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