root reached the deep subcutis, clearly indicating the
growth of inner and outer root sheath of hair. The epidermal
cell differentiation into hair follicles and growth of hair fol-
licles was observed in the skins of the PC and other groups by
Toluidine blue staining. Although the follicles in the PS and
TT groups were not fully induced to the extent of in the LA or
OZ groups, downward growth of follicles from the dermis and
increased follicles length were observed, which were different
aspects from those of NC group.
To conrm the effect on hair growth cycle following appli-
cation of major components of RB-SCE, the formation of hair
follicles in each group were compared in the hematoxylin-
eosin-stained sections (Fig. 8). The numbers of hair follicles of
the PS and TT groups were 6 and 8 count/mm2, respectively,
which were not signicantly different from those of the NC
group. However, the number of hair follicles in the LA and
OZ groups was 30 and 31 count/mm2, respectively, which
were signicantly higher than that of PC group and NC group
(p=0.000, Fig. 8).
Effect of RB -SCE Major Components on mRNA Ex-
pression Levels of Growth Factors To investigate the effect
of the major components in RB-SCE as hair restoration or
loss inhibition, the mRNA expression levels of VEGF, IGF-I,
KGF, and TGF-β on the dorsal skin tissue of sacriced mice 4
weeks after treatment were determined using real-time PCR
(Fig. 9). The expression level of VEGF of the mouse skin
tissue treated with PC and major components of RB-SCE was
signicantly higher than that of NC (p=0.002). The expres-
sion levels of IGF-1 and KGF of the mouse skin tissue treated
with PC and major components of RB-SCE were also signi-
cantly higher than that of NC ( p=0.000 for IGF-1 and 0.001
for KGF). In addition, in all tested group, the expression level
of TGF-β was lower than that in the NC group ( p=0.006).
Based on these results, the hair growth-promoting activity
of RB-SCE likely resulted from changes in expression levels
of growth factors, such as increased expression of VEGF,
Table 2. Concentrations and Compositions of RB-SCE Major Compo-
nents
Components Sub-components Composition
(%)
Total
concentration
Fatty acids Myristic acid 1.6 83.9
(g/100 g oil)
Palmitic acid 16.3
Stearic acid 1.6
Oleic acid 41.1
Linoleic acid 36.4
Arachidic acid 0.6
γ-Linolenic acid 0.6
Linolenic acid 1.4
Arachidonic acid 0.4
Nervonic acid 0.1
Policosanols Tetra(C24) 3.5 92.33
(mg/100 g oil)
Hexa(C26) 26.2
Hepta(C27) 5.8
Octa(C28) 2.4
Tria(C30) 62.0
Tocols α-Tocopherol 7.6 46.88
(mg/100 g oil)
β-Tocopherol 5.9
γ-Tocopherol 10.0
δ-Tocopherol 0.9
α-Tocotrienol 5.5
γ-Tocotrienol 66.8
δ-Tocotrienol 3.3
γ-Oryzanol 0.74
(g/100 g oil)
Phytosterols Campesterol 20.1 655.68
(mg/100 g oil)
Stigmasterol 15.1
β-Sitosterol 49.9
Cycloatenol 8.8
24-Methyl-cycloartanol 6.2
Squalene 141.04
(mg/100 g oil)
Fig. 6. Hai r Growth Index of C57BL/6 Mice af ter Topical Application of Exper imental Mater ials during 4 Weeks
Group 1: NC (negative control), group 2: PC (positive cont rol; 3% minoxidil), group 3: LA (lin oleic acid; 11.1 mg/mL), group 4: PS (policosanol; 0.03 mg/mL), group 5:
OZ (γ-or yzanol; 0.22 mg /mL), group 6: TT (γ-toc otrie nol; 0.0 093 mg/ mL).
50 Vol. 37, No. 1
KGF, and IGF-I genes and decreased expression of TGF-β,
induced from the major components LA, OZ, PS, and TT,
and from the differentiation and proliferation of hair follicles
induced by LA and OZ.
DISCUSSION
Generally, hair follicles are known to renew cyclically
through 3 phases: anagen, catagen, and telogen. Hair follicles
in each phase have distinct mor phological characteristics.
During anagen, the hair bulb is enlarged and it encloses the
dermal papilla. The follicles show downward growth from
the dermis via the panniculus adipisus to the panniculus car-
nosus.30) Hair matrix cells proliferate and differentiate into
daughter cells, which move upwards to form the inner root
sheath and hair shaft. The length of hair follicle increases
continuously as the hair bulb enters the hypodermis. The skin
becomes thicker and the number and size of hair follicles in-
crease during this stage.31) During the catagen phase, follicles
involute and skin thickness decreases. Through the telogen
phase, both the follicles and the skin are at rest. When stem
cells in hair follicles are activated, the follicle enters a new
anagen phase and a new hair shaft is produced.
Various cytokines and growth factors play important roles
in hair growth control. To promote hair growth and initiate
anagen, it is essential that expression of factors maintaining
anagen is increased, such as IGF-1, basic broblast growth
factor (bFGF), KGF, and VEGF, while decreasing expression
of cytokines promote apoptosis, such as TGF-β, and IL-1.32,33)
VEGF plays a central role in promoting angiogenesis as
well as inuencing diverse cell functions including cell sur-
vival, proliferation, and generation of nitric oxide and prosta-
cyclin.34) The expression of VEGF is related to the formation
of blood vessels. VEGF mRNA expression during the hair
cycle was variable, increasing during the anagen phase and
then regressing during the catagen and telogen phases. IGF-1
is a peptide hormone that promotes the growth, sur vival, and
differentiation of cells in various organ and tissues, including
skin. IGF-I is critically involved in promoting hair growth
by regulating cellular proliferation and migration during hair
follicle development.35) KGF also has been shown to be ex-
pressed in the dermis and to regulate epidermal proliferation
and differentiation via a paracrine mechanism, stimulating
wound healing, and hair growth.36) In contrast, TGF-β induces
apoptosis in keratinocytes against cell death, indicating that
TGF-β is involved in apoptosis-driven catagen development.37)
In this st udy, the shaved back skins of 7-week-old C57BL/
6 mice were topically treated with RB-SCE for 4 weeks.
Because the rst cycle in mouse skin is synchronized, 6–7
week-old C57BL/6 mice are specically known to be in the
stable telogen phase. Thus, skin of 7 week-old C57BL/6 mice
Fig. 7. Hematoxylin–Eosin (Magnicat ion ×40: a, b, c, d, e, f. Magnication ×100: g, h, I, j, k, l) and Toluidine Blue (Magnication ×40: m, n, o,
p, q, r. Magni cation ×100: s, t, u, v, w, x) Staining of the Skin Sections
The test mater ials were applied topically on the back ski n of the mice once per d ay for 4 weeks. G roup 1: NC (negative control; a, g, m, s), group 2: PC (positive control;
3% minoxid il; f, l, r, x), group 3: LA (linoleic acid; 11.1 mg /mL; b, h, n, t), gr oup 4: PS (policosanol; 0.03 mg/mL; c, i, o, u), group 5: OZ (γ-or yzanol; 0.22 mg /mL; d, j, p,
u), group 6: TT (γ-t ocotr ienol; 0.00 93 mg /mL; e, k, q, w).
Fig. 8. Comparisons of Follicle Number in C57BL/6 Mice after Topical
Application of Experimental Mater ials
Group 1: NC (negative control), group 2: PC (po sitive cont rol; 3% minox idil),
group 3: LA (linoleic acid ; 11.1 mg/m L), group 4: PS (pol icosanol; 0.03 mg/mL),
group 5: OZ (γ-o ryza nol; 0.22 mg/mL), group 6: TT ( γ-tocot rienol; 0.0093 mg/mL)
for 4 weeks. The asterisk indicates a statistic ally signicant differen ce compared
with negative control (*** p<0.0 01) .
January 2014 51
was used for examining the effects of hair growth-promotion
in hair follicle cycling.
The macroscopic and histological alterations in the skin of
mice were evaluated for examining the effects of hair growth-
promotion as a result of the treatments of RB-SCE. The
growth rate of hair in R B-SCE treated group was higher than
in NC group and similar with that of minoxidil group (Figs. 1,
2). In the histological evaluation, most hair follicles were fully
induced and the hair root reached at deep subcutis, distinctly
revealing the growing inner and outer root sheath of hair due
to RB-SCE application (Figs. 3, 4). These results were similar
to those of minoxidil. The action mode of minoxidil on the
hair growth effect was not completely elucidated. However,
mechanisms underlying hair growth stimulated by minoxidil
have been reported. Otomo38) proposed that minoxidil func-
tions as a sulfonylurea receptor (SUR) activator and prolongs
the anagen phase of hair follicles through by inducing cell
growth factors such as VEGF, HGF, and IGF-1.
The mRNA expression levels of VEGF, IGF-I, and KGF
following RB-SCE treatment were higher than those of the
negative control group were. Particularly, the expression level
of KGF by RB-SCE treatment was signicantly higher than
that of minoxidil (Fig. 5).
The present results showed that during the treatment with
RB-SCE, an increase in the number of hair follicles, and in-
creased expression levels of cell growth factors such as VEGF,
HGF, and IGF-1, and decreased expression of TGF-β were
conrmed, suggesting that RB-SCE may induce differentia-
tion and proliferation of hair follicles through the expression
of cell growth factors and resulting in the induction of the
early anagen phase.
To conrm the main factors promoting hair growth in
RB-SCE, the major components of RB-SCE were ana-
lyzed. The lipids of rice bran were comprised mainly of TG
(80.6–86.0 wt%), free fatty acid (4.2–9.0 wt%), and phos-
pholipids (5.5–6.7 wt%), while other components were also
detected in minor proportions (0.2–2.1 wt%).39) According to
the results of Manosroi et al., 19) the raw rice bran oil contains
both unsaturated and saturated fatty acids, in which palmitic
acid is a major saturated fatty acid (C18:0, 12–26%, w/w, typi-
cally 18%, w/w). Unsaturated fatty acids primarily included
oleic acid (C18:1, 35– 46%, w/w, typically 42%, w/w) and
linoleic acid (C18:2, 25–38%, w/w, typically 37%, w/w) with
traces of γ -linolenic acid (C18:3, 0.4–3.8%, w/w). Rice bran
is known to contain signicant levels of tocols up to 300 mg/
kg.40) Rice bran also contains 3000 mg/kg OZ, which is a mix-
ture of 10 ferulate esters of triterpene alcohol.41) In our study,
the fatty acid prole of RB-SCE was similar to those stated in
previous reports. However, the minor components of rice bran
differed compared with those stated in previous reports, which
was likely due to the difference in rice strains and extraction
conditions used based on supercritical CO2 extraction.
Among the main components, LA, OZ, PS, and TT were
selected to examine the hair growth-promoting activity of RB-
SCE. In particular, the unsaturated fatty acids, such as γ-LA,
LA, and oleic acid, have been shown to have anti-hair loss
activity by inhibiting the 5-α-reductase enzyme in androgen
responsive organs.42) Γ-Oryzanol has several important physi-
cal effects, including hypocholesterolemic, anti-inammatory,
and antioxidant activities.43) More than 50 studies indicate that
policosanol decreases serum cholesterol, while other studies
failed to reproduce this effect,44) and tocotrienol possess po-
Fig. 9. mRNA Expression Levels of Growth Factors in t he Skin of C57BL/6 Mice after Topical Application of Exper iment al Materials for 4 Weeks
(a) VEGF, (b) IGF-1, (c) KGF, and (d) TGF-β; Group 1: NC (negative cont rol), grou p 2: PC (posit ive contr ol; 3% minoxid il), grou p 3: LA (linoleic a cid; 11.1 mg/m L),
group 4: PS ( policosa nol; 0.03 mg /mL), group 5: OZ (γ- oryz anol; 0.22 mg/mL), group 6: TT (γ-tocotrie nol; 0.0093 mg/mL). The asteri sk indicat es a s tatis tically signicant
differ ence compared w ith cont rol (** p<0.01, ***<0.0 01).
52 Vol. 37, No. 1
tent antioxidant activity.45)
Upon examining the hair growth-promoting effects of the
major components in RB-SCE, LA and OZ exhibited out-
standing hair growth-promoting potential, showing similar
results to that of 3% minoxidil based on macroscopic and his-
tological evaluation (Fig. 6–8). Especially, in the LA and OZ
groups, the number of hair follicles was markedly increased
compared to the negative control group. In addition, mRNA
expression levels of VEGF, IGF, and KGF in groups treated
with LA and OZ were signicantly higher than those of NC
and mRNA expression levels of TGF-β were signicantly
lower than those of NC (Fig. 9).
And although PS and TT tested in this study were able to
up-regulate the expression levels of VEGF, IGF-1, and KGF
mRNA and to down-regulate expression level TGF-β mRNA,
in terms of hair growth promoting actions, they were not
able to positively affect the hair growth. This disagreement
between histological evaluation and mRNA expression levels
of growth factors in PS and TT treated groups may be able to
explain with the concentration differences of treated materi-
als. To conrm the major active components in RB-SCE, the
corresponding concentrations of LA, OZ, PS, and TT included
in 3% RB-SCE were applied to the dorsal skin of C57BL/6
mice, which were 11.1 mg/mL, 0.22 mg/mL, 0.03 mg/mL, and
0.0093 mg/mL, respectively. The concentrations of applied PS
and TT were lower than that of LA and OZ, approximately
from 10−1 folds to 10−3 folds. Accordingly, further studies are
necessar y to reconcile this disagreement between histological
evaluation and mRNA levels in PS and TT treated groups,
through adjusting the concentration levels of treatment com-
ponents.
Nevertheless, the increase of hair index, the abundance of
hair follicles in mice skin, and the induction of the change in
expression levels of growth factors via RB-SCE, LA and OZ
applications clearly supported that LA and OZ may act as the
main factors for hair growth promoting and resulting earlier
telogen-to-anagen conversion in RB-SCE.
A previous report showed that LA has strong hair growth-
promoting effect in vitro.22) According to our results, LA and
OZ showed outstanding hair growth-promoting effects. This
is the rst report that LA and OZ have hair growth-promoting
effects in vivo.
In conclusion, this study provides potent evidence that RB-
SCE, which contains LA and OZ, exhibited outstanding hair
growth-promoting potential and suggests that these substances
can be applied as hair loss treatments.
Acknowledgments This work was supported by a Grant
(No. 311014-03) from the Ministr y for Food, Agriculture,
Forestry and Fisheries, Republic of Korea. JSC was also sup-
ported by the Global Healthcare Industr y RIS Center from the
Ministry of Knowledge Economy, Republic of Korea.
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