1. Naturally-occurring Phytoestrogen
Precursors Are Converted to Estrogenic
Compounds by Human Intestinal Bacteria
Masao Hattori
Emeritus Professor, University of Toyama
On Plant Isoflavones and Lignans
In an epidemiologic study, the prevalence of breast cancer
and prostate cancer was lower in Japan and Finland
compared it with U.S.A
As a low factor of the prevalence, a Japanese soy bean and
Finnish rye bread were considered as the candidates, which
contain so called phytoestrogens, isoflavonoids and lignans.
Since phytoestrogens structurally resemble estradiol, they
have estrogenic and anti-estrogenic activities and an
incidence of prostate cancer or breast cancer may decrease
depending on the blood concentrations of phytoestrogens
(reverse correlation).

2. METABOLISM OF SDG BY HUMAN INTESTINAL FLORA
METABOLISM OF ARCTIIN BY HUMAN
INTESTINAL FLORA

3. CIRCULAR DICHROISM OF (+)- AND (-)-ENTEROLACTONES
MIRROR IMAGES OF (+)- AND (-)-ENTEROLACTONES
(+)-(2S,3S)-Enterolactone (-)-(2R,3R)-Enterolactone
L R

4. ENANTIO SELECTIVE DEHYDROXYLATION
R
L
ENANTIO SELECTIVE OXIDATION OF DIOLS
R
L

5. DEMETHYLATION PROCEEDS IN EITHER
ENANTIOMER
ORAL ADMINISTRATION OF A. LAPPA
DECOCTION TO HUMAN SUBJECTS
Subject: (BMI, 18.6 ± 2.1 kg/m2 22.6 ±
0.4 kg/m2).
Drug: (14.5 mM arctiin and 7.5 mM arctigenin)
Dose: 1.0 ml/kg (= 0.5 g of /kg )
1
-2 day 0 day 4 day
Collection of samples:
Sample pretreatment: C18-Sep Pak
Analysis: LC/MS/MS

6. URINARY METABOLITES OF HUMAN SUBJECTS
AFTER ORAL ADMINISTRATION OF A. LAPPA
DECOCTION
% of dosed arctiin and ARG
e
lit
bo
ta
Me
A B C D E F G H I J K L M Mean
Female Male
Individual Dose, 0.5 ml/kg weight
Structural Similarity between Tracheloside from
Safflower and Arctiin from Burdock
Tracheloside Arctiin

7. Time Course of Metabolite Formation after Anaerobic
Incubation of Trachelogenin with Human Intestinal Bacteria
Trachelogenin 0hr
72hr
1
96hr
120hr
4
332
2 5 6
168hr
7
Estimation of Trachelogenin Metabolite on the Basis
of Mass Spectral Data
m/z
Difference
Elimination
Trachelogenin
388
M1 374
-14
-Me x1
M2
360
-28
-Me x2
M3
360
-28
-Me x2
M4
346
-42
-Me x3
M5
330
-58
-Me x3 -OH x1
M6
314
-74
-Me x3 -OH x2
M7
298
-90
-Me x3 -OH x3

8. Metabolic Process of Trachelogenin
- Me - Me
1 2
Trachelogenin ‐Me - Me
- Me
3 4
- OH
- OH - OH
(-)-Enterolactone, (7) 6 5
Expression of Estrogenic Action
Physiological Action
Co-activator Complex
Estrogen
CBP/p300
SRC-1 TF
RNA pol
ER ER mRNA Gene Products
TBP
ERE TATA Target Gene
• Through a complex formation of estrogen receptors, estrogen, co-
activators such as SCR-1, transcription of target genes is
accelerated to induce physiological actions.
• Two types of estrogen receptors ERα and ERβ are reported and
phytoestrogens are more strongly bound to ERβ than ERα in
general.

9. B/Bmax
0
10
20
30
40
50
60
-10
Arctiin
Arctigenin
Tracheloside
Tracherogenin
1
TMB
2
3
4
5
ERα agonist
(-)-Dihydroxyenterolactone
6
B/Bmax % of control (estradiol)
*
(-)-Enterolactone( 7)
(+)-Enterolactone
(-)-Enterodiol
(+)-Enterodiol
peroxidase
B/Bmax
0
10
20
30
40
50
60
-10
Arctiin
SRC-1
Arctigenin
Ligand Activity against Erα
Tracheloside
ER
Tracherogenin
1
Principle of Assay
2
Detection antibody horseradish
3
4
Erβ
5
ER β agonist
(-)-Dihydroxyenterolactone
6
TMB
(-)-Enterolactone( 7)
*
*
(+)-Enterolactone
(-)-Enterodiol
± SE
TMB
(+)-Enterodiol
Oxidation product

10. STEPWISE DEGRADATION OF PINORESINOL
Enterococcus faecalis strain PDG-1
MOST OF LIGNANS ARE PRECURSORS OF ENTERODIOL
AND ENTEROLACTONE

11. CONVERSION OF PUERARIN
TO EQUOL
Equol
Strain PUE
Strain DZE
Daidzein
Jin et al.: Biol.Pharm. Bull., 31, 1621-1625 Estradiol
(2008)
Characterization of a Human Intestinal Bacterium
that Transforms Puerarin to Daidzein
Strain PUE
Daidzein
Puerarin
Dendrograms of phylogenic affiliation of strain PUE based on 16S rRNA gene

12. DETECTION OF DUTERIUM-LABELED GLUCOSE
DERIVATIVE BY LC/MS
Conclusions
1. Trachelogenin was demonstrated to convert estrogenic
enterolactone as well as secoisolarisiresinol diglucoside
(SDG), arctiin and pinoresinol by human intestinal bacteria
(HIB).
trachelogenin
and arctiin to (-)-enterolactone by HIB.
ERα ERβ.
4. Dehydroxylation and oxidation of lignans by HIB were
enantioselective as substrates.
5. Puerarin (C-glucosyl) was converted to estrogenic equol
through daidzein by HIB.

13. Coworkers