1. Anti-metastatic effect of intestinal bacterial
metabolites of ginseng saponins and their molecular
mechanism of action
University of Toyama
Ikuo SAIKI, Executive Vice President
Biological activities of saponins extracted from
the 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-induced
angiogenesis
• anti-tumor activity
• alleviation of some types of
inflammatory diseases
21
Glc 1- 6 Glc-O 22 24 26 Glc-O
OH 20 25
OH
23
11 12 13 17
27
19 18 16
1 14
2 9
10 8 15
3 30
4 5 6 7 HO
Glc 1 -6 Glc -O
28 29 H O- Glc
Protopanaxadiol-type Protopanaxatriol-type

2. Isolation and chemical structure of ginsenosides
antheir intestinal bacterial metabolites
White Ginseng
21
Glc 1- 6 Glc-O 22 24 26 Arap 1-6 Glc -O Araf - Glc -O Glc-O Glc -O
OH OH OH OH OH
20 23 25
11 12 13 17
27
1 19 18 14 16
2 9
10 8 15
3 30
4 5 6 7 O Glc 1-6 Glc -O HO HO
Glc 1 -6 Glc -O Glc 1 -6 Glc - 2 1
28 29 H H H O- Glc O- G lc - Rha
Ginsenoside Rb1 Rb2 Rc Ginsenoside Rg1 Re
Protopanaxadiol-type Protopanaxatriol-type
Red Ginseng
(Mild-acid treatment)
Ginsenoside Rg3
Hydrolysis by Intestinal bacteria
Glc -O
OH
Ginsenoside Rh1
Ginsenoside Rh2
HO
HO
H OH
Compound K
or M1
20(S)-protopanaxatriol!
20(S)-protopanaxadiol HO
OH or M4
Effect of oral or i.v. administration of ginsenosides and
their metabolites on lung metastasis of B16-BL6 cells
p.o. Intestinal
Bacteria
i.v.

3. Anti-metastatic effect by oral administration of ginsenosides may be primarily
mediated by their intestinal bacterial metabolites (M1 or M4)
Glc -O
OH
Intestinal
bacteria M1
M1 HO
H
Protopanaxadiol-type and M4 HO
OH
Protopanaxatriol-type
ginsenosides
(Rb1, Rb2, Rc, Rg1, Re) M4
M1 M4
HO
OH
Lung metastasis
Oral administration
Ginsenoside Rb1 M1 ! Inhibition
!
B16-BL6
!"#$% Metabolite M1 M1 ! Inhibition
Intravenous administration
Ginsenoside Rb1 "
Metabolite M1 M1
! Inhibition
Time course of Rb1 and/or M1 level in the serum
obtained from mice administered orally with Rb1 or M1
Rb1 M1
M1
M1
M1
M1
M1 M1
M1
Concentration in blood (&g/ml)
12 M1
10
: M1
8 : Rb1
6
4
2
0
0 !2 4 8 16 24 0 !2 4 8 16 24
Hours after oral administration

4. Relationship between parent and litters in Rb1'(1
transformation rate
#
Intestinal bacteria
Rb1 M1 low high
25 Parent
Intestinal bacteria 0-20% 20-50% 50-70%
20
Number of mice
Litters
15 Rb1 M1
transformation rate (%)
(at 6 week-old)
10
5
0
0- 10- 20- 30- 40- 50- 60- 70- 80- 0- 10- 20- 30- 40- 50- 60- 70- 80- 0- 10- 20- 30- 40- 50- 60- 70- 80-
10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90
Rb1 M1 transformation rate (%)
Effect of oral administration of Rb1 on Rb1'(1
transformation rate
#
mice with low Rb1 M1
Parent
Rb1-hydrolysing potential
10 25±11% Intestinal bacteria
8 Ginseng extract, Mean transformation rate
150 mg/kg x 14d (10 % <)
Number of mice
None 25± 11 %
6 Rb1 M1 Rb1-treated 51± 26 %
transformation rate (%)
4
2
0
0- 10- 20- 30- 40- 50- 60- 70- 80- 90- 0- 10- 20- 30- 40- 50- 60- 70- 80- 90-
10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100
Rb1 M1 transformation rate (%)

5. Relationship between anti-metastatic potential and Rb1
metabolic activity
##
LLC Lung
metastasis
day 21
day 0 Rb1, 25 mg/kg, p.o.
Rb1 metabolic activity 60
50
Anti-metastatic activity
Rb1'(1ransformation rate
M1 40
(% Inhibition)
M1
M1 mice 30 Rb1-hydrolysing potential
M1 with high hydrolyzing 46.8 ± 10.3 %
M1
potential 20
M1 M1
10
M1 0 Rb1-hydrolysing potential
mice 4.8 ± 3.2 %
with low hydrolyzing
M1 -10
potential
0 10 20 30 40 50 60 70
Rb1 M1 transformation rate (%)
Effect of ginsenosides and their metabolites (M1, M4) on the
growth of B16-BL6 melanoma, HT-1080 fibrosarcoma, and MLF
fibroblastic cells in vitro

6. Effect of ginsenosides or their metabolites (M1, M4) on the
invasion of B16-BL6 melanoma and HT-1080 fibrosarcoma cells
into Matrigel
B16-BL6 melanoma
Adhesion to
24 well
subendothelial matrix cluster plate
HT-1080 fibrosarcoma
Enzymatic degradation
of extracellular matrix Tumor cell
Matrigel
Microporous
membrane
Tumor cell migration (8 µm pore size)
Laminin or
Fibronectin
0.1%BSA in Medium
(Lower compartment)
Morphological changes of B16-BL6 melanoma and Lewis lung
carcinoma (LLC) cells treated with ginsenoside-Rb1 and its
metabolite M1
M1 (20 "M) Control
Control M1 (40 "M)
LLC cells
Rb1 (40 "M)
B16-BL6 cells

7. M1-induced DNA fragmentation of LLC cells and the
cell morphology
Control
M1 (40 "M)
Caspase-3 activity in LLC cells treated with M1 for
various time periods
***, p < 0.001
activated-caspase-3
DEVD pNA
DEVD
pNA

8. Effect of caspase-3 inhibitor on the M1-induced
Growth inhibition of LLC cells
120
100 **
**
% of control
**
80
*
60
40
20
0
Control 0 5 10 20 40
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 in
LLC cells

9. Cell cycle regulation
c-Myc
Phosphorylation
Transcriptional
activation
Inhibition
Inhibition
Inhibition
Western blot analysis of p27Kip1, c-Myc and cyclin D1 in
B16-BL6 cells treated with M1

10. Cell cycle regulation
M1
c-Myc
Phosphorylation
Transcriptional
activation
Inhibition
Inhibition
Inhibition
Intracellular distribution of M1 after the incubation of
tumor cells with fluorescent-lablled dansyl M1
TLC profile of dansyl M1 and Fluorescent microscopy of
its parent compound B16-BL6 cells
treated with dansyl M1

11. Proposed mechanism of M1-induced inhibition of
tumor growth
Distribution of M1 after intravenous or oral
administration to mice
i.v. p.o.

12. TLC profiles of M1-metabolites in the liver after
administration of M1
i.v. administration i.v. administration oral administration
of M1 of M1 of M1
M1 0 10 20 40 80 160 min M1 Liver Contents M1 – +
Livers after of small Livers
administration intestine after
(at 40 min) administration
( at 2 h)
Putative metabolic pathways of
ginsenosides
in the body after oral administration
EM1
M1 M1
M1
EM4
M4 M4