2 하토리교수

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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).

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METABOLISM OF SDG BY HUMAN INTESTINAL FLORA

METABOLISM OF ARCTIIN BY HUMAN INTESTINAL FLORA

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CIRCULAR DICHROISM OF (+)- AND (-)-ENTEROLACTONES

MIRROR IMAGES OF (+)- AND (-)-ENTEROLACTONES

(+)-(2S,3S)-Enterolactone (-)-(2R,3R)-Enterolactone

L R

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ENANTIO SELECTIVE DEHYDROXYLATION

R

L

ENANTIO SELECTIVE OXIDATION OF DIOLS

L

R

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DEMETHYLATION PROCEEDS IN EITHER ENANTIOMER

ORAL ADMINISTRATION OF A. LAPPA DECOCTION TO HUMAN SUBJECTS

Dose: 1.0 ml/kg 体重 (= 0.5 g of 牛蒡子/kg 体重)

4 day -2 day 0 day

Drug: 牛蒡子煎剤 (14.5 mM arctiin and 7.5 mM arctigenin)

LC/MS/MS

尿

Subject: 女 8人、 男5人, 年齢 20-32 歳 (BMI, 女 18.6 ± 2.1 kg/m2 、男 22.6 ± 0.4 kg/m2).

1回経口投与

Collection of samples:

Sample pretreatment: 酵素水解、C18-Sep Pak 抽出

Analysis:

血清

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URINARY METABOLITES OF HUMAN SUBJECTS AFTER ORAL ADMINISTRATION OF A. LAPPA

DECOCTION

A B C D E F G H I J K M L Mean

Individual

Metab

olite

% o

f dos

ed a

rctii

n an

d A

RG

Dose, 0.5 ml/kg weight Female Male

Structural Similarity between Tracheloside from Safflower and Arctiin from Burdock

Arctiin (牛蒡子由来)

Tracheloside （紅花種子由来）

紅花種子

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Time Course of Metabolite Formation after Anaerobic Incubation of Trachelogenin with Human Intestinal Bacteria

Trachelogenin

4 5 6

7

3 2

0hr

72hr

96hr

120hr

168hr

1

2 3

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

Estimation of Trachelogenin Metabolite on the Basis of Mass Spectral Data

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Metabolic Process of Trachelogenin

1 2

3 4

5 6 (-)-Enterolactone, (7)

‐Me - Me

- OH

- Me - Me

- Me

- OH - OH

Trachelogenin

Expression of Estrogenic Action

• 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.

ER ER

ERE TATA Target Gene

mRNA Gene Products

SRC-1

CBP/p300

TF

TBP

RNA pol

Co-activator Complex

Estrogen

Physiological Action

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Principle of Assay

SRC-1 ER E2 or Lignans Antibody TMB

Detection antibody （horseradish peroxidase）　

TMB

TMB

Oxidation product

Ligand Activity against Erα and Erβ (SD ± SE)

ER α agonist

-10

0

10

20

30

40

50

60

B/B

max

ER β agonist

-10

0

10

20

30

40

50

60 *

B/B

max

Arc

tiin

Arc

tigen

in

Trac

helo

side

Trac

hero

geni

n

代謝物

1

代謝物

2

代謝物

3

代謝物

4

代謝物

5

(-)-D

ihyd

roxy

ente

rola

cton

e

代謝物

6

(-)-E

nter

olac

tone

(代謝物

7)

(+)-E

nter

olac

tone

(-)-E

nter

odio

l

(+)-E

nter

odio

l

Arc

tiin

Arc

tigen

in

Trac

helo

side

Trac

hero

geni

n

代謝物

1

代謝物

2

代謝物

3

代謝物

4

代謝物

5

(-)-D

ihyd

roxy

ente

rola

cton

e

代謝物

6

(-)-E

nter

olac

tone

(代謝物

7)

(+)-E

nter

olac

tone

(-)-E

nter

odio

l

(+)-E

nter

odio

l

*

*

B/Bmax % of control (estradiol)

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STEPWISE DEGRADATION OF PINORESINOL

Enterococcus faecalis strain PDG-1

MOST OF LIGNANS ARE PRECURSORS OF ENTERODIOL AND ENTEROLACTONE

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CONVERSION OF PUERARIN TO EQUOL

Estradiol

Equol

Strain PUE Strain DZE

Jin et al.: Biol.Pharm. Bull., 31, 1621-1625 (2008)

Daidzein

Dendrograms of phylogenic affiliation of strain PUE based on 16S rRNA gene

Puerarin Daidzein

Characterization of a Human Intestinal Bacterium that Transforms Puerarin to Daidzein

Strain PUE （グラム陰性偏性嫌気性桿菌）

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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).

2.  SDG was converted to (+)-enterolactone, while trachelogenin and arctiin to (-)-enterolactone by HIB.

3.  Metabolites of trachelogenin bound with both ERα and 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.

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Coworkers