U.S. patent application number 10/539012 was filed with the patent office on 2007-07-05 for agent for controlling bcl-2 expression comprising ginsenoside f1 as an active component.
Invention is credited to Hui-Kyoung Chang, Si-Young Cho, Duck-Hee Kim, Su-Jung Kim, Eun-Hee Lee, Tae-Ryong Lee, Eui-Seok Shin, Young-Chul Sim, Kwang-Sik Woe, Myeong-Hoon Yeom.
Application Number | 20070155829 10/539012 |
Document ID | / |
Family ID | 36241075 |
Filed Date | 2007-07-05 |
United States Patent
Application |
20070155829 |
Kind Code |
A1 |
Cho; Si-Young ; et
al. |
July 5, 2007 |
Agent for controlling bcl-2 expression comprising ginsenoside f1 as
an active component
Abstract
The present invention relates to an agent for controlling Bcl-2
expression comprising ginsenoside F1
(20-O-.beta.-D-glucopyranosyl-20(S)-protopanaxatriol) represented
by the following formula 1 as an active component. [Formula 1]
##STR1##
Inventors: |
Cho; Si-Young; (Yongin-si,
KR) ; Lee; Eun-Hee; (Yongin-si, KR) ; Kim;
Su-Jung; (Yongin-si, KR) ; Shin; Eui-Seok;
(Yongin-si, KR) ; Chang; Hui-Kyoung; (Yongin-si,
KR) ; Kim; Duck-Hee; (Seoul, KR) ; Yeom;
Myeong-Hoon; (Yongin-si, KR) ; Woe; Kwang-Sik;
(Chungju-si, KR) ; Lee; Tae-Ryong; (Suwon-si,
KR) ; Sim; Young-Chul; (Seongnam-si, KR) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Family ID: |
36241075 |
Appl. No.: |
10/539012 |
Filed: |
December 27, 2003 |
PCT Filed: |
December 27, 2003 |
PCT NO: |
PCT/KR03/02859 |
371 Date: |
April 27, 2006 |
Current U.S.
Class: |
514/468 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 35/04 20180101; A61P 39/00 20180101; A61P 17/16 20180101; A61P
43/00 20180101; A61K 31/70 20130101 |
Class at
Publication: |
514/468 |
International
Class: |
A61K 31/365 20060101
A61K031/365 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 28, 2002 |
KR |
10-2002-0085716 |
Claims
1. An agent for controlling Bcl-2 expression comprising ginsenoside
F1 as an active component.
2. The agent according to claim 1, wherein the Bcl-2 expression is
controlled because the ginsenoside F1 controls Brn-3a
expression.
3. Apoptosis inhibitor comprising gensenoside F1 as an active
component, which inhibits apoptosis induced by low-dose ultraviolet
radiation.
4. The apoptosis inhibitor according to claim 1, wherein the
apoptosis is inhibited because the ginsenoside F1 inhibits decrease
of Bcl-2 expression.
5. Apoptosis promoter comprising gensenoside F1 as an active
component, which promotes apoptosis induced by high-dose
ultraviolet radiation.
6. An agent for controlling Brn-3a expression comprising
ginsenoside F1 as an active component.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to an agent for controlling
Bcl-2 expression comprising ginsenoside F1
(20-O-.beta.-D-glucopyranosyl-20(S)-protopanaxatriol) represented
by the following formula 1 as an active component. ##STR2##
BACKGROUND OF THE INVENTION
[0002] Ultraviolet radiation is a part of solar rays with 200-400
nm of wavelength and is a part of the electromagnetic spectrum;
especially UVB (Ultraviolet-B) with 280-320 nm of wavelength is the
major part of the ultraviolet radiation to cause skin aging leading
to skin-burn and skin cancer. When the skin is exposed to
ultraviolet radiation, DNA, proteins, lipids, etc., in cells are
damaged and thereby generate sunburn-cells. These sunburn-cells
undergo apoptosis, accompanied by DNA fragmentation, activation of
caspase, and the like. High-dose radiation on the cell causes
serious DNA damages that are not recovered from; and the apoptosis
prevents the cell from developing to a tumor by inducing the cell
to die. Therefore, in order to prevent cancer and maintain cell
homeostasis, it is very important to induce or to prevent apoptosis
of the cell selectively according to the degree of cell
damages.
[0003] Bcl-2 plays a very important role in the process of
apoptosis of the skin cell. The Bcl-2 gene encodes 26 kDa protein
present in a nuclear membrane and an outer membrane of
mitochondria. Bcl-2 is a protein that inhibits apoptosis of a cell
by adhering to a protein such as Bax, which accelerates the
apoptosis, to inhibit its function. Therefore, apoptosis of a cell
can be determined by the concentration ratio of Bcl-2 and Bax.
[0004] UVB irradiation has been known to decrease Bcl-2 expression
of human keratinocyte. Furthermore, Bcl-2-transfected HaCaT cells
or Bcl-2-overexpressing transgenic mice were shown to be resistant
to UVB-induced apoptosis. However, over-expression of Bcl-2
prevents apoptosis of a cell with serious DNA damage and thereby
causes a cancer. Therefore, it is very important to control the
expression of Bcl-2 selectively.
[0005] Up to now, techniques or methods for controlling the
expression of Bcl-2 have not been widely disclosed compared with
the functions of Bcl-2. Only some transcription factors such as
pRb, c-myb and Brn-3a in nerve cells have been disclosed. In
particular, it is reported that Bm-3a, a type IV POU domain
transcription factor, adheres to a P2 promoter of Bcl-2 and
controls the expression of a Bcl-2 gene to protect the nerve cells
from apoptosis.
[0006] However, mechanisms or factors to control the expression of
Bcl-2 in HaCaT cells derived from human skin have not yet been
disclosed.
[0007] In addition, a material for controlling the expression of
Bcl-2 that is not toxic and is easily applied to the human body
have not yet been disclosed.
SUMMARY OF THE INVENTION
[0008] Under theses circumstances, the present inventors found that
ginsenoside F1, which obtained from purified ginseng saponin,
protects human HaCaT cells from UVB-induced apoptosis by
maintaining constant levels of Bcl-2. That is, the present
inventors found that ginsenoside F1 controls the expression of
Bcl-2 and thereby inhibits the apoptosis of cells, and so
accomplished the present invention.
[0009] Therefore, an object of the present invention is to provide
an agent for controlling Bcl-2 expression comprising ginsenoside F1
as an active component.
[0010] Another objection of the present invention is to provide a
promoter or an inhibitor of apoptosis comprising ginsenoside F1 as
an active component.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0011] The present invention provides an agent for controlling
Bcl-2 expression comprising ginsenoside F1 represented by the
following formula 1 as an active component. ##STR3##
[0012] Ginsenoside F1 protects these cells against low-dose
radiation of UVB-induced apoptosis by maintaining constant levels
of Brn-3a and the corresponding inhibition of Bcl-2 downregulation.
That is, ginsenoside F1 itself does not promote the Bcl-2
expression, but inhibits the downregulation of Bcl-2 caused by
ultraviolet radiation. On the contrary, under the high-dose
radiation of ultraviolet rays, it induces the decrease of Bcl-2
expression to promote apoptosis.
[0013] In conclusion, ginsenoside F1 inhibits the decrease of Bcl-2
expression under low-dose radiation of ultraviolet rays resultingly
to prevent apoptosis of cells; however induces apoptosis under the
high-dose radiation of ultraviolet rays, on the contrary; and
thereby prevents skin cancer. Therefore, ginsenoside F1 may be used
as an anti-aging material inhibiting cell damages.
[0014] The present invention confirmed that ginsenoside F1 controls
the expression of Brn-3a, a transcription factor of Bcl-2, and
thereby can maintain the degree of Bcl-2 expression to a normal
level.
[0015] Therefore, ginsenoside F1 can prevent apoptosis by
maintaining the degree of Bcl-2 expression in a cell to desirable
level. However, ginsenoside F1 itself does not promote the Bcl-2
expression. These show that ginsenoside F1 has the efficacies of
preventing apoptosis and cell damages caused by the radiation of
ultraviolet rays without generating skin cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a graph showing viabilities of HaCaT skin cells
obtained from the result of MTT assay, wherein cells treated with
ginsenoside F1 and exposed to ultraviolet radiation were compared
with a control (cells) untreated with ginsenoside F1.
[0017] FIG. 2 shows the changes of shapes of the cells, wherein
cells treated with ginsenoside F1 and exposed to ultraviolet
radiation were compared with a control untreated with ginsenoside
F1.
[0018] FIG. 3 shows the degrees of DNA fragmentation of cells,
wherein cells treated with ginsenoside F1 and exposed to
ultraviolet radiation were compared with a control untreated with
ginsenoside F1.
[0019] FIG. 4 shows the degrees of PARP segmentation in cells
obtained from the result of Western blotting, wherein cells treated
with ginsenoside F1 and exposed to ultraviolet radiation were
compared with a control untreated with ginsenoside F1.
[0020] FIG. 5 shows the degrees of Bcl-2 expression with mRNA
level, wherein cells treated with ginsenoside F1 and exposed to
ultraviolet radiation were compared with a control untreated with
ginsenoside F1.
[0021] FIG. 6 shows the degrees of the expression of Bm-3a, a
transcription factor of Bcl-2, obtained from Western blotting,
wherein cells treated with ginsenoside F1 and exposed to
ultraviolet radiation were compared with a control untreated with
ginsenoside F1. Hsp 70 means that a same amount of protein was
used.
PREFERRED EMBODIMENT OF THE INVENTION
[0022] Hereinafter, the present invention is described in detail
with reference to examples. However, the scope of the present
invention is not restricted by the examples.
REFERENCE EXAMPLE 1
Preparation of Purified Ginseng Saponin
[0023] 2 kg of Red ginseng (KT&G Corporation, 6 year old red
ginseng) was added into 4 l of methanol containing water, and
refluxed 3 times then deposited for 6 days at 15.degree. C.
Residues and remainders were separated by filtration and
centrifugation, then the remainders were concentrated under reduced
pressure to obtain extract. The extract was suspended into the
water and re-extracted with 1 l of ether 5 times to remove
pigments, then water layer thereof was extracted with 500 ml of
1-butanol 3 times. The above-obtained 1-butanol layer was treated
with 5% of KOH and washed with distilled water then concentrated
under reduced pressure to obtain 1-butanol extract. The extract
(1-butanol extract) was dissolved in a small amount of methanol,
and a large amount of ethylacetate was added thereto to obtain
precipitation. The precipitation was dried to obtain 70 g of
purified ginseng saponin extract.
REFERENCE EXAMPLE 2
Preparation of Gensenoside F1
[0024] 10 g of purified ginseng saponin obtained from the Reference
Example 1 was dissolved in 1000 ml of citrate buffer solution (pH
4.0), 15 g of naringinase obtained from penicillium sp. was added
thereto and reacted for 48 hours while stirring at 40.degree. C.
After the reaction, reaction mixture was heated for 10 minutes to
inactivate the enzyme, then the reaction mixture was extracted 3
times with same amount of ethylacetae and concentrated. The product
obtained underwent column chromatography (chloroform:methanol=9:1)
and finally separated 1.5 g of ginsenoside F1.
EXAMPLE 1
Inhibition of Apoptosis of HaCaT Cells Induced by Ultraviolet
Radiation, when Treated with Ginsenoside F1
<Step 1> Cell Line and Cell Culture
[0025] Human keratinocyte, HaCaT (obtained from Dr. Fusenig in
German Cancer Research Center (DKFZ)) was cultured in Dulbecco's
modified Eagle's Medium (DMEM; Gibco 1210-0038) containing 10% of
fetal bovin serum at 37.degree. C. and 5% of CO.sub.2.
<Step 2> Inhibition of Apoptosis of Cells Induced by
Ultraviolet Radiation, when Treated with Ginsenoside F1
[0026] Cells cultured in step 1 were treated with trypsin to obtain
single cell suspension, and seeded into 6-well flask with
2.times.10.sup.5/well, then cultured for 24 hours. After that, the
cells were cultured again in a new DMEM not containing fetal bovin
serum for 24 hours, then treated with 1, 5, 10 .mu.M of ginsenoside
F1. Ginsenoside F1 was dissolved in 100% ethanol and added to be
1/1000 of the medium concentration. After 24 hours of treatment
with ginsenoside F1, the cell cultures were washed with phosphate
buffered saline (PBS), and exposed to 60-120 mJ/cm.sup.2 of UVB
radiation with a state containing PBS. Then, the PBS was removed
and medium was changed with a fresh one containing the same
concentration of ginsenoside F1. In addition, the cells untreated
with ginsenoside F1 were cultured as a control. After 24 hours of
UVB radiation, 3-[4,5-dimethyl tetrazole]-2,5-diphenyltetrazolium
bromide (MTT, Sigma) was added to all the cells treated and
untreated with ginsenoside F1, then cultured 4 hours at 37.degree.
C. After culture, the cells were dissolved with dimethylsulfoxide,
then optical density (OD) of formazan dye generated at 540 nm was
measured with ELISA reader (Thermo Max, Molecular Devices Co.). OD
value of the cells not-exposed to the UVB was given 100%, then the
relative values of the other cells were calculated and determined
as viabilities thereof. The results are shown in FIG. 1. The cells
treated with ginsenoside F1 showed 1.5 times more inhibition of
apoptosis compared with those untreated, when exposed to the UVB.
However, under the 120 mJ/cm.sup.2 of UVB radiation, the cells
treated with ginsenoside F1 showed more apoptosis than those
untreated.
EXAMPLE 2
Inhibition of DNA Fragmentation of HaCaT Cells Induced by
Ultraviolet Radiation, when Treated with Ginsenoside F1
<Step 1> Cell Line and Cell Culture
[0027] Same procedures of step 1 of Example 1 were performed.
<Step 2> Inhibition of DNA Fragmentation of HaCaT Cells
Induced by Ultraviolet Radiation, when Treated with Ginsenoside
F1
[0028] Cells cultured in step 1 were treated with trypsin to obtain
single cell suspension, and seeded into 6-well flask with
2.times.10.sup.5/well, then cultured for 24 hours. After that the
cells were cultured again in a new DMEM not containing fetal bovin
serum for 24 hours, then treated with 5 .mu.M of ginsenoside F1.
After 24 hours of treatment with ginsenoside F1, the cultures were
washed with phosphate buffered saline (PBS), and exposed to 60
mJ/cm.sup.2 of UVB radiation with a state containing PBS. Then, the
PBS was removed and medium was changed with a fresh one containing
the same concentration of ginsenoside F1. In addition, the cells
untreated with ginsenoside F1 were cultured as a control.
[0029] After 24 hours of UVB radiation, all the cells treated and
untreated with ginsenoside F1 were washed with phosphate buffered
saline (PBS), and treated with phosphate buffered saline containing
4% of paraformaldehyde for 15 minutes to fix the cells. Then the
cells were washed with phosphate buffered saline (PBS), and reacted
in phosphate buffered saline (PBS) containing 0.05% of tween 20 and
0.2% of BSA for 15 minutes. Terminal deoxynucleotide transferase
reaction solution (TUNEL Apoptosis detection kit, Upstate, USA) was
added thereto and reacted for 1 hour. Reaction termination buffer
(TUNEL Apoptosis detection kit, Upstate, USA) was added to stop the
reaction, and blocking solution (TUNEL Apoptosis detection kit,
Upstate, USA) was added thereto, then reacted for 20 minutes.
Avidin-FITC (TUNEL Apoptosis detection kit, Upstate, USA) was added
thereto and reacted for 30 minutes, then washed with phosphate
buffered saline.
[0030] Counterstain was performed with 500 nM of propidium iodide
solution and observed with microscope. By observing about 200
cells, the number of DNA fragmentation in the total cells was
calculated. The cells treated with ginsenoside F1 showed 2.4 times
decrease of DNA fragmentation compared with those untreated, when
exposed to the UVB. The results are shown in FIG. 3.
EXAMPLE 3
Inhibition of PARP Protein Segmentation Induced by Ultraviolet
Radiation, when Treated with Ginsenoside F1
<Step 1> Cell Line and Cell Culture
[0031] Same procedures of step 1 of Example 1 were performed.
<Step 2> Inhibition of PARP Protein Segmentation Induced by
Ultraviolet Radiation, when Treated with Ginsenoside F1
[0032] Cells cultured in step 1 were treated with trypsin to obtain
single cell suspension, and seeded into 6-well flask with
2.times.10.sup.5/well, then cultured for 24 hours. After that the
cells were cultured again in a new DMEM not containing fetal bovin
serum for 24 hours, then treated with 5 .mu.M of ginsenoside F1.
After 24 hours of treatment with ginsenoside F1, the cell cultures
were washed with phosphate buffered saline (PBS), and exposed to 60
mJ/cm.sup.2 of UVB radiation with a state containing PBS. Then, the
PBS was removed and medium was changed with a fresh one containing
5 .mu.M of ginsenoside F1. In addition, the cells untreated with
ginsenoside F1 were cultured as a control.
[0033] After 24 hours of UVB radiation, all the cell cultures
treated and untreated with ginsenoside F1 were washed with
phosphate buffered saline (PBS), and cells were collected by
treating with trypsin, and which were treated with 8M of urea, 2%
of CHAPS, 50 mM of DTT, 2M of thiourea, 2 mM of PMSF and 500 .mu.l
of protein extract buffer solution of 100 .mu.g/ml leupeptine, then
left at room temperature for 10 minutes; and then centrifuged with
15,000 g of gravity for 10 minutes at 4.degree. C., and supernatant
was collected then protein was quantitated with BIO-Rad Protein Dye
Reagent.TM.. 20 .mu.g of protein was separated according to sizes
thereof with 8% SDS-PAGE, and blotted on PDF (BioRad) membrane with
50V for 12 hours. The blots obtained were blocked with 5% of
fat-free milk solution for 1 hour and performed Western blotting
with enhanced chemiluminescence (ECL) kit of Amersham Bioscience by
using polyclonal anti-PARP (Santa Cruz) as primary antibody and
using anti-rabbit IgG (amersham) combined with horse radish
peroxidase as secondary antibody.
[0034] The blots reacted were exposed to X-ray Fuji film and
developed to observe the degree of protein expression. Bands on the
film were scanned with PowerLook 2100 XL (umax) and analyzed with
an image-analyzing program of ImageMaster 2D Elite (Amersham
Bioscience). The amount of PARP protein segmentation was estimated
with a relative value compared with that of control. The cells
treated with ginsenoside F1 showed 1.4 times decrease of PARP
protein segmentation compared with those untreated, when exposed to
the UVB. The results are shown in FIG. 4.
EXAMPLE 4
Inhibition of Decrease of Bcl-2 Expression Induced by Ultraviolet
Radiation, when Treated with Ginsenoside F1
<Step 1> Cell Line and Cell Culture
[0035] Same procedures of step 1 of Example 1 were performed.
<Step 2> Inhibition of Decrease of Bcl-2 Expression Induced
by Ultraviolet Radiation, when Treated with Ginsenoside F1
[0036] Cells cultured in step 1 were treated with trypsin to obtain
single cell suspension, and seeded into 6-well flask with
2.times.10.sup.5/well then cultured for 24 hours. After that the
cells were cultured again in a new DMEM not containing fetal bovin
serum for 24 hours, then treated with 5 .mu.M of ginsenoside F1.
After 24 hours of treatment with ginsenoside F1, the cell cultures
were washed with phosphate buffered saline (PBS), and exposed to 60
mJ/cm.sup.2 of UVB radiation with a state containing PBS. Then, the
PBS was removed and medium was changed with a fresh one containing
5 .mu.M of ginsenoside F1. In addition, the cells untreated with
ginsenoside F1 were cultured as a control.
[0037] After 24 hours of UVB radiation and excluding UVB radiation
on a predetermined time interval, all the cell treated and
untreated with ginsenoside F1 were washed with phosphate buffered
saline (PBS), and mRNAs were extracted with Oligotex Direct mRNA
kits (QIAGEN, Hilden, Germany) then performed quantitative reverse
transcription PCR (RT-PCR). The sequences of Bcl-2 and
glyceraldehydes-3-phosphate dehydrogenase (GAPDH) primer pairs to
performed the quantitative RT-PCR of are shown in table 1.
TABLE-US-00001 TABLE 1 Bcl-2 and GAPDH primers to performed
quantitative RT-PCR Primer Sequence Bcl-2 forward primer
5'-TACGATAACCGGGAGATAGTGA-3' (nucleotides 56-77 of human Bcl-2
cDNA) Bcl-2 reverse Primer 5'-CAGGTGCCGGTTCAGGTACT-3' (nucleotides
566-586 of human Bcl-2 cDNA) GAPDH forward primer
5'-CAACTACATGGTTTACATGTTCC-3' (nucleotides 174-194 of human GAPDH
cDNA) GAPDH reverse primer 5'-GGACTGTGGTCATGAGTCCT-3' (nucleotides
570-589 of human GAPDH cDNA)
[0038] 1 .mu.g of mRNA was added to 25 .mu.l of reverse
transcription reaction buffer solution with 50 mM of Tris-HCl (pH
8.3), 75 mM of KCl, 3 mM of MgCl.sub.2, 0.1M of DTT, 10 mM of dNTP
and 40units/ml of RNase inhibitor, then 0.5 .mu.g/ml
oligo(dT).sub.16 primer and 200 units of SuperScript II (GiboBRL)
reverse transcription polymerase were added thereto and reacted for
1 hour at 42.degree. C. 2.5 .mu.l of reverse transcription reaction
solution was mixed to 50 .mu.l PCR buffer solution with 50 mM of
KCl, 10 mM of tris-HCl (pH 8.3), 5 mM of MgCl.sub.2 and 100 .mu.M
of dNTP, and 10 .mu.M of primer and 0.5U of Taq DNA polymerase were
added thereto, then repeated 30 cycles of; 30 sec at 95.degree. C.,
30 sec at 58.degree. C. and 30 sec at 72.degree. C. PCR products
were electrophoresed through agarose gels and scanned, then
analyzed with the image-analyzing program of ImageMaster 2D Elite
(Amersham Bioscience). The degree of Bcl-2 expression was estimated
with a relative value compared with that of GAPDH.
[0039] When cells were treated with ginsenoside F1 only, expression
of Bcl-2 according to time-passage was not different from that of
the cells untreated. When UVB was radiated, the expression of Bcl-2
in the cells untreated with ginsenoside F1 was decreased, and after
24 hours later Bcl-2 was hardly expressed. However, in the cells
treated with ginsenoside F1, the expression of Bcl-2 was maintained
almost the same even when UVB was radiated compared when UVB was
not radiated. That is, when the cells were treated with ginsenoside
F1, the expression of Bcl-2 increased 3 times compared with that
untreated. The results are shown in FIG. 5.
EXAMPLE 5
Inhibition of Decrease of Brn-3a Expression Induced by Ultraviolet
Radiation, when Treated with Ginsenoside F1
<Step 1> Cell Line and Cell Culture
[0040] Same procedures of step 1 of Example 1 were performed.
<Step 2> Inhibition of Decrease of Brn-3a Expression Induced
by Ultraviolet Radiation, when Treated with Ginsenoside F1
[0041] Cells cultured in step 1 were treated with trypsin to obtain
single cell suspension, and seeded into 6-well flask with
2.times.10.sup.5/well, then cultured for 24 hours. After that the
cells were cultured again in a new DMEM not containing fetal bovin
serum for 24 hours, then treated with 5 .mu.M of ginsenoside F1.
After 24 hours of treatment with ginsenoside F1, the cell cultures
were washed with phosphate buffered saline (PBS), and exposed to 60
mJ/cm.sup.2 of UVB radiation with a state containing PBS. Then, the
PBS was removed and medium was changed with a fresh one containing
5 .mu.M of ginsenoside F1. In addition, the cells that are not
treated with ginsenoside F1 were cultured as a control.
[0042] After 24 hours of UVB radiation, all the cell cultures
treated and untreated with ginsenoside F1 were washed with
phosphate buffered saline (PBS), and cells were collected by
treating with trypsin, and which were treated with 8M of urea, 2%
of CHAPS, 50 mM of DTT, 2M of thiourea, 2 mM of PMSF and 5000 of
protein extract buffer solution of 100 .mu.g/ml leupeptine, then
left at room temperature for 10 minutes; and then centrifuged with
15,000 g of gravity for 10 minutes at 4.degree. C., and supernatant
was collected then protein was quantitated with BIO-Rad Protein Dye
Reagent.TM.. 20 .mu.g of protein was separated according to sizes
thereof with 8% SDS-PAGE, and blotted on PDF (BioRad) membrane with
50V for 12 hours. The blots obtained were blocked with 5% of
fat-free milk solution for 1 hour and performed Western blotting
with enhanced chemiluminescence (ECL) kit of Amersham Bioscience by
using polyclonal anti-PARP (Santa Cruz) as primary antibody and
using anti-rabbit IgG (amersham) combined with horse radish
peroxidase as secondary antibody.
[0043] The blots reacted were exposed to X-ray Fuji film and
developed to observe the degree of protein expression. Bands on the
film were scanned with PowerLook 2100 XL (umax) and analyzed with
an image-analyzing program of ImageMaster 2D Elite (Amersham
Bioscience). The expression of Bm-3a decreased when UVB was
radiated, however the expression was recovered when treated with
ginsenoside F1. The results are shown in FIG. 6.
[0044] As described above, ginsenoside F1 of the present invention
inhibits the decrease of Bcl-2 expression caused by ultraviolet
radiation to inhibit apoptosis induced from ultraviolet radiation,
on the contrary under the high-dose ultraviolet radiation,
ginsenoside F1 promotes apoptosis of cell to prevent the cell from
developing as a cancer cell.
Sequence CWU 1
1
4 1 22 DNA Artificial Sequence Bcl-2 forward primer 1 tacgataacc
gggagatagt ga 22 2 20 DNA Artificial Sequence Bcl-2 reverse Primer
2 caggtgccgg ttcaggtact 20 3 23 DNA Artificial Sequence GAPDH
forward primer 3 caactacatg gtttacatgt tcc 23 4 20 DNA Artificial
Sequence GAPDH reverse primer 4 ggactgtggt catgagtcct 20
* * * * *