Chemistry and isoflavonoids

Stephen Barnes, PhDPurdue-UAB Botanicals Center

University of Alabama at Birmingham

Isoflavone workshopOxford, MS August 21, 2006

Major issues in use of isoflavones

• Does it matter which isoflavones (aglycones) are in the product?

• Does it matter whether the isoflavones are O- or C-glycosides?

• Does it matter whether the isoflavone glycoside conjugates are esters, and if so, should this appear on the label?

• Should the weight of isoflavones be given aglycone equivalents?

• Does it matter which isoflavones (aglycones) are in the product?

• Does it matter whether the isoflavones are O- or C-glycosides?

• Does it matter whether the isoflavone glycoside conjugates are esters, and if so, should this appear on the label?

• Should the weight of isoflavones be given aglycone equivalents?

Keys to life on planets and moons

Earth and the Saturn moon Titan have atmospheres thatare largely nitrogen and smaller amounts of carbonaceous gases - mostly CO2 on Earth and methane on Titan

Earth and the Saturn moon Titan have atmospheres thatare largely nitrogen and smaller amounts of carbonaceous gases - mostly CO2 on Earth and methane on Titan

So, where is this?

It’s not a Swiss lake in winter, but lakeside on Titan (courtesy of NASA-ESA Cassini-Huygens)

So, where is this?

It’s not a Swiss lake in winter, but lakeside on Titan (courtesy of NASA-ESA Cassini-Huygens)

Making nitrogen accessibleThe key to our form of life was/is converting non-reactive N2 gas to fixed nitrogen (ammonia, amino acids, etc.). Before life appeared, this occurred from lightning and volcanic action.

The key to life was nitrogen-fixing archeal bacteria and then algae (the latter also fix CO2).

In the “modern” era, it is the symbiosis between plants (legumes such as clover and the soybean) and soil bacteria that fix nitrogen. Plants also convert CO2 with the aid of sunlight to carbohydrates and O2.

Plant-bacterium dilemmaPlants have to defend themselves against microorganisms. But the legumes have a symbiotic relationship with rhizobia sp. How does thisoccur?The key is molecular signals from the plant to the bacteria and bacteria to plant.Bacteria have nodulation (Nod) genes. These are activated by compounds released by the root of the legume -for the soybean, these compounds are isoflavonoids.

Plants have to defend themselves against microorganisms. But the legumes have a symbiotic relationship with rhizobia sp. How does thisoccur?The key is molecular signals from the plant to the bacteria and bacteria to plant.Bacteria have nodulation (Nod) genes. These are activated by compounds released by the root of the legume -for the soybean, these compounds are isoflavonoids. Genistein

DaidzeinGlycitein

Chalcone synthase( ) x 3

OH

O

O

HHO

OH

CO.SCoACOOH

NH2

OH

OOC.CH2.COSCoA

phenylalanine

4-hydroxycinnamoyl CoA

Naringenina flavanoid

O

O

HO

OHOH

Genistein,an isoflavonoid

Chalcone isomerase

Biosynthesis of isoflavonoids

Numbering scheme for isoflavonoids

O

O

HO

OH

OH

2

345

6

7

8

2’3’

4’

5’

6’

Genistein = 5,7,4’-trihydroxyisoflavoneGenistein = 5,7,4’-trihydroxyisoflavone

Classes of isoflavonoids

O

OR

R

Isoflavone, R=Hdaidzein, genistein

O

OR

R

Isoflavonol, R=OH

O

OR

R

Isoflavanone, R=HIsoflavanol, R=OH

O

R

R

Isoflavan, R=HEquol, bacterial metabolite

Note that these compounds have an assymetric carbon atoms and are optically active

Note that these compounds have an assymetric carbon atoms and are optically active

What’s up duck?Superposition of genistein (green) and 17β-estradiol (blue) in the ligand binding pocket of ERβ

− note that the phenolic B-ring of this isoflavone is in the position occupied by the aromatic A-ring of 17β-estradiol

From Pike et al. (1999)

Superposition of genistein (green) and 17β-estradiol (blue) in the ligand binding pocket of ERβ

− note that the phenolic B-ring of this isoflavone is in the position occupied by the aromatic A-ring of 17β-estradiol

From Pike et al. (1999)

Genistein “flipped and spun around” is the right way

O

OHOH

HO

O

O

O

HO

OHOH

O

HO

OH

OHO

In this presentation

In plant journals

If genistein is an estrogen

Soy isoflavones

O

O

HO

OH

O

O

HO

OH

OH

O

O

HO

OH

MeO

Daidzein7,4’-dihydroxyisoflavone

Daidzein7,4’-dihydroxyisoflavone

Genistein5,7,4’-trihydroxyisoflavone

Genistein5,7,4’-trihydroxyisoflavone

Glycitein7,4’-dihydroxy-6-methoxyisoflavone

Glycitein7,4’-dihydroxy-6-methoxyisoflavone

Methylated isoflavonoids

O

O

HO

OHOMe

Biochanin AO

O

HO

OMe

Formononetin

Leung & Foster, 1996

Red clover (Trifolium pratense)

Prenylated isoflavones

OO

OH OOH

OH

Osajin

Pomiferin

O

OH OOH

O

From the Osange orange fruit

From the Osange orange fruit

O

OH OOH

HO

8-prenyldaidzein in hop-based beer

8-prenyldaidzein in hop-based beer

Genistein glucosides in soy products and supplements

fermentation

OH

HO

OOH

genisteinO

OOH

HO

HO

OH

O

OgenistinOH

OH

hot water extraction

O

OOH OH

6"-O-malonylgenistin

OHO

O-OOC.CH2.COO

HOOH

O

OHOOH

6"-O-acetylgenistinO

O

HOOH

OH

CH3COO

dry heat (100oC); -CO2

O

Tofu and soy milk

Tofu and soy milk

MisoMiso

Whole soybean or unheated soy flour

Whole soybean or unheated soy flour

SoyLife™ or TVP

SoyLife™ or TVP

Two non-identical soy protein products

Soy flourDefatted, low heat

SoyLife™Roasted soy hypocotyl

DN

GL

GNmDN

aDN

Both different with regard to proportion of isoflavones and in glucoside composition

“Purified Isoflavones (from GMO-Free Soybeans and Other Plants) 80.0 mg”

“Soy and other plants, sources of natural estrogens, for hormonal balance.”

• The problems with the equine estrogens extract (Premarin™) have led women to turn to “soy”estrogens for estrogen replacement therapy

• But soy estrogens are not what’s in the bottle - it’s the kudzu isoflavones

Lessons in labeling• The problems with the

equine estrogens extract (Premarin™) have led women to turn to “soy”estrogens for estrogen replacement therapy

• But soy estrogens are not what’s in the bottle - it’s the kudzu isoflavones

Kudzu (Pueraria lobata)

O

OHO

O

HO

OHOH

OH

HO

The new player on the shelves

Puerarin - daidzein 8-C-glucosidePuerarin - daidzein 8-C-glucoside

Overall view of isoflavonoid uptake and metabolism

Genistein

Genistein GlcA

Genistein GlcA

Genistein GlcA

ColonColon

Duodenum - jejunum - ileumDuodenum - jejunum - ileum

Liver

GenisteinOther metabolitesOther metabolites

Possible puerarin uptake and metabolism

puerarin

puerarin

puerarin

? puerarin

Colon

Duodenum - jejunum - ileum

Liver

Puerarin?Other metabolites?

?

??

Isoflavone bacterial metabolismO

O

HO

OH

O

O

HO

OHOH

OH

O

HO

OH

OH

O

HO

OH

OHO

OH

HO

O-desmethylangolensins

Dihydro metabolites

equol

dihydrodaidzein dihydrogenistein

Acknowledgements

• Jeevan Prasain, PhD• Ali Arabshahi• Lori Coward• Michelle Smith-Johnson• Marion Kirk• D. Ray Moore II

NCCAM/ODS P50 AT00477-07Purdue-UAB

Botanicals Center (C.M. Weaver, PI)

NCRR Shared Instrument grants

S10 RR06487S10 RR17261S10 RR19231

NCCAM/ODS P50 AT00477-07Purdue-UAB

Botanicals Center (C.M. Weaver, PI)

NCRR Shared Instrument grants

S10 RR06487S10 RR17261S10 RR19231