jae-myo yu, hyeon-jeong, kim , eun-su lee, yong-hun cho ...herbnoori.img14.kr/img/lab/kaw1.pdf ·...
Post on 27-Mar-2020
9 Views
Preview:
TRANSCRIPT
Jae-myo Yu, Hyeon-jeong, Kim1, Eun-su Lee, Yong-hun Cho, Jae-yoon Jang, Seul-ah Seo, Hyeon-guk Jeong, Yu-hyeon
Shin1, Young-jae Cho2, Yong-ha Jeong3, Bong-jeun An*
Dept. of Cosmeceutical Science, Daegu Haany University, 1Institute of technology, Herbnoori.Co.,Ltd., 2School of Food Science & Biotechonology / Food & Bio-Industry Research Institute, Kyungpook National
University, 3Laboratory , Thuringen Korea
This study investigated about the whitening activity of
the solvent fraction from 70% acetone extract of Kaki
Calyx. Fraction layer using four solvents which are
chloroform (KCC), ethyl acetate (KCE), n-buthyl
alchol (KCB) and water (KCH) were separated. The
cell viability of KCB fraction identified each over 90%
from 5 µg/mL to 100 µg/mL concentration. The cell
tyrosinase activity of KCB fraction was reduced from
95% to 84% as the concentration increase. The melanin
contents of KCE, KCB, KCH fractions demonstrated
less than 50% at a concentration of 100 µg/mL.
Inhibition cAMP levels of KCB fraction was
significantly reduced from 50 µg/mL concentration.
Western Blotting and PCR experiments of whitening
effect reduced in a dose-dependently. The immunofluor-
escence were identified as to be effective in whitening.
Fig. 1. The procedure for extraction from Kaki Calyx. Fig. 2. The procedure for fraction from Kaki Calyx extracted with
70% acetone.
Extraction
(70% methanol room
temperature, 24h)
Supernatant
Extraction
(Distilled water,
85 °C, 3h)
Kaki Calyx (100g)
Extraction
(70% acetone room
temperature, 24h)
Filtration
Freeze drying
Ethanol extract
Extraction
(70% ethanol room
temperature, 24h)
Evaporation
Supernatant Supernatant Supernatant
Filtration Filtration Filtration
Evaporation Evaporation Evaporation
Freeze drying Freeze drying Freeze drying
Water extract Methanol extract Acetone extract
Precipitate Precipitate Precipitate Precipitate
0
20
40
60
80
100
120
Con 5 10 50 100
Rea
ctiv
e m
RN
A l
evel
s
(% o
f G
AP
DH
)
TRP-1
GAPDH
TRP-1
GAPDH
α-MSH + + + + +
Kaki Calyx (µg/mL)
α-MSH + + + + +
Kaki Calyx (µg/mL)
0
20
40
60
80
100
120
Con 5 10 50 100
Rea
ctiv
e p
rote
in l
evel
s
(% o
f G
AP
DH
)
Fig. 7. TRP-1 protein and mRNA expression rate of solvent fraction
from Kaki Calyx extracts on melanoma cell (B16F10).
B16F10 were treated Kaki Calyx n-BuOH layer for 24hrs. Histogram show the
densitometric of TRP-1 protein normalized to GAPDH. One-way ANOVA was used for
comparisons of multiple group means followed by t-test (significant as compared to
control. *p<0.05, **p<0.01).
0
20
40
60
80
100
120
Con 5 10 50 100
Rea
ctiv
e m
RN
A l
evel
s
(% o
f G
AP
DH
)
MITF
GAPDH
MITF
GAPDH
α-MSH + + + + +
Kaki Calyx (µg/mL)
α-MSH + + + + +
Kaki Calyx (µg/mL)
0
20
40
60
80
100
120
Con 5 10 50 100
Rea
ctiv
e p
rote
in l
evel
s
(% o
f G
AP
DH
)
Fig. 8. MITF protein and mRNA expression rate of solvent fraction
from Kaki Calyx extracts on melanoma cell (B16F10).
B16F10 were treated Kaki Calyx n-BuOH layer for 24hrs. Histogram show the
densitometric of MITF protein normalized to GAPDH. One-way ANOVA was used for
comparisons of multiple group means followed by t-test (significant as compared to
control. *p<0.05, **p<0.01).
Kaki Calyx
Aceton extract
CHCl3 - Fr H2O - Fr
EtOAc - Fr
n-BuOH - Fr
H2O - Fr
H2O - Fr
Added : EtOAc (1:1)
Added : CHCl3 (1:1)
Added : n-BuOH
(1:1)
Fig. 5. Inhibition melanin synthesis of solvent fraction from
Kaki Calyx extracts on melanoma cell (B16F10).
KCA : Kaki Calyx extracted with acetone.
KCC : CHCl3 layer of Kaki Calyx extracted with acetone.
KCE : EtOAc layer of Kaki Calyx extracted with acetone.
KCB : n-BuOH layer of Kaki Calyx extracted with acetone.
KCH : H2O layer of Kaki Calyx extracted with acetone.
Results are ± means S.D. of triplicate date .
0
20
40
60
80
100
120
Con 5 10 50 100
Mel
an
in c
on
ten
ts (
%)
Concentration (μg/mL)
KCA KCC KCE KCB KCW
Fig. 4. Inhibition rate of solvent fraction from Kaki Calyx
extracts on melanoma cell (B16F10) originated tyrosinase.
KCA : Kaki Calyx extracted with acetone.
KCC : CHCl3 layer of Kaki Calyx extracted with acetone.
KCE : EtOAc layer of Kaki Calyx extracted with acetone.
KCB : n-BuOH layer of Kaki Calyx extracted with acetone.
KCH : H2O layer of Kaki Calyx extracted with acetone.
Results are ± means S.D. of triplicate date .
0
20
40
60
80
100
120
Con 5 10 50 100
Tyro
sin
ase
act
ivit
y (
%)
Concentration (µg/mL)
KCA KCC KCE KCB KCW
Fig. 9. Indirect immunofluorescence and confocal microscopy analysis demonstrating the effect of
n-BuOH extracted from Kaki Calyx on α-MSH-induced sub cellular localization of MITF in
B16F10 cells. A: blue channel (DAPI), B: green channel (FITC), C:red channel and the merge. Results are representatives of three independent
experiments.
Fig. 6. Inhibition cAMP levels of solvent fraction from Kaki
Calyx extracts on melanoma cell (B16F10).
KCB : n-BuOH extracted from Kaki Calyx, After B16F10 cells (1X106 cells)
were started in serum free medium for 1 h the cells were treated with 5, 10,
50 and 100 µg/mL of n-BuOH extracted of Kaki Calyx for 48h. One-way
ANOVA was used for comparisons of multiple group means followed by t-
test(significant as compared to control. *p<0.05, **p<0.01).
Fig. 3. Cell viability of solvent fraction from Kaki Calyx
extracts on melanoma cell (B16F10).
KCC : CHCl3 layer of Kaki Calyx extracted with acetone.
KCE : EtOAc layer of Kaki Calyx extracted with acetone.
KCB : n-BuOH layer of Kaki Calyx extracted with acetone.
KCH : H2O layer of Kaki Calyx extracted with acetone.
Results are ± means S.D. of triplicate date .
0
20
40
60
80
100
120
5 10 50 100 500 1000
Cel
l via
bil
ity
(%
)
Concentration (µg/mL)
KCC KCE KCB KCH
1. Whitening activity were evaluated in inhibition of intracellular tyrosinase activity,
inhibition of melanin synthesis, inhibition cAMP levels, western blotting, PCR and
immunofluorescence. Overall, the whitening activities of n-BuOH fraction showed
a higher than those of CHCl3, EtOAc, H2O fractions.
2. Cell tyrosinase activity inhibition rate was reduced to a dose-dependent manner by
KCB. Then, KCB fraction showed about 84% at 100 µg/ml.
3. Inhibition melanin synthesis of solvent fraction was reduced in all sectors. Melanin
contents of KCE, KCB and KCH fractions showed inhibition rate in each of 51%,
44% and 49% at 100 µg/mL.
4. Inhibition cAMP levels of KCB fraction investigated about 60% at 50 µg/mL.
5. mRNA expression rate of MITF was about 80% at 100 µg/mL.
A Merge C B
Con
Nor
KCB
100
0
20
40
60
80
100
120
Con 5 10 50 100
cAM
P l
evel
s (%
)
Concentration (µg/mL)
* * * *
1. Cell viability
: measured by Carmichael1)
2. Protein expression rate
: measured by Western blot
3. mRNA expression rate
: measured by RT-PCR
4. cAMP levels
: measured by cAMP immunoassay kit (Cayman,
Ann Arbor, MI, USA)
* *
* * * * * * * *
1) Carmichael J, DGraff WG, Gazdar AF, Minna JD and Mitchell JB (1987)
Evaluation of a tetrazolium based semiautomated colorimetric assay : assessment
of chemosensitivity testing. Cancer Res. 47(4), 936-942.
top related