biological significance of the gut microbial ellagic acid

Juan Carlos Espín de Gea Research Group on Quality, Safety and

Bioactivity of Plant Foods.CEBAS-CSIC. (Murcia, Spain)

E-mail: [email protected]

Biological significance of the gut microbial ellagic acid-derived

metabolites urolithins

CONTENT

BIOLOGICAL ACTIVITYInflammatory bowel disease (IBD) Mechanisms of actionRole of urolithins as anti-inflammatory compounds

MetabolismUrolithins in Nature

ELLAGITANNINS, ELLAGIC ACID AND UROLITHINS

TARGET ORGANSHuman prostate as target organ for urolithins

CONCLUSIONS

O

OO

O O OH

OHOH

O

OHOH

OH

O

O

O

O

OO

O

OHOH

OH

OHOH

OH

O

O

OH

OH

O

OH OH

OH

OH

OHOH

OO

OO

OHOH

OH

OHOH

OH

O

O

O

OHOH

OH

OHOH

OH

OHOH

OH

O

O

O

OO

O OO

OH

OHOH

O

OH

OH OH

OH

OO

OH

OH

OHOH

OH

O

O

OH

OH

OH

OH

OHO

O

OO

O

O

OH

OH OHOHOH

OH

O

OH

OO

ELLAGITANNINS

Ellagitannin-containing foodstuffs(animal models and humans)

-Cancer

-Diabetes

-Cardiovascular

-Alzheimer’s

-…….

Multitarget action (antioxidant,

anti-inflammatory, anticarcinogen…),

BUT…

Are ellagitannins or ellagic acid the real active molecules in vivo?

O

O O

O OH

OH

OH

O O

OHOH

OHOH

OH

O O

OHOH

OH

O O

OH

OH

OH

OH

O O

OH

OH

O OOH

OH

O O

OH

OHO OOH

OHOH

OH

OHOH

OH

O

O

O

OO

OHO O

O

OH

OHOH

O

OH

OH OH

Ellagitannin

O

O O

O OHOH

OHOH

Ellagic acid

Ellagitannins are metabolized to urolithins

Accumulation of ellagitannins and ellagic acid can be toxic in animals.

Urolithin A Urolithin B

Complex-toothed

flying

squirrel

(Trogopterus xanthipes)

The Urolithins

Jeong et al., 2000, Planta Med. 66, 76-77.

Doyle B, Griffiths LA (1980). The metabolism of ellagic acid in the rat. Xenobiotica, 10, 247-256

Cerdá et al., 2003, Eur. J. Nutr. 42, 18-28.

Cerdá et al., 2003, J. Agric. Food Chem. 51, 3493-3501.

González-Sarrías et al., 2009, J. Agric. Food Chem. 57, 5623-5632.

Larrosa et al., 2009, J. Nutr. Biochem. 21, 717-725.

RAT

Cerdá et al., 2004, Eur. J. Nutr. 43, 205-220.

Cerdá et al., 2005a, J. Agric. Food Chem. 53, 227-235.

Cerdá et al., 2005b, J. Agric. Food Chem. 53, 5571-5576.

Cerdá et al., 2006, Eur. J. Clin. Nutr. 63, 245-253.

González-Sarrías et al., 2010, Mol. Nutr. Food Chem. 54, 311-322.

HUMANS

PIG Espín et al., 2007, J. Agric. Food Chem. 55, 10476-10485.

BEAVER, MICE, SHEEP, COW….González-Barrio et al. 2010, J. Agric. Food Chem. Submitted

Urolithins in the phylogenetic scaleUrolithins are produced

by mammals. Not

found

in birds

and

insects

BA

CTE

RIA

L M

ETA

BO

LISM

Tri-hidroxy-

-benzopyran-6-one(URO-C)

OO

OHOH

OH

Urolithin A

(URO-A)

OO

OHOH

METABOLISM OF ELLAGITANNINS (What do we know?)Ellagitannins

Ellagic acid

O

OO

OHOH

OOH

OH

Espín et al. (2007). The Iberian pig as a model to clarify obscure points in the bioavailability and metabolism of ellagitannins in humans. J. Agric. Food Chem. 55, 10476–10485

Tetra-hidroxy-

-benzopyran-6-one(URO-D)

OO

OHOH

OHOH

Urolithin B

(URO-B)

OO

OH

ABSORPTION AND METABOLISM OF ELLAGITANNINS (Key points)

Ellagitannins are not

absorbed but

hydrolyzed

to yield

ellagic acid.

Ellagic acid

is

very

poorly

absorbed and

mainly metabolized

by gut

microbiota to

yield

hydroxy-dibenzo-pyran-6-one

derivatives

(urolithins).

Urolithins can reach

high

micromolar concentrations

in the

colon (aglycones) and

in the

bloodstream

(glucuronides)

Human subjects

can be divided

into

high

and

low-urolithin producers

(due

to

their

microbiota)

CONTENT





CONCLUSIONS

(Trogopterus xanthipes)

BIOLOGICAL ACTIVITY OF UROLITHINSTRADITIONAL CHINESE MEDICINE

-Hyaluronidase

inhibitor

(metastasis, bacterial invasion…)-Abdominal pain-Hematological

disorders

and

ischemia……

http://images.google.es/imgres?imgurl=https://www.drshen.com/images/plumflower%2520photos/Shen%2520Tong%2520Zhu%2520Yu%2520Wan.jpg.bmp&imgrefurl=https://www.drshen.com/plumflowerherbs.htm&usg=__HanuvX71z44URjEqKHG5DKowLtE=&h=220&w=120&sz=78&hl=es&start=6&um=1&tbnid=xuuYTDmwynysaM:&tbnh=107&tbnw=58&prev=/images%3Fq%3Dtrogopterus%2Bxanthipes%26hl%3Des%26rlz%3D1T4GGLR_esES228ES229%26sa%3DN%26um%3D1


O

OH

OH

O 10.7 Å

6 Å

Urolithin A

OHOH

12.8 Å

6 Å

17--Estradiol

O

OH

O

6 Å

Urolithin B

Structure-activity relationship studies suggest that urolithins might exhibit weak estrogenic and/or antiestrogenic

activity

BIOLOGICAL ACTIVITY OF UROLITHINS

OOH

HO

O

Urolithin A

OH

HO

Estradiol

Larrosa et al. (2006). Urolithins, ellagic acid-derived

metabolites produced by human colonic microflora, exhibit estrogenic and antiestrogenic

activities in MCF-7 breast cancer cells. J. Agric. Food Chem. 54, 1611-1620

Urolithins bind ER

and ER

Estrogenic/Antiestrogenic(MCF-7 assay)

POMEGRANATE EXTRACTS IN INFLAMMATORY BOWEL DISEASE (IBD): The

role of

urolithins

Fisher 344 rats

-Oxidative stress-Inflammation markers-Colon microbiota-Gene expression (microarrays)-Metabolism-Histological analyses

Larrosa

et al. (2010). Anti-inflammatory properties of a pomegranate extract and its metabolite urolithin-A in a colitis rat model and the effect of colon inflammation on

the phenolic metabolism. J. Nutr. Biochem. 21, 717-725.

Chronic

inflammation

increases

CRC risk

in IBD patients

20 d ías 5 días20 days 5 daysDSS

Control

DSS SD SD + 5% DSS

DSS -PE SD + PESD + 5% DSS+ PE

n = 8

n = 8

n = 8

DSS -UROA SD + UROASD + 5% DSS+ UROAn = 8

Standard Diet (SD)

5 days20 days

25 days

Pre-treatment

HED=2.5 g/day

HED=150 mg/day

Histological

analyses

of

colon samples

Control DSS

Uro-A PE

-Crypts

damaging

(a)-Epithelium

loss

(b)-Infiltration

of

inflammatory

cells

(c)

DSS

Uro-A Pomegranate

extract

(PE)

PE and

Uro-A protected

colon from

tissue

damageLarrosa



Inflammatory

markers

in colon mucosa

Prostaglandins, Nitric

Oxide (NO)

PE and

Uro-A decreased

NO and

prostaglandins

by downreglating

the

enzymes

involved

in their

synthesis

Larrosa



Uro-A Pomegranate

extract

(PE)

DSSGene expression

and

protein

level

Cyclooxygenase-2 (COX-2)

Inducible

Nitric

Oxide synthase (iNOS)Prostaglandin

synthase (PTGES)

Days

0 5 10 15 20 25 30

Clo

strid

ium

spp

. (Lo

g 10 C

FU/g

)

8.0

8.5

9.0

9.5

Lact

obac

illi (

Log 10

CFU

/g)

8.0

8.5

9.0

9.5

Bifid

obac

teria

(Log

10 C

FU/g

)

8.0

8.5

9.0

9.5

DSS groupDSS-PE groupDSS-UROA group DSS

DSS

DSS

(A)

(B)

(C)

Bifidobacteria

Lactobacilli

Clostridia

Effect

of

PE and

Uro-A on

gut

microbiota

Larrosa

et al. (2010).

J. Nutr. Biochem. 21, 717-725.

HED=2.5 g/day

HED=154 mg/day

20 días 5 días20 days 5 daysDSS

Control

DSS SD SD + 5% DSS

DSS-PE SD + PESD + 5% DSS+ PE

n = 8

n = 8

n = 8

DSS-UROA SD + UROASD + 5% DSS+ UROAn = 8

Standard Diet (SD)

5 days20 days

25 days

SD + PE

SD + UROA

n = 4

n = 4

PE

UROA

DSS DSS-PE DSS-UROA

Log 1

0 Inc

reas

e (N

/N0)

0

1

2

3

4E. coli (LSD, 0.47)Enterobacteria (LSD, 0.54) Total aerobic bacteria (LSD, 0.41)

Both

PE and

Uro-A modulate

gut

microbiota by increasing

bifidobacteria, lactobacilli

and

clostridia

and

decreasing

enterobacteria growth

The

first

in vivo evidence

of

gut

microbiota modulation by pomegranate. Uro-A critically

contributes

to

this

effect.

Gut

microbiota could

be involved

in the

anti-inflammatory effects

observed

0

250

500

750

mAbs

(305

nm

)

0

250

500

750

Healthy colon

+DSS (inflammed colon)

Effect

of

DSS on

pomegranate

polyphenols metabolism

Uro-AEA

Punicalagin

Colon

Imbalance

in gut

microbiota prevents

normal urolithin formation

Urolithin A: a promising

targeting

active molecule

to

the

colon

Larrosa

et al. (2010).

J. Nutr. Biochem. 21, 717-725.

PE

PE

mAb

s(3

05 n

m)

Retention time (min)

0 10 20 30 400

250

500

7500

250

500

750

+DSS (inflammed colon)

Healthy colon Uro-A

Uro-A

Gene expression

in colon mucosa(transcriptomic)

DSS-PE vs DSS DSS-UroA vs DSS

Down-regulated probes 329 3,008

Up-regulated probes 1,728 3,987

2,057 genes 6,995 genes

667 common

genes

Both

PE and

Uro-A modulate

gene profile

of

colon mucosaLarrosa

et al. (2010). J. Nutr. Biochem. 21, 717-725.

Differential

expression

at least

2-fold, P<0.001(colon mucosa)

Affymetrix: Approx. 22,000 human genes

Common: PE-UroA

(667 genes)

URO-A

PE

Functional

analysis

(top

ten). (Ingenuity

Software)

Pomegranate

extract

(PE) Urolithin A

Cell

deathCellular

growth

and

proliferationCancerGastrointestinal disease

Organismal

survivalCell

cycle

CELL DEATHCELLULAR GROWTH AND PROLIFERATIONCANCERGASTROINTESTINAL DISEASEORGANISMAL SURVIVALCELL CYCLE

Pomegranate

extract Urolithin A

Up-regulated

Down-regulated

MOLECULAR MECHANISMS OF CANCER

AKT

Rb

p53

p53

BclXL

Inflammation: Mechanistic

studies. Confirming

the

responsible

González-Sarrías, A.; Larrosa, M.; Tomás-Barberán, F. A.; Dolara, P.; Espín, J.C. (2010). NF-κB

dependent anti-inflammatory activity of gut microbiota ellagic acid derived metabolites, urolithins, in human colonic fibroblasts. Brit. J. Nutr. 104, 503-512.

COX-2iNOSmPGESCytokines…

mRNA

ERK JNK p38

Oxidative

stress/inflammatory

stimuli

(p)-MAPKs

NFB

NFB IB

Prostaglandins

(PGE2)

Fever, pain, inflammation….

COX-2

Prostaglandin

synthases

Arachidonic

acid

‘First

in vivo and

then, in vitro’

-PGE2

-COX-2 and

mPGES-1 (RT-PCR and

WB)-NF-B

activation-MAPKs

pathways

activation-Cell

metabolism

0

2.000

4.000

6.000

8.000

10.000

12.000

PGE

2(p

g/m

L)

Quer +Ct IL- UroB EA + ResvUroA

a b

a b

b

a

aa

a

a b

a b

a bb

10 M1 M

0

2.000

4.000

6.000

8.000

10.000

12.000

PGE

2(p

g/m

L)

Quer +Ct IL- UroB EA + ResvUroA Quer +Ct IL- UroB EA + ResvUroA Quer +Ct IL- UroB EA + ResvUroACt IL- UroB EA + ResvUroA

a b

a b

b

a

aa

a

a b

a b

a bb

10 M1 M

IL-

0

2.000

4.000

6.000

8.000

10.000

12.000

PGE

2(p

g/m

L)

Quer +Ct IL- UroB EA + ResvUroA

a b

a b

b

a

aa

a

a b

a b

a bb

10 M1 M

0

2.000

4.000

6.000

8.000

10.000

12.000

PGE

2(p

g/m

L)

Quer +Ct IL- UroB EA + ResvUroA Quer +Ct IL- UroB EA + ResvUroA Quer +Ct IL- UroB EA + ResvUroACt IL- UroB EA + ResvUroA

a b

a b

b

a

aa

a

a b

a b

a bb

10 M1 M

IL-

Prostaglandins

-EA, Uro-A or

Uro-B (10 and

1M) and 1ng/mL IL-1

CCD-18Co

Inflammation

of

human colon normal cells

with

IL-1

Effects

on

COX-2 and

mPGES-1

mPGES -1

COX -2

0,00

0,20

0,40

0,60

0,80

1,00

1,20

1,40

IL-b UroA Uro-B EA

mPGES -1

COX -2

mPGES -1

COX -2

Rat

io tr

eatm

entv

s. IL

-1

Gene expression

Proteins

COX-2

m-PGES-1

González-Sarrías, A.; Larrosa, M.; Tomás-

Barberán, F. A.; Dolara, P.; Espín, J.C. (2010). NF-κB


NF-B

and

MAPKs

pathways

aa a

a

a a

bb

bb

aa

b bb

a,b

aa

a,b a,b

aa

b ba,b

a,b

a,ba

NFk

Bac

tivat

ion

(Abs

. 450

nm

)

0,0

0,2

0,4

0,6

0,8

1,0

1,2

2h 4h

C IL-1 Uro-A Uro-B EA JurkatIL-1

aa a

a

a a

bb

bb

UroA, UroB, EA: 10 M

González-Sarrías, A.; Larrosa, M.; Tomás-

Barberán, F. A.; Dolara, P.; Espín, J.C. (2010). NF-κB


Rat

io p

-ER

K /

ERK

1,0

1,2

1,4

1,6

1,8

2,0

aa

aa,b

a,b

(A)

C IL-1 Uro-A Uro-B EA PD98059IL-1

ERK

1,0

1,1

1,2

1,3

1,4

1,5

Rat

io p

-p38

/ p3

8

C IL-1 Uro-A Uro-B EA SB203580IL-1

a a

ba,b

(C)

p38

a,b

1,0

1,2

1,4

1,6

1,8

2,0

a

a,b

aa,b

a,b

C IL-1 Uro-A Uro-B EA PD98059IL-1

JNK(B)

Rat

io p

-JN

K /J

NK

Retention time (min)

0 5 10 15 20 25 30

mA

bs(3

05 n

m)

0

6

12

18

mA

bs(3

05 n

m)

0

200

400

600

800

1000

1200

(A)

(B)

Cell

medium

Cell

lysate

Uro-A

Uro-A

Urolithins metabolism

in coloncells

Cell

receptor-mediated mechanism????

-Very

low

metabolism

(characteristic

in ‘normal’

cells)

-Absence

of

conjugated

metabolites

-Trace amount

inside

the

cells

(pM)

González-Sarrías, A.; Larrosa, M.; Tomás-Barberán, F. A.; Dolara, P.; Espín, J.C. (2010). NF-κB


In vitro assays

confirm

in vivo effects: Uro-A is

the main

anti-inflammatory

compound

The

anti-inflammatory

activity

is

NFkB-dependent

Uro-A exerts

the

activity

at lower

concentrations than

those

found

in the

colon lumen

Ellagic acid

(the

precursor of

urolithins) does

not exert

anti-inflammatory

activity

CONTENT





CONCLUSIONS

63 PATIENTS with

BPH or

PCaFasting(18h)

Control n = 30

n = 14

n = 19

Walnuts

35g/day

Pomegranate

juice

(200mL/day)

3 INTAKES (three

days, one

per

day)

Blood

and

urine

samples

(baseline)

HPLC-MS-MSBlood

and

urine

samples

Surgery Prostate

samples

HPLC-MS-MS

Pomegranate

juice

and

prostate

cancer: Could urolithins be behind

these

effects? The

human prostate

as target

organ

González-Sarrías

et al. (2010). Occurrence of urolithins, gut microbiota ellagic acid-derived metabolites, in the human prostate gland upon consumption of walnuts and pomegranate juice. Mol. Nutr. Food Res. 54, 311-352.

Analyses

of

human prostates-

High

interindividual variability

- Metabolites

in 8 prostate

samples

(high

urolithin producers): 24% of

patients

- Uro-A glc: 6 samples

(0.5-2 ng/g tissue) UV, MS, MS/MS

- Uro-B glc: 2 samples

MS and

MS/MS

-

Dimethyl

ellagic acid

(DMEA): 4 samples

MS and

MS/MSEIC 403 -All MS

0.0

1.0

2.0x105

Intens.

5 10 15 20 25 30 35 40 Time [min]

112.8

174.7226.8

-MS2(403), 18.8min

0

500

1000

1500

2000

Intens.

100 200 300 400 500 600 700 800 900 m/z

O OH

OHOH

COOH

O

O

O

OH

EIC 387 -All MS

0.0

0.5

1.0

1.5x105

5 10 15 20 25 30 35 40 Time [min]

112.7

174.6

210.7

341.0

-MS2(387), 23.0min

0

250

500

750

1000

1250Intens.

100 200 300 400 500 600 700 800 900 m/z

O OH

OHOH

COOH

O

O

O

EIC 329 -All MS

0

1

2

3x105

5 10 15 20 25 30 35 40 Time [min]

313.3

170.7

228.9

298.9

-MS2(329), 29.8min

0

1000

2000

3000

4000

5000Intens.

100 200 300 400 500 600 700 800 900 m/z

O

O O

O OH

OH

OMe

OMe

No correlation

was

observed

between

type

of

tissue (prostate

cancer

or

benign

hyperplasia) and

metabolites

detection

Metabolites

were

detected

at very

low

concentration: Fasting

period

before

the

surgery?

González-Sarrías


Sacrifice

Prostate

samples

HPLC-MS-MS

n = 2

n = 2

n = 2

n = 2

Fasting18h

Control n= 4

n= 4

n= 4

n= 4

n= 4

Control n= 4

n= 4

n= 4

n= 4

n= 4

Sacrifice

HED PE = 10 g/day

HED PE = 2 g/day

HED Uro-A = 1 g/day

HED Uro-A = 0.2 g/day

Standard diet

Consumption

for

three

days

Analyses

of

rat

prostates: Influence

of

the fasting

period

González-Sarrías


PE

Uro-A

Urolithins (mainly

Uro-A glucuronide) can reach

the

human prostate

upon

ingestion

of

ellagitannins-rich

foodstuffs

These

metabolites

could

be involved

in the

protective

effects of

pomegranate

juice

intake

against

prostate

cancer

(Without

fasting) The

presence

of

higher

urolithins levels cannot

be discarded

in the

human prostate

In both

groups, urolithin A glucuronide was

only

detected

in rats

with

free access

to

feed, with

no fasting

period

CONTENT





CONCLUSIONS


UROLITHINS: MULTITARGET MOLECULESPRODUCED BY THE GUT MICROBIOTA

(anti-inflammatory, cancer cell regulation….)

CELL CYCLE ARREST(IN VITRO AND IN VIVO)

REGULATION OF MAPKSSIGNALLING(IN VITRO AND IN VIVO)

INHIBITION OF NF-KBACTIVATION

(IN VITRO AND IN VIVO)

ESTROGENIC/ANTIESTROGENICACTIVITY (IN VITRO)

INHIBITION OF PROSTAGLANDIN SYNTHESIS(IN VITRO AND IN VIVO)

REGULATION OF GUTMICROBIOTA

(IN VIVO)

REGULATION OF GENE EXPRESSION:-tumor suppressor

genes,-transcription

factors,-COX-2, mPGES-1, iNOS……(IN VITRO AND IN VIVO)

HIGH BIOAVAILABILITY

HIGH CONCENTRATION IN THE GUT

THE HUMAN PROSTATE

AS TARGET ORGAN

(IN VIVO)

‘Systemic’ effect of urolithin conjugates: cardiovascular, other cancers….

Urolithins as an iceberg: A long way to go for this emerging topic…

Identification of the microbiota involved in urolithins production

To study in depth the role of urolithins in colon inflammation and cancer: Many important markers

Metabolism of ellagitannins in very low urolithin producers: What happens? Toxicity? Other effects?

Funding Opportunity Announcement issued by NCI: “The Role of Microbial Metabolites in Cancer Prevention and Etiology” (November 15, 2011)

(Prof. J.C. Espin: [email protected])

AcknowledgementsCEBAS-CSICF. Tomás-BarberánM.T. García-ConesaM. LarrosaA. González SarríasM.J. YáñezM. AzorínF. VallejoJ.A. GiménezJ. Tomé-CarneiroB. CerdáR. González Barrio

Collaborations:Hospital V. Arrixaca

Univ. Complutense-Madrid

Univ. Murcia

Hospital Reina Sofia

Clinic Veterinary Hospital (Murcia)

(Public-competitive) Funding: EU; CICYT; CSIC; CARM

InternationalInternationalConferenceConferenceonon PolyphenolsPolyphenolsandand HealthHealth

55thth

UROLITHINS: Multitarget molecules produced by the gut microbiota

CELL CYCLE ARREST

REGULATION OF MAPKSSIGNALLING

INHIBITION OF NF-KBACTIVATION ESTROGENIC/ANTIESTROGENIC

ACTIVITY

INHIBITION OF PROSTAGLANDIN

SYNTHESIS

REGULATION OF GUT MICROBIOTA

REGULATION OF GENE EXPRESSION:

HIGH BIOAVAILABILITY

HIGH CONCENTRATION IN THE GUT

THE HUMAN PROSTATE AS TARGET ORGAN


THANK YOU FOR YOUR ATTENTION!

Juan Carlos Espín de GeaE-mail: [email protected] Group on Quality, Safety

and Bioactivity of Plant Foods.CEBAS-CSIC. (Murcia, Spain)

biological significance of the gut microbial ellagic acid

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