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In Vivo Hair Growth-Promoting Effect of Rice Bran Extract Prepared by Supercritical Carbon Dioxide Fluid


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The potential hair growth-promoting activity of rice bran supercritical CO2 extract (RB-SCE) and major components of RB-SCE, linoleic acid, policosanol, γ-oryzanol, and γ-tocotrienol, were evaluated with the histological morphology and mRNA 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.
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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
deciency, 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 signicant
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 efcacy prole. 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 benecial 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,
nonammable, and inexpensive. In addition, it can be easily
removed from the nal products.25)
* To whom correspondence should be addressed. e-mail:;
The authors declare no conict 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.
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 esterication 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 identied using authentic standards, and peaks
were quantied using digital integration according to the
American Oil Chemists’ Society ofcial 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 reux condenser. The solution was saponied
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 identied 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 conrm 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
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. Briey, 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 sacriced. Their dorsal skins were removed and xed
in 4% formaldehyde solution and embedded in parafn. 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
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 signicantly 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 inammatory
cell inltrations and so on (data not shown). The dorsal skin
fragments of sacriced 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 Amplica-
tion in This Study
Growth factor Primer sequence
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 conrm 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 signicantly 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 sacriced 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 signicantly 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 signicantly higher than
that in the NC group. In addition, expression was signicantly
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 (Magnication ×40: a, b, c. Magn ication ×100: d, e, f) and Toluidine Blue (Magnication ×40: g, h, i. Magnication
×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
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
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 sacriced 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 icant 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 signicant 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 conrm 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 signicantly 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 signicantly 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 sacriced 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
signicantly 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-
Components Sub-components Composition
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.
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 inuencing 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 specically known to be in the
stable telogen phase. Thus, skin of 7 week-old C57BL/6 mice
Fig. 7. Hematoxylin–Eosin (Magnicat ion ×40: a, b, c, d, e, f. Magnication ×100: g, h, I, j, k, l) and Toluidine Blue (Magnication ×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 signicant 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 signicantly 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
conrmed, 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 conrm 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 signicant 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 prole 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-inammatory,
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 signicant
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 signicantly higher than those of NC
and mRNA expression levels of TGF-β were signicantly
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 conrm 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 101 folds to 103 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-
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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... The dorsal skin hairs of mice were shaved completely using an electric clipper and a hair waxing cream containing 80% thioglycolic acid (NiClean TM , Ildong Pharmaceutical Co., Seoul, Korea) for inducing anagen synchronization, as described elsewhere [28][29][30]. The next day, a total of 48 mice was divided into eight groups (n = 6 per group) based on their body weights; a control group with DW as a vehicle (DW), six treatment groups with fRGM (fRGM400, fRGM200 and fRGM100) or PH (PH400, PH200 and PH100) at three different doses of 400, 200 and 100 mg/kg in DW. ...
... The dorsal skin image was obtained using a digital camera, and hair growth was assessed as darkening of the dorsal skin, as described previously [28,29]. After treatments for 2 weeks, mice were euthanized using CO2 gas, and the dorsal skin area was sampled. ...
... The sections were stained with hematoxylin and eosin (H&E) or immunostained [32]. In H&E stain, histomorphometric analyses were examined for dermal thickness (μm/skin) and numbers of hair follicles (follicles/mm 2 ) in the cross sections, and hair shafts and follicular sizes with hair root sheath (μm/follicle) in the longitudinal sections, using an image analysis program (iSolution FL ver 9.1, IMT isolution Inc.), as described previously [28,30]. The analyses were performed at least in three histological fields in each section by a histopathologist blinded to the groups. ...
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An abnormal hair-growth cycle induces hair loss, which affects psychological distress and impairs life quality. Red ginseng marc (RGM) is usually discarded as a byproduct after extracting red ginseng, but several studies have shown that the RGM still has bioactive components including ginsenosides. Therefore, the hair-growth effects of fermented RGM (fRGM) and traditional polyherb formula (PH) were examined in C57BL/6 mice. The dorsal hairs of mice were depilated, and they were topically treated with fRGM or PH at 400, 200 and 100 mg/kg or the combination of both middle doses (combi) once a day for two weeks. The hair-covering regions were significantly increased with higher doses of fRGM and PH and in combi groups, compared with the control treated with distilled water. Hair length, thickness and weight also increased in the treatment groups. In particular, the fRGM and PH increased the anagen-phased hair follicles, the follicular diameters and the dermal thickness. Immunostains for Ki-67 showed the anagen-phased cell division in the treatment groups. The beneficial effects were greater in the high doses of fRGM and PH and the combi groups. These suggest hair-growth-promoting effects of fRGM, PH and the combination by enhancing the hair-growth cycle.
... Excessive DHT results in androgen-dependent disorders which can cause pathogenetic conditions such as prostate cancer (Pham & Ziboh, 2002), benign prostatic hyperplasia, alopecia, hirsutism, and acne (Kettyle & Arky, 1998;Norman & Litwack, 1997). In contrast, linoleic acid and γ-oryzanol, which are the major biological compounds of rice bran, demonstrate inhibition of 5αreductase (Choi et al., 2014). ...
... On the other hands, the maceration method using 50% ethanol as a high polarity solvent was poorly to snatch lipophilic compounds like fatty acids from the raw materials. Linoleic acid and oleic acid were confirmed as a marker for 5α-reductase inhibition activity by correlation coefficient study (Choi et al., 2014;Ruksiriwanich et al., 2011). ...
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Rice bran is an essential source of natural bioactive compounds with widely applications. This study aimed to determine the effects of extraction methods on the bioactivities of the extracts. Jasmine and Riceberry rice bran were extracted using 50% ethanolic maceration, cold pressed and subcritical dimethyl ether (DME) extraction. The extracts were analyzed for antioxidant, pro-collagen type I synthesis and 5α-reductase inhibition activities. The results showed that Riceberry rice bran extract exhibited superior antioxidant activity than Jasmine rice bran extract. The highest pro-collagen type I synthesis of Jasmine and Riceberry rice resulted from subcritical DME extraction (50.91%) and maceration (46.99%) respectively. It is a newly found that the subcritical DME extract of Jasmine rice bran displayed the strongest 5α-reductase inhibition activity (73.14 %inhibition) resulting from the biological markers linoleic acid (22.39±0.61 %w/w) and oleic acid (39.48±1.07 %w/w). Therefore, subcritical DME extraction is suitable for preparing rice bran extract for 5α-reductase inhibitor application.
... These properties are especially present in RGO obtained by supercritical CO 2 fluid extraction. Compared to conventional methods that use organic solvents, supercritical CO 2 fluid extraction, which uses CO 2 as a solvent, has the following advantages: minimisation of thermal degradation of valuable constituents; decreased contamination by solvent residues; and reduced formation of undesirable byproducts [9]. This method is also cheap, safe, and environmental friendly because CO 2 is recyclable, nontoxic, and nonflammable [10]. ...
This study aimed to investigate the underlying mechanisms of red ginseng extract (RGE) on regulating hair growth and hair follicle development. Results from in vitro studies showed that RGE treatment simultaneously enhanced viability and inhibited apoptosis in human hair dermal papilla cells. Moreover, RGE administration promoted telogen-to-anagen transition, prolonged anagen in hair follicular cycling, and increased the size of hair follicles and skin thickness in a C57BL/6 mouse model. Furthermore, RGE treatment significantly upregulated the expression of β-catenin, phospho-glycogen synthase kinase 3β, cyclin D1, cyclin E, and Bcl-2, phospho-extracellular signal-regulated protein kinase, and phospho-Akt, which are associated with promoting hair growth. In addition, RGE enhanced skin health by activation of antiox-idant defense systems. Our data demonstrates that hair regenerative mechanisms of RGE may be mediated by stimulating dermal papilla cell proliferation and enhancing skin functions.
... Rice bran, with a protein content of approximately 15% [1], is the main by-product of rice processing [2]. Rice bran protein (RBP) is the main nutrient component in rice bran [3]. ...
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Ultrasound technology was used to treat rice bran protein (RBP), and the structural and functional properties of ultrasonically treated RBP (URBP) and its chlorogenic acid (CA) complex were studied. When ultrasonic power of 200 W was applied for 10 min, the maximum emission peak λmax of the URBP-CA complex in the fluorescence spectrum was red-shifted by 3.6 nm compared to that of the untreated complex. The atomic force microscope (AFM) analysis indicated that the surface roughness of the complex was minimized (3.89 nm) at the ultrasonic power of 200 W and treatment time of 10 min. Under these conditions, the surface hydrophobicity (H0) was 1730, the contents of the α-helix and β-sheet in the complex were 2.97% and 6.17% lower than those in the untreated sample, respectively, the particle size decreased from 106 nm to 18.2 nm, and the absolute value of the zeta-potential increased by 11.0 mV. Therefore, ultrasonic treatment and the addition of CA changed the structural and functional properties of RBP. Moreover, when ultrasonic power of 200 W was applied for 10 min, the solubility, emulsifying activity index (EAI), and emulsion stability index (ESI) were 68%, 126 m2/g, and 37 min, respectively.
... FGF-2 and FGF-7 (identified as Keratinocyte growth factor (KGF)) positively stimulate the hair growth cycle of mice [46] while FGF-5 acts as an inhibitor of hair growth during anagen phase [47] . Lycopene isolated from rice bran supercritical CO 2 extract and other major components (linoleic acid, c-oryzanol) [48] , Lycopersicon esculentum extracts [49] , myristoleic acid from Malva verticillata seeds extract [50] , and Carthamus tinctorius extract [51] are beneficial in the treatment of hair loss because of their property to stimulate KGF. ...
Cosmetic products are complex mixtures of ingredients with different physical, chemical and functional properties formulated in order to obtain the product with desired activity and quality. This paper provides a comprehensive review on the effectiveness of the environmentally friendly extraction technique based on the application of GRAS solvent CO2 and high pressures which present many advantages over the conventional processes. Supercritical CO2 (SCO2) proved to be a promising extraction solvent in this process and has the potential to replace the organic toxic solvents. Due to its unique properties, the desirable texture, absence of water or solvent traces, chemical stability and presence of bioactive agents, SCO2 extracts can be easily incorporated into the cosmetic products. Depending on the plant material, the operation parameters and the chemical profile, the obtained extracts can be used as the common cosmetic ingredients (thickeners, emollients, waxes, fragrances, and colorants). Moreover, SCO2 provides a high concentration of bioactive compounds in the final extracts which exhibit many bioactivities in the cosmetic products acting as antioxidants, anti-ageing, photo-protective agents or providing antibacterial activity and inhibition of enzymes, such as tyrosinase, lipoxygenase, collagenase and hyaluronidase. This review highlights the potential application of the SCO2 extracts in the cosmetic industry. The bioactive properties of SCO2 extracts are discussed and their capability for skin health is evaluated.
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Red ginseng oil (RGO), rather than the conventional aqueous extract of red ginseng, has been receiving much attention due to accumulating evidence of its functional and pharmacological potential. In this review, we describe the key extraction technologies, chemical composition, potential health benefits, and safety of RGO. This review emphasizes the proposed molecular mechanisms by which RGO is involved in various bioactivities. RGO is mainly produced using organic solvents or supercritical fluid extraction, with the choice of method greatly affecting the yield and quality of the end products. RGO contains a high unsaturated fatty acid levels along with considerable amounts of lipophilic components such as phytosterols, tocopherols, and polyacetylenes. The beneficial health properties of RGO include cellular defense, antioxidation, anti-inflammation, anti-apoptosis, chemoprevention, hair growth promotion, and skin health improvement. We propose several molecular mechanisms and signaling pathways that underlie the bioactivity of RGO. In addition, RGO is regarded as safe and nontoxic. Further studies on RGO must focus on a deeper understanding of the underlying molecular mechanisms, composition–functionality relationship, and verification of the bioactivities of RGO in clinical models. This review may provide useful information in the development of RGO-based products in nutraceuticals, functional foods, and functional cosmetics.
Improvement of antioxidant and anti-inflammatory functions is believed to be an effective strategy for protection against various diseases such as cancer, aging, and neurodegenerative disease. This study focused on investigating antioxidant and anti-inflammatory abilities of Zingiber montanum oil (ZMO) extracted by the supercritical CO2 fluid system in HepG2 cells and lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Ten predominant constituents of ZMO were identified, in which triquinacene, 1,4-bis (methoxy), terpinen-4-ol, triquinacene, 1,4,7-tris (methoxy), α-terpinene, sabinene hydrate, and (E and Z)-1-(3,4-dimethoxyphenyl)butadiene account for 86.47%. ZMO exhibited anti-inflammatory capacity by inhibiting the formation of pro-inflammatory markers such as nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein-1 in LPS-treated macrophages. The LPS-induced stimulation of nuclear factor-kappa B, signal transducer and activator of transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) pathways as evident from increased phosphorylation of IKKα/β, IκBα, p65, Stat3, ERK, JNK, and p38 MAPK was also suppressed by ZMO pretreatment. Further, ZMO enhanced the expression of nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase-1 (HO-1), and concurrently, reduced intracellular reactive oxygen species accumulation in LPS-treated RAW 264.7 cells. In addition, ZMO treatment markedly upregulated the expression of Nrf2 as well as its target genes, HO-1 and NAD(P)H:quinone oxidoreductase 1 in HepG2 cells. These data propose that ZMO may be a potent candidate for prevention and/or treatment of inflammatory and oxidative conditions.
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Washing rice water (WRW) refers to the sewage produced by rice washing in China and other parts of Asia people's daily life. As in the WRW is rich a variety of nutrients, microorganisms are prone to multiply and pollute the environment. In this article, high-throughput sequencing is used to describe the microbial diversity in different fermentation time WRW. The results showed that the sequencing depth effectively covered the microbial species in the samples, and the bacterial community structure in the samples of WRW at different fermentation periods was rich in diversity. Preominant taxa included Proteobacteria (62%), Firmicutes (28%), approximately Cyanobacteria (10%) and Bacteroidetes (0.5%). The core WRW microbiome comprises Trabulsiella, Pseudomonas, Serratia, Lactobacillus, Erwinia, Enterobacter, Clostridium and Acinetobacter, some of which are potential beneficial microbes. The change of microbial community composition with the change of habitat was assessed. It was found that environmental factors had significant influence on the assembly structure of microbial community.
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Background A wide range of environmental factors, such as diseases, nutritional deficiencies, ageing, hormonal imbalances, stress, and ultraviolet (UV) radiation, may affect the structure and function of the skin that covers the entire surface of the human body. In this study, we investigated roles of red ginseng oil (RGO) in enhancing skin functions, including hair growth and skin protection, using mouse models. Methods For hair growth experiment, shaved dorsal skins of C57BL/6 mice were topically applied with vehicle, RGO, RGO’s major compounds, or minoxidil for consecutive 21 days and skin tissues were examined the hair growth promoting capacity. For skin protection experiment, SKH-1 hairless mice were topically applied with vehicle or RGO twice a day for three days prior to exposure to UVC radiation at 20 kJ/cm². Skin tissues were collected to evaluate skin protective effects of RGO. Results Topical application of RGO to C57BL/6 mice effectively promoted hair regeneration by inducing early telogen-to-anagen transition and significantly increasing the density and bulb diameter of hair follicles. Major compounds, including linoleic acids and β-sitosterol, contributed to RGO-promoted hair growth. Treatment with RGO as well as its major components upregulated expression of hair growth–related proteins. Furthermore, in SKH-1 hairless mice, RGO had a protective effect against UVC-induced skin damage by inhibiting inflammation and apoptosis, as well as inducing cytoprotective systems. Conclusion These data suggest that RGO may be a potent agent for improving skin health and thereby preventing and/or treating hair loss and protecting skin against UV radiation.
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Policosanol comprises a mixture of long-chain aliphatic alcohols from sugarcane wax. More than 50 studies indicate that policosanol decreases serum cholesterol, while others failed to reproduce this effect. The objective of this investigation was to assess the bioavailability of esterified policosanol and non-esterified policosanol (NEP), in relation to their hypocholesterolemic effects. Sprague Dawley rats were given a daily oral dose of 100 mg/kg of NEP, 117 mg kg<sup>–1</sup> of butyric acid esterified policosanol (BAEP), or 164 mg kg<sup>–1</sup> of oleic acid esterified policosanol (OAEP). Policosanol absorption was evaluated in plasma between 0 and 3 hours after ingestion. To assess changes in total cholesterol, LDL-cholesterol, HDLcholesterol and triacylglycerols in plasma and liver 3-hydroxy- 3-methylglutaryl coenzyme A reductase (HMG- CoA red) phosphorylation, the rats were supplemented with nonesterified or esterified policosanol for 5 weeks. The results indicate that policosanol absorption was significantly greater in OAEP-treated rats than in those subjected to NEP or BAEP administration. OAEP significantly reduced plasma total and LDL-cholesterol in rats, in addition to a 5.6-fold increase (P < 0.05) in the hepatic content of phosphorylated HMG-CoA red over the control values. In conclusion, esterification of policosanol with oleic acid enhances policosanol bioavailability, and significantly improves the serum lipid profile in normocholesterolemic rats in association with the inactivation of HMG-CoA red controlling cholesterogenesis. Los Policosanoles están formados por una mezcla de alcoholes alifáticos de cadena larga y se obtienen de las ceras de la caña de azúcar. Más de cincuenta estudios indican que los policosanoles reducen el colesterol sérico, mientras que otros no logran reproducir este efecto. El objetivo de esta investigación fue evaluar la biodisponibilidad de policosanoles esterificados y no esterificados (NEP), en relación con sus efectos hipocolesterolémicos. Para ello, a ratas Sprague Dawley se les administró una dosis oral diaria de 100 mg kg<sup>–1</sup> de NEP, 117 mg kg<sup>–1</sup> de policosanoles esterificados con ácido butírico (BAEP), ó 164 mg kg<sup>–1</sup> de policosanoles esterificados con ácido oleico (OAEP). La absorción de los policosanoles se evaluó en el plasma entre 0 y 3 horas después de la ingestión. Para evaluar los cambios en el colesterol total, colesterol-LDL, colesterol-HDL y triglicéridos en el plasma y en la fosforilación de la hígado 3-hidroxi-3-metilglutaril coenzima A reductasa (HMG-CoA red), la ingesta de las ratas fue suplementada con policosanoles no esterificados o esterificados durante 5 semanas. Los resultados indicaron que la absorción de los policosanoles fue significativamente mayor en las ratas tratadas con los OAEP que en las sometidas a los NEP o las que se les administró BAEP. Los OAEP redujeron significativamente el colesterol plasmático total y el colesterol- LDL de las ratas, además de aumentar 5.6 veces (P < 0,05) sobre los valores control, en el contenido hepático de la HMG-CoA fosforilada red. En conclusión, la esterificación de policosanoles con ácido oleico aumenta la biodisponibilidad de los policosanoles, y mejora significativamente el perfil de lípidos séricos en ratas normocolesterolémicos en asociación con la inactivación de la colesterogénesis control HMG-CoA red.
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To isolate oryzanol from the by-product of rice bran oil refinning, experiment of solvent fractional crystallization was carried out at various conditions with the dark oil obtained by acidifying the soap stock of micella refinning process and the pitch obtained from vacuum distillation of the dark oil. The impurity interfering the crystallization process such as waxes can be removed as precipitates by cooling the 1:1 mixtrue of acetone and dark oil to , From the dewaxed dark oil, oryzanol concentrate with 51.3% purity was obtained by fractional crystallization at with the mixture of 8 part volume of hexane and 1 part of the dewaxed dark oil. The concentrate was recrystallized at room temperature with 20 part volume of methanol to yield oryzanol crystal of 98.3% purity. The optimum condition of vacuum distillation was temperature of with 2% steam sparging. At this condition, the free fatty acid in the dark oil was removed as distillate without thermal deomposition to yield 82.3% of oryzanol as the pitch of 27.3% purity. After concentration from the pitch with 20 part volume of hexane to yield yellow powder of 75.4% purity, the yellow powder was recrystallized in methanol at room temperature to obtain the crystal containing 99.0% oryzanol. The overall oryzanol yield from the dark oil and the pitch was 9.5 and 28.5%, respectively. The change of the composition of sterols and triterpenoid alcohols in the compounds isolated during fractionation was analyzed by GC-MS.
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Vitamin E and γ-oryzanol display a wide range of biological activities including hypocholesterolemic, anti-inflammatory and antioxidant activities. Although white rice is far more popular worldwide, consumption of brown rice is increasing, partially on account of the presence of bioactive compounds; however, the effects of parboiling, storage and cooking on them are not well-characterized. The effects of parboiling and a 6-month storage period on the contents of vitamin E and γ-oryzanol in three brown rice cultivars grown in three different locations in Brazil were investigated. Also, their levels in branded non-parboiled and parboiled brown rice were monitored before and after cooking. Vitamin E homologues and γ-oryzanol were separated by RP-HPLC equipped with PDA and fluorescence detectors. The average levels of total tocols and γ-oryzanol in the raw brown rice cultivars studied were 25 and 188mg/kg, respectively. Of the vitamin E homologues, γ-tocotrienol contributed with 74% of total tocols, followed by α-tocopherol, α-tocotrienol and γ-tocopherol in minor quantities. The combined processes, parboiling, storage and cooking, led to an approximate 90% reduction in tocols and only γ-tocotrienol was detectable after any of the processes. Parboiling followed by storage resulted in an approximate 40% loss of γ-oryzanol. Cooking had almost no further effect over γ-oryzanol levels in parboiled rice previously stored for 6months.
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The three crude extracts including Oryza sativa (bran) from supercritical carbon dioxide fluid (scCO2) process which gave the highest unsaturated fatty acid contents and biological activities including the antioxidative, tyrosinase inhibition, stimulation index on human normal skin fibroblast were selected from ten edible plants to prepare the semi-purified fractions. Fraction No. 3 of the O. sativa bran crude extract gave the highest content of unsaturated fatty acids and 5α-reductase (type 1) inhibition activity (5AR). Its linoleic acid (LN) and total unsaturated fatty acid (TUC) contents were significantly positive and linear correlated to 5AR on DU-145 cell line (at r of 1.00, p0.9 (p
We have examined the role that individual TGF-beta isoforms, and in particular TGF-beta3, play in control of epidermal homeostasis. Mice with a knockout mutation of the TGF-beta3 gene die a few hours after birth. A full-thickness skin grafting approach was used to investigate the postnatal development and homeostatic control of the skin of these mice. Grafted skin of mice with a disruption of the TGF-beta3 gene developed similarly to grafts of wild type and TGF-beta1 knockout animals. However, a strikingly different response was observed after acute treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). When exposed to TPA, the grafted skin of wild type and TGF-beta1 knockout mice underwent a hyperplastic response similar to that of normal mouse skin. In marked contrast, TPA treatment of TGF-beta3 knockout grafts induced widespread areas of keratinocyte cell death. Analysis of cultured keratinocytes treated with purified TGF-beta isoforms revealed that TGF-beta3 plays a direct and specific function in protecting keratinocytes against TPA-induced cell death. The protective function of TGF-beta3 on TPA-induced cell death was not because of general suppression of the signaling pathways triggered by this agent, as ERK1/2 activation occurred to a similar if not greater extent in TGF-beta3-treated versus control keratinocytes. Instead, TGF-beta3 treatment led to a significant reduction in TPA-induced c-Jun N-terminal kinase activity, which was associated and possibly explained by specific counteracting effects of TGF-beta3 on TPA-induced disruption of keratinocyte focal adhesions
The 5α-reductase inhibitor finasteride blocks the conversion of testosterone to dihydrotestosterone (DHT), the androgen responsible for male pattern hair loss (androgenetic alopecia) in genetically predisposed men. Results of phase III clinical studies in 1879 men have shown that oral finasteride 1 mg/day promotes hair growth and prevents further hair loss in a significant proportion of men with male pattern hair loss. Evidence suggests that the improvement in hair count reported after 1 year is maintained during 2 years’ treatment. In men with vertex hair loss, global photographs showed improvement in hair growth in 48% of finasteride recipients at 1 year and in 66% at 2 years compared with 7% of placebo recipients at each time point. Furthermore, hair counts in these men showed that 83% of finasteride versus 28% of placebo recipients had no further hair loss compared with baseline after 2 years. The clinical efficacy of oral finasteride has not yet been compared with that of topical minoxidil, the only other drug used clinically in patients with male pattern hair loss. Therapeutic dosages of finasteride are generally well tolerated. In phase III studies, 7.7% of patients receiving finasteride 1 mg/day compared with 7.0% of those receiving placebo reported treatment-related adverse events. The overall incidence of sexual function disorders, comprising decreased libido, ejaculation disorder and erectile dysfunction, was significantly greater in finasteride than placebo recipients (3.8 vs 2.1%). All sexual adverse events were reversed on discontinuation of therapy and many resolved in patients who continued therapy. No other drug-related events were reported with an incidence ≥1% in patients receiving finasteride. Most events were of mild to moderate severity. Oral finasteride is contraindicated in pregnant women because of the risk of hypospadias in male fetuses. Conclusions:Oral finasteride promotes scalp hair growth and prevents further hair loss in a significant proportion of men with male pattern hair loss. With its generally good tolerability profile, finasteride is a new approach to the management of this condition, for which treatment options are few. Its role relative to topical minoxidil has yet to be determined. Pharmacodynamic Properties Finasteride specifically inhibits the type II 5α-reductase enzyme which converts testosterone to dihydrotestosterone (DHT), the androgen responsible for the development of male pattern hair loss in genetically predisposed men. Oral finasteride 1 mg/day significantly reduced serum DHT levels by a median 68.4% in men with male pattern hair loss treated for 1 year. A corresponding 9.1% (median) increase in testosterone levels from baseline was reported, but these levels remained within the normal physiological range. Oral finasteride 0.2 to 5 mg/day for 4 to 6 weeks reduced DHT levels by up to 65% in the scalp, the desired site of action of finasteride in men with male pattern hair loss. Finasteride did not affect serum luteinising hormone or follicle-stimulating hormone responses to gonadotropin-releasing hormone stimulation in healthy volunteers, and therefore does not appear to influence the hypothalamic-pituitary-testicular axis. In addition, the drug did not alter serum prolactin, sex hormone-binding globulin, aldosterone or cortisol levels in healthy volunteers. Slight increases in serum estradiol levels were seen but, like testosterone levels, these remained within the normal physiological range and the ratio of testosterone to estradiol was unaltered. Finasteride 1 mg/day does not appear to significantly affect ejaculate volume or other measures of testosterone-mediated semen production such as sperm motility, morphology and number. In addition, this dosage of finasteride had no clinically significant effects on prostate volume in healthy men aged ≤41 years, but caused a slight reduction in serum prostate-specific antigen levels. Evidence suggests that finasteride has no adverse effects on lipid or bone metabolism. Pharmacokinetic Properties Peak plasma concentrations of finasteride (9.2 µg/L) were reached 1 to 2 hours after drug administration in healthy volunteers who received a 1 mg/day dosage of finasteride for 17 days. Modest accumulation of finasteride in plasma was reported with repeated administration, but trough concentrations appeared to reach steady state within 3 days. The oral bioavailability of finasteride (80%) is not affected by the presence of food. Finasteride undergoes wide tissue distribution (volume of distribution = 76L), with ≈90% of circulating finasteride being protein bound. The drug has been detected in nanogram quantities in seminal fluid but these low levels have no clinical significance. After oral administration, finasteride is extensively metabolised in the liver to compounds which are then eliminated in the bile (56.8%) and the urine (39.1%). Virtually no unchanged drug is recovered after an oral dose of finasteride. The mean terminal elimination half-life of finasteride 1 mg/day after repeated administration is 4.8 hours. The elimination of finasteride is slower in elderly (≥70 years) than in younger (45 to 60 years) volunteers, but no dosage adjustment is warranted in the former age group, nor in patients with renal impairment. Reduced renal excretion of finasteride is compensated for by an increase in faecal elimination. There are currently no data on the pharmacokinetic properties of finasteride in patients with hepatic impairment. Although finasteride is principally metabolised by cytochrome P450 3A4 enzymes within the liver, no clinically significant interactions have been reported between finasteride and digoxin, propranolol, aminophylline, warfarin, glibenclamide (glyburide) or antipyrine. Therapeutic Efficacy Oral finasteride 1 mg/day has shown efficacy in men with male pattern hair loss. The clinical use of this dosage of finasteride has been assessed in 3 phase III studies involving 1879 men with vertex or frontal hair loss who were treated for up to 2 years. Finasteride produced statistically significant increases in scalp hair growth from baseline within several months of starting treatment, a finding documented by hair count, patient self-assessment, investigator assessment and pre- and post-treatment clinical assessment based on standardised photos. Importantly, finasteride prevented the further hair loss seen in placebo recipients. In men with vertex hair loss, the mean improvement in hair count reported after 1 year with finasteride was maintained during a further 12 months’ treatment. Global photographs of the vertex area showed improvement in hair growth in 48% of finasteride recipients at 1 year (versus 7% with placebo) and in 66% at 2 years (versus 7% with placebo). Furthermore, vertex hair counts showed that 83% of finasteride versus 28% of placebo recipients had no further hair loss compared with baseline after 2 years. Hair growth was enhanced in patients who switched from placebo to finasteride after 12 months but declined progressively in those switched from finasteride to placebo. Tolerability Available data from 1879 patients with male pattern hair loss who received either finasteride 1 mg/day or placebo for 1 year in phase III studies show that finasteride is generally well tolerated. Overall, 7.7% of finasteride and 7.0% of placebo recipients reported mild to moderate treatment-related adverse events (1.4 and 1.6% withdrew). The only events reported more frequently in finasteride than placebo recipients were sexual disorders (3.8 vs 2.1%; p = 0.041), which comprised decreased libido (1.8 vs 1.3%), ejaculation disorders (1.2 vs 0.7%) and erectile dysfunction (1.3 vs 0.7%). These resolved in many men who reported them but remained on therapy and in all men who discontinued therapy because of these adverse events. No other drug-related events were reported with an incidence ≥1% in finasteride recipients. The incidence of drug-related laboratory events was similar in finasteride and placebo groups (2.6 vs 2.4%). Finasteride had no significant effects on non-scalp body hair. Dosage and Administration Finasteride is indicated for the treatment of men with male pattern hair loss. The recommended dosage of finasteride in male pattern hair loss is 1 mg/day, taken with or without food. Daily treatment for 3 months or more is necessary before results are seen, and continued treatment is essential to sustain benefit. Furthermore, the effects of the drug are reversed within 12 months after treatment cessation. There are no current data to support the use of finasteride in women with androgenetic alopecia. Moreover, pregnant women should not be directly exposed to finasteride by using or handling crushed tablets because of the risk of hypospadias developing in a male fetus.
Studies have shown an association between oxidative stress and alopecia. Patients with alopecia generally exhibit lower levels of antioxidants in their scalp area as well as a higher lipid peroxidation index. Tocotrienols belong to the vitamin E family and are known to be potent antioxidants. Hence, a study was conducted to investigate the effect of tocotrienol supplementation on hair growth in volunteers suffering from hair loss. Twenty one volunteers were randomly assigned to orally receive 100 mg of mixed tocotrienols daily while 17 volunteers were assigned to receive placebo capsule orally. The volunteers were monitored for the number of hairs in a pre-determined scalp area as well as the weight of 20 strands of 1 cm length hair clippings at 0 (before supplementation), 4 and 8 months. The number of hairs of the volunteers in the tocotrienol supplementation group increased significantly as compared to the placebo group, with the former recording a 34.5% increase at the end of the 8-month supplementation as compared to a 0.1% decrease for the latter. Nevertheless, the cumulative weight of 20 strands of hair clippings did not differ much from the baseline for both supplementation groups at the end of the study period. In conclusion, this trial demonstrated that supplementation with tocotrienol capsules increases hair number in volunteers suffering from hair loss as compared to the placebo group. This observed effect was most likely to be due to the antioxidant activity of tocotrienols that helped to reduce lipid peroxidation and oxidative stress in the scalp, which are reported to be associated with alopecia.
Two decades ago hair growth promoters were nonexistent. From a medical perspective, little could be offered to patients with common hair loss problems. Today there are new classes of hair growth promoters with proven efficacy. A hair growth-promoting agent may prolong the anagen phase or increase matrix girth by influencing growth controls. For example, in androgenetic alopecia, drug targets may include steroid receptors, steroid metabolizing enzymes, growth factors, or cytokines, which are implicated in controlling cell cycling and conversion of terminal to miniaturized hairs. Presently, scientists are just beginning to identify the molecular control mechanisms and their location within the follicle. Further understanding of this cascade of orchestrated events is crucial for the development of more effective agents. This article reviews the current state of the art for two of the most common forms of hair loss encountered in clinical practice, androgenetic alopecia and alopecia areata. Current strategies are based on recent advances in the understanding of disordered hair growth.
Endogenous tocochromanols in extracted lipids from rice brans of the five cultivars were determined by high-performance liquid chromatography, and were investigated in relation to the fatty acid (FA) distribution of triacylglycerols (TAG) and phospholipids (PL). The dominant tocols were α-tocopherol and γ-tocotrienol, followed by α-tocotrienol and with much smaller amounts of γ-tocopherol and δ-tocotrienol. The lipids of these rice brans comprised mainly TAG (80.6–86.0wt.%), free FA (4.2–9.0wt.%), and phospholipids (5.5–6.7wt.%), whilst other components were also detected in minor proportions (0.2–2.1wt.%). The PL components included phosphatidyl choline (31.8–46.8wt.%), phosphatidyl ethanolamine (25.0–38.9wt.%) and phosphatidyl inositol (20.2–23.2wt.%). Comparison of these different cultivars showed, with a few exceptions, no significant differences (P>0.05) in FA distribution. FA distribution of TAG among the five cultivars was evident in the rice brans: unsaturated FA were predominantly concentrated at the sn-2 position and saturated FA primarily occupying the sn-1 or sn-3 position. These results suggest that the tocopherol content, lipid component, and FA distribution in rice brans are not dependent on the cultivation areas during the growing season.
High-purity gamma-oryzanol was obtained from crude rice bran oil using a normal-phase preparative scale HPLC. A reverse-phase HPLC method was used for separating the individual components of gamma-oryzanol present in rice bran oil. Ten fractions were isolated and collected using the reverse-phase HPLC method, and their structures were identified. Identification was accomplished using GC/MS with an electron impact mass spectrum after components were transformed into trimethylsilyl ether derivatives. The 10 components of gamma-oryzanol were identified as Delta(7)-stigmastenyl ferulate, stigmasteryl ferulate, cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, Delta(7)-campestenyl ferulate, campesteryl ferulate, Delta(7)-sitostenyl ferulate, sitosteryl ferulate, compestanyl ferulate, and sitostanyl ferulate. Three of these, cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, and campesteryl ferulate, were major components of gamma-oryzanol.