Date post: | 20-Jun-2015 |
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University of Toyama
Ikuo SAIKI, Executive Vice President
Anti-metastatic effect of intestinal bacterialmetabolites of ginseng saponins and their molecular
mechanism of action
Biological activities of saponins extracted fromthe root of Panax ginseng C. A. MEYER
• anti-diabetic effect• anti-hyperlipidemic effect• management of psychosomatic
diseases (indefinite complaints,stress ulcer, anxiety neurosis)
• inhibition of tumor-inducedangiogenesis
• anti-tumor activity• alleviation of some types of
inflammatory diseases
HO
OH
-OGlc
O- GlcH28 29
26
-O1-6 GlcGlc
OH-O
1
2
34 5 6 7
89
10
11 12 13
14
15
16
17
1819
20
2122
23
24
25
27
30
Glc 1- 6Glc
Protopanaxadiol-type Protopanaxatriol-type
20(S)-protopanaxatriol!
or M4
Isolation and chemical structure of ginsenosides antheir intestinal bacterial metabolites
HO
OH
H
-OGlc
HO
OHHO
OH
Compound K
or M1
HO
OH
-OGlc
O- Glc
HO
OH
-OGlc
O-Glc -2 1Rha
Ginsenoside Rg1 ReH28 29
26
-O1-6 GlcGlc
OH-O
1
2
34 5 6 7
89
10
11 12 13
14
15
16
17
1819
20
2122
23
24
25
27
30
Glc1- 6Glc
H
OH-O
O-1-6 GlcGlc
GlcArap 1-6
H
OH
Araf - Glc -O
-O1-6 GlcGlc
Ginsenoside Rb1 Rb2 Rc
Ginsenoside Rg3(Mild-acid treatment)
Ginsenoside Rh2
20(S)-protopanaxadiol
Red Ginseng
Ginsenoside Rh1
Protopanaxadiol-type Protopanaxatriol-type
White Ginseng
Hydrolysis by Intestinal bacteria
p.o. i.v.
Effect of oral or i.v. administration of ginsenosides andtheir metabolites on lung metastasis of B16-BL6 cells
IntestinalBacteria
Intestinal bacteria
Protopanaxadiol-type and
Protopanaxatriol-type
ginsenosides
(Rb1, Rb2, Rc, Rg1, Re)
Oral administration
B16-BL6!"#$%
Ginsenoside Rb1
Anti-metastatic effect by oral administration of ginsenosides may be primarily
mediated by their intestinal bacterial metabolites (M1 or M4)
HO
-OGlc
HO
OHHO
OH
H
OH
M4
M1
M4
M1
M4
M1
Intravenous administration
Lung metastasis
Metabolite M1
!
!
!
!
"
Inhibition M1
M1
M1
Ginsenoside Rb1
Metabolite M1
Inhibition
Inhibition
0 !2 4 8 16 24 0 !2 4 8 16 24
Hours after oral administration
Co
nc
en
tra
tio
n i
n b
loo
d (&
g/m
l)
12
10
8
6
4
2
0
: M1
: Rb1
Rb1 M1
M1
M1
M1
M1
M1
M1M1
M1
Time course of Rb1 and/or M1 level in the serumobtained from mice administered orally with Rb1 or M1
Relationship between parent and litters in Rb1'(1transformation rate
Rb1 M1 transformation rate (%)
Nu
mb
er
of
mic
e
70-80
0-10
10-20
20-30
30-40
40-50
50-60
60-70
80-90
0
5
10
15
20
25
0-20% 20-50% 50-70%
Litters
Parent
Rb1 M1 transformation rate (%)
(at 6 week-old)
70-80
0-10
10-20
20-30
30-40
40-50
50-60
60-70
80-90
70-80
0-10
10-20
20-30
30-40
40-50
50-60
60-70
80-90
Rb1 M1
Intestinal bacteria
#
Intestinal bacteria
low high
Effect of oral administration of Rb1 on Rb1'(1transformation rate
0-10
10-20
20-30
30-40
40-50
50-60
60-70
70-80
80-90
90-100
0
2
4
6
8
10
Rb1 M1 transformation rate (%)
Nu
mb
er
of
mic
e
25±11%
Rb1 M1 transformation rate (%)
Ginseng extract, 150 mg/kg x 14d
Parent
Mean transformation rate(10 % <)
None 25± 11 %
Rb1-treated 51± 26 %
0-10
10-20
20-30
30-40
40-50
50-60
60-70
70-80
80-90
90-100
Rb1 M1
Intestinal bacteria
#
mice with low
Rb1-hydrolysing potential
0 10 20 30 40 50 60 70
-10
0
10
20
30
40
50
60
Rb1 M1 transformation rate (%)
An
ti-m
eta
sta
tic
ac
tiv
ity
(%
In
hib
itio
n)
Rb1-hydrolysing potential46.8 ± 10.3 %
Rb1-hydrolysing potential4.8 ± 3.2 %
##
Relationship between anti-metastatic potential and Rb1metabolic activity
Rb1 metabolic activity
day 0day 21
Lungmetastasis
LLC
Rb1, 25 mg/kg, p.o.
Rb1'(1ransformation rate
M1
M1
M1M1
M1
M1
M1
M1
M1
mice
with high hydrolyzing
potential
mice
with low hydrolyzing
potential
Effect of ginsenosides and their metabolites (M1, M4) on thegrowth of B16-BL6 melanoma, HT-1080 fibrosarcoma, and MLF
fibroblastic cells in vitro
Effect of ginsenosides or their metabolites (M1, M4) on theinvasion of B16-BL6 melanoma and HT-1080 fibrosarcoma cells
into Matrigel
B16-BL6 melanoma
HT-1080 fibrosarcoma
Matrigel
0.1%BSA in Medium (Lower compartment)
Laminin or Fibronectin
24 well cluster plate
Tumor cell
Adhesion to subendothelial matrix
Enzymatic degradation of extracellular matrix
Tumor cell migration
Microporous membrane (8 µm pore size)
Morphological changes of B16-BL6 melanoma and Lewis lungcarcinoma (LLC) cells treated with ginsenoside-Rb1 and its
metabolite M1
B16-BL6 cells
LLC cells
Control M1 (40 "M)
M1 (20 "M) Control
Rb1 (40 "M)
M1-induced DNA fragmentation of LLC cells and thecell morphology
Control
M1 (40 "M)
Caspase-3 activity in LLC cells treated with M1 forvarious time periods
activated-caspase-3
DEVD pNA
DEVDpNA
***, p < 0.001
Effect of caspase-3 inhibitor on the M1-inducedGrowth inhibition of LLC cells
0
20
40
60
80
100
120
Control 0 5 10 20 40
% o
f co
ntr
ol
Pretreatment with caspase-3 inhibitor:
Z-DEVD-FMK (µM) before the addition
of 30 µM of M1
*
****
**
Effect of M1 on the expression of caspase-3 mRNA inLLC cells
Cell cycle regulation
Inhibition
Inhibition
Inhibition
Phosphorylation
Transcriptional
activation
c-Myc
Western blot analysis of p27Kip1, c-Myc and cyclin D1 inB16-BL6 cells treated with M1
Cell cycle regulation
Inhibition
Inhibition
Inhibition
Phosphorylation
Transcriptional
activation
c-Myc
M1
Intracellular distribution of M1 after the incubation oftumor cells with fluorescent-lablled dansyl M1
TLC profile of dansyl M1 and
its parent compound
Fluorescent microscopy of
B16-BL6 cells
treated with dansyl M1
Proposed mechanism of M1-induced inhibition oftumor growth
Distribution of M1 after intravenous or oraladministration to mice
i.v. p.o.
TLC profiles of M1-metabolites in the liver afteradministration of M1
M1 0 10 20 40 80 160 min
Livers after
administration
– + Livers
afteradministration
( at 2 h)
M1
(at 40 min)
Liver Contents
of small
intestine
M1
i.v. administrationof M1
i.v. administrationof M1
oral administrationof M1
M1 M1
EM1
M4
M1
M4EM4
Putative metabolic pathways ofginsenosides
in the body after oral administration