Preparation of Loquat Leaf Extract and Corosolic Acid

Molecular Regulation of Hair Growth Factors and Cytokines
by Loquat Leaf Extract and Its Key Component, Corosolic Acid
Nobuaki Ohto, Akinori Kiso, Yoshihito Kawashima,
Naoko Kishida, Keiko Tohi, Toshiyuki Murakami,
Yasuo Miyake, Jun Nojima and Toshimitsu Kambara
COOH
HO
HO
MARUZEN PHARMACEUTICALS CO, LTD.
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Introduction
Scientific Name: Eryobotrya japonica Lindley
Family:
Rosaceae
English Name:
Loquat
Part:
Leaf
Corosolic acid
HO
COOH
HO
Loquat (Eriobotrya japonica) leaf extract has been widely used as a cosmetic
ingredient because of its anti-inflammatory and astringent effects. To add another
application of plant extracts to cosmetics, we performed a fundamental research of a
number of plant extracts for hair growth, and found that loquat leaf extract and its key
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component, corosolic acid, are promising ingredients.
Hair Growth Cycle and Hair Growth Cycle Related Cytokines
成長期
毛母細胞
初期成長期
毛乳頭細胞
退行期
休止期
BMP Bone Morphogenetic Protein
VEGF Vascular endothelial Growth Factor
FGF
HGF
IGF
Fibroblast Growth Factor
Hepatocyto Growth Factor
Insulin Growth Factor
TGF Transforming Growth Factor
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Finished Product 1: Focused on Hair Growth Cytokines
ADENOGEN (SHISEIDO)
Quasi-drug
Active principle: adenosine
Up-regulate the FGF-7 expression
INNOVATE (LION)
Quasi-drug
One of the active principle: 6-benzyl aminopurine
Up-regulate: BMP expression
RiUP (TAISHO PHARMACEUTICAL CO., LTD.)
OTC drug
Active principle: minoxidil
Up-regulate the VEGF expression
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Finished Product 2: Focused on Hair Growth Cytokines
GROWING SHOT ETOI
(POLA)
Quasi-drug
One of the active principle:
Evolvulus alsinoides extract
Up-regulate the VEGF expression
FURORIN LIVE ACT (SHISEIDO)
Quasi-drug
One of the active principle: Sophora extract
Down-regulate the TGF-β2 expression
MOUGA SYNERGY-X (TSUMURA)
Quasi-drug
One of the active principle: bark of Morus root extract
TGF-β1 Receptor blocker
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Preparation of Loquat Leaf Extract and Corosolic Acid
The dried leaves of E. japonica were extracted with 80 % EtOH under
reflux for 2 hours. The loquat leaf extract was obtained after an
activated charcoal treatment and concentration. It was purified using
ODS-column, SiO2 column and finally by HPLC to give a corosolic acid
yield of 2.5% from the loquat leaf extract. (Fig.1)
Corosolic acid
HO
COOH
HO
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Experiments In this Study
Proliferative Effects on Human Follicle Dermal Papilla Cells
and Normal Human Epidermal Keratinocytes
Inhibitory Effect on Testosterone 5α-reductase Activity
Effects on Hair Growth Cycle on Gene Expressions in
Cultured Dermal Papilla Cells
Effects on Hair Growth Cycle on Protein Production in
Cultured Dermal Papilla Cells
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Assays for DPC and NHEK Proliferation
Normal Human Follicle Dermal Papilla Cells (DPC)
Normal Human Epidermal Keratinocytes (NHEK)
Add Loquat leaf extract or Corosolic acid
Culture
Add MTT
Extract the blue formazan
Measurement Absorbance at 570-650 nm
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Proliferative Effect on Dermal Papilla Cells (毛乳頭細胞）
Loquat leaf extract increased the DPC proliferation significantly (p<0.05);
112 % and 122 % at 2.5 µg/mL and 10 µg/mL, respectively. Corosolic
acid enhanced the significant proliferation at 1 µg/mL.
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Proliferative Effect on Epidermal Keratinocytes
Loquat leaf extract also increased the NHEK proliferation significantly (p<0.05);
112 % and 113 % at 0.156 µg/mL and 0.625 µg/mL, respectively. Corosolic acid
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enhanced the significant proliferation at 0.0625 and 0.0156 µg/mL.
Experiments In This Study
Proliferative Effects on Human Follicle Dermal Papilla Cells
and Normal Human Epidermal Keratinocytes
Inhibitory Effect on Testosterone 5α-reductase Activity
Effects on Hair Growth Cycle on Gene Expressions in
Cultured Dermal Papilla Cells
Effects on Hair Growth Cycle on Protein Production in
Cultured Dermal Papilla Cells
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Testosterone 5α-reductase
Testosterone 5αreductase
Testosterone
Transition to
the Catagen
phase
DHT
Cytokines
Androgen
Receptor
DHT
Dermal papilla cell
Hair loss
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Assay for TSR Activity
Substrate solution (testosterone + 5mM Tris-HCl buffer, 1mg/mL NADPH)
test sample or vehicle
S-9 (enzyme source)
Incubate (37oC, 30min)
Add CH2Cl2 (methylene chloride )
Centrifuge (3000rpm, 10min)
Gas chromatographic analysis
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Inhibitory Effects on TSR Activity
IC50 value
Loquat leaf extract
556 µg/mL
Corosolic acid
558 µg/mL
Loquat leaf extract and corosolic acid showed the inhibitory effect on TSR
activity in a dose dependent manner. Inhibition of TSR contributes to keep
the hair cycle on the anagen phase.
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Experiments In This Study
Proliferative Effects on Human Follicle Dermal Papilla Cells
and Normal Human Epidermal Keratinocytes
Inhibitory Effect on Testosterone 5α-reductase Activity
Effects on Hair Growth Cycle on Gene Expressions in
Cultured Dermal Papilla Cells
Effects on Hair Growth Cycle on Protein Production in
Cultured Dermal Papilla Cells
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Assay for Hair Growth Cycle on Gene Expressions
Normal Human Follicle Dermal Papilla Cells (DPC)
add test sample
Incubation for 2 hours
Isolation of total RNA
Evaluation of mRNA levels by RT-PCR
RT-PCR: Reverse Transcription-Polymerase Chain Reaction
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Effects on Hair Growth Cycle Concerning Gene Expressions
µg/mL
Significant changes
Up-regulated gene:
FGF-7, VEGF and BMP-2
Down-regulated gene:
FGF-5
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Effects on Hair Growth Cycle on Gene Expressions
FGF-7 (A), VEGF (B) and BMP-2 (C) mRNA
expressions were up-regulated by 130 %, 138 % and
147 % with treatment of loquat leaf extract,
respectively, and corosolic acid also increased the
same levels that of these factors at low concentrations.
On the other hand, the mRNA expression level of
FGF-5 (D) was down-regulated by 80 % and 78 %
with treatment of loquat leaf extract and corosolic acid,
respectively.
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Experiments in This Study
Proliferative Effects on Human Follicle Dermal Papilla Cells
and Normal Human Epidermal Keratinocytes
Inhibitory Effect on Testosterone 5α-reductase Activity
Effects on Hair Growth Cycle Concerning Gene
Expressions in Cultured Dermal Papilla Cells
Effects on Hair Growth Cycle on Protein Production in
Cultured Dermal Papilla Cells
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Assay for Hair Growth Cycle on Protein Production
Normal Human Follicle Dermal Papilla Cells (DPC)
Add test sample
Incubation for 72 hours
Evaluation of promoting activity on FGF-7 by ELISA
method using anti-human FGF-7 antibody (R&D system)
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Effects on FGF-7 Protein Production
Loquat leaf extract and Corosolic acid surely up-regulated the protein
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level of FGF-7.
Conclusion
Loquat leaf extract and its component, corosolic acid,
increased the proliferation of DPC and NHEK significantly.
Loquat leaf extract and corosolic acid inhibited the
testosterone 5α-reductase activity in a dose dependent
manner.
Loquat leaf extract and corosolic acid up-regulated the
mRNA expression levels of FGF-7, VEGF and BMP-2,
down-regulated that of FGF-5 in dermal papilla cells.
Loquat leaf extract and its key component, corosolic acid,
have hair growth potential and can be used in hair
growing cosmetic products.
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Five Approaches for Hair Growth Promotion
Stimulation of
FGF-7, BMP-2 expression
FGF-7 and BMP-2 induces
the proliferation of hair matrix cells
and contributes to the extension
of anagen phase.
Inhibition of TSR activity
TSR converts testosterone into
DHT which triggers catagen phase.
Inhibition of TSR contributes to keep
the hair cycle on anagen phase.
Suppression of
FGF-5 expression
FGF-5 expression triggers the
transition from anagen phase to
catagen phase. Suppression of
FGF-5 expression leads to keep
the hair cycle on anagen phase.
Proliferation of
Dermal papilla cells
Proliferation of DPC contributes to
enlarge the dermal papilla which
relate to thick and strong hair.
Stimulation of VEGF expression
VEGF plays an important role in the improvement of vascular network.
VEGF is also known to not only autocrine growth factor for dermal papilla cells,
but also paracrine growth factor for hair matrix cells.
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Product Information
■About Product
INCI NAME
Content
ERIOBOTRYA JAPONICA LEAF EXTRACT
LOQUAT LEAF EXTRACT 1.00 %
1,3-Butylene Glycol
99.00 %
■Safety Test
Test Items
Human patch test
Dosage
Evaluation
10%
Negative
We were engaged in finding a hair growth material using
several biological assay methods, and found Loquat leaf
extract with different mechanisms of action.
We hope loquat leaf extracts and their active principles,
corosolic acid, will be useful ingredients for total hair care
cosmetic products.
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THANK YOU FOR YOUR TIME AND ATTENTION.
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