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Phytoestrogen content of fruits and vegetables commonly consumed in the UK based on LC–MS and 13C-labelled standards

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Phytoestrogens are a group of non-steroidal secondary plant metabolites with structural and functional similarity to 17β-oestradiol. Urinary and plasma phytoestrogens have been used as biomarkers for dietary intake, however, this is often not possible in large epidemiological studies or to assess general exposure in free-living individuals. Accurate information about dietary phytoestrogens is therefore important but there is very limited data concerning food contents. In this study, we analysed the phytoestrogen (isoflavone, lignan and coumestrol) content in more than 240 different foods based on fresh and processed fruits and vegetables using a newly developed sensitive method based on LC–MS incorporating 13C3-labelled standards. Phytoestrogens were detected in all foods analysed with a median content of 20 μg/100 g wet weight (isoflavones: 2 μg/100 g; lignans 12 μg/100 g). Most foods contained less than 100 μg/100 g, however, 5% of foods analysed contained more than 400 μg/100 g, in particular soya-based foods and other legumes. The results published here will contribute to databases of dietary phytoestrogen content and allow the more accurate determination of phytoestrogen exposure in free-living individuals.
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Analytical Methods
Phytoestrogen content of fruits and vegetables commonly consumed in the UK
based on LC–MS and
13
C-labelled standards
Gunter G.C. Kuhnle
a,*
, Caterina Dell’Aquila
a
, Sue M. Aspinall
a
, Shirley A. Runswick
a
,
Annemiek M.C.P. Joosen
a
, Angela A. Mulligan
b
, Sheila A. Bingham
a
a
MRC Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK
b
EPIC, Department of Public Health and Primary Care, Institute of Public Health, University of Cambridge, Worts Causeway, Cambridge, UK
article info
Article history:
Received 15 August 2008
Received in revised form 21 February 2009
Accepted 1 March 2009
Keywords:
Phytoestrogens
Fruits
Vegetables
Lignans
Isoflavones
Coumestrol
LC/MS
abstract
Phytoestrogens are a group of non-steroidal secondary plant metabolites with structural and functional
similarity to 17b-oestradiol. Urinary and plasma phytoestrogens have been used as biomarkers for die-
tary intake, however, this is often not possible in large epidemiological studies or to assess general expo-
sure in free-living individuals. Accurate information about dietary phytoestrogens is therefore important
but there is very limited data concerning food contents. In this study, we analysed the phytoestrogen
(isoflavone, lignan and coumestrol) content in more than 240 different foods based on fresh and pro-
cessed fruits and vegetables using a newly developed sensitive method based on LC–MS incorporating
13
C
3
-labelled standards. Phytoestrogens were detected in all foods analysed with a median content of
20
l
g/100 g wet weight (isoflavones: 2
l
g/100 g; lignans 12
l
g/100 g). Most foods contained less than
100
l
g/100 g, however, 5% of foods analysed contained more than 400
l
g/100 g, in particular soya-based
foods and other legumes. The results published here will contribute to databases of dietary phytoestro-
gen content and allow the more accurate determination of phytoestrogen exposure in free-living
individuals.
Ó2009 Elsevier Ltd. All rights reserved.
1. Introduction
Phytoestrogens are a group of non-steroidal polyphenolic plant
metabolites that induce biological responses and can mimic or
modulate the action of endogenous oestrogens, often by binding
to oestrogen receptors (Committee on Toxicity of Chemicals in
Food, 2003). The bioactivity of these compounds is based on their
structural similarity with 17b-oestradiol (Branham et al., 2002;
Martin, Horwitz, Ryan, & McGuire, 1978; Setchell & Adlercreutz,
1988; Verdeal, Brown, Richardson, & Ryan, 1980) and their ability
to bind to oestrogen receptors (Shutt & Cox, 1972). Apart from
their effect on oestrogen receptors, phytoestrogens can also act
as antioxidants (Wei, Bowen, Cai, Barnes, & Wang, 1995) and inhib-
itors of enzymes such as tyrosine kinase (Akiyama et al., 1987) and
DNA topoisomerase (Markovits et al., 1989). As a result of their
bioactivity, these compounds have received increasing attention
for potentially beneficial effects for a wide range of human condi-
tions such as cancer (Adlercreutz, 2002; Duffy, Perez, & Partridge,
2007; Peeters, Keinan-Boker, van der Schouw, & Grobbee, 2003;
Stark & Madar, 2002), cardiovascular disease (Anthony, 2002; Stark
& Madar, 2002), osteoporosis (Dang & Lowik, 2005; Stark & Madar,
2002) menopausal symptoms (Krebs, Ensrud, MacDonald, & Wilt,
2004; Stark & Madar, 2002), male infertility (Phillips & Tanphai-
chitr, 2008), obesity and type 2 diabetes (Bhathena & Velasquez,
2002). However, elevated endogenous sex hormone levels are gen-
erally associated with an increased risk of breast cancer in women
(The Endogenous Hormones and Breast Cancer Collaborative,
2002) and not all studies have shown a beneficial effect on breast
cancer risk associated with increased exposure to phytoestrogens
in Western societies (Grace et al., 2004; Ward et al., 2008). There
are also strong gene–nutrient interactions between phytoestro-
gens and oestrogen receptor polymorphisms (ESR1 and NR1I2)
(Low et al., 2005b, 2007), polymorphisms in the gene for the sex-
hormone binding globulin (SHBG) (Low et al., 2006) and probably
polymorphisms in the gene encoding aromatase (CYP19) (Low
et al., 2005a) which influence their bioactivity. Despite the large
number of studies conducted, there is still no clear evidence
whether phytoestrogen intake has a beneficial or detrimental ef-
fect on human health and the UK Committee on Toxicity (COT)
has recommended further research (Committee on Toxicity of
Chemicals in Food, 2003).
Exposure to phytoestrogens can be determined either directly
by measuring diet or indirectly by using biomarkers in plasma or
urine (Grace et al., 2004). Although biomarkers are often more reli-
able due to the limitations in dietary assessment (Day, McKeown,
0308-8146/$ - see front matter Ó2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.03.002
*Corresponding author. Tel.: +44 1223 252769; fax: +44 1223 252765.
E-mail address: ggck2@cam.ac.uk (G.G.C. Kuhnle).
Food Chemistry 116 (2009) 542–554
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Table 1
Phytoestrogen content of fruits and vegetables analysed. The data is the average of three samples analysed in duplicate and given in
l
g/100 g wet weight. Isoflavones are the sum of daidzein, genistein, glycitein, biochanin A and
formononetin, lignans the sum of secoisolariciresinol and matairesinol. Unless stated otherwise, food analysed was unprepared.
Food (taxonomic
name)
Preparation Variety Family Phytoestrogens Isoflavones Lignans Daidzein Genistein Glycitein Biochanin A Formononetin Secoisolariciresinol Matairesinol Coumestrol
Apple (Malus
domestica)
Cored Cox Rosaceae 4 2 2 <1 <1 1 <1 2 <1 <1
Apple (Malus
domestica)
Cored Golden
Delicious
Rosaceae 5 2 3 <1 <1 <1 <1 3 <1 <1
Apple (Malus
domestica)
Cored Granny
Smith
Rosaceae 4 2 2 <1 <1 <1 1 2 <1 <1
Apple (Malus
domestica)
Cored Red
dessert
Rosaceae 3 1 2 <1 <1 <1 <1 <1 2
Apple (Malus
domestica)
Peeled, cored &
cooked
Cooking
apple
Rosaceae 9 7 2 2 <1 <1 4 <1 2 <1
Apple (Malus
domestica)
Peeled & cored Cooking
apple
Rosaceae 5 3 2 1 <1 <1 1 <1 1 <1
Apple (Malus
domestica)
Peeled & cored Cox Rosaceae 5 2 2 <1 <1 <1 <1 2 <1 <1
Apple (Malus
domestica)
Peeled & cored Golden
Delicious
Rosaceae 5 3 2 <1 <1 <1 2 <1 2 <1
Apple (Malus
domestica)
Peeled & cored Granny
Smith
Rosaceae 4 2 2 <1 <1 <1 <1 2 <1 <1
Apple (Malus
domestica)
Peeled & cored Red
dessert
Rosaceae 2 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1
Apricot (Prunus
armeniaca)
Stoned Rosaceae 53 1 52 <1 <1 <1 <1 51 <1
Apricot (Prunus
armeniaca)
Dried Rosaceae 443 12 431 – 8 4 430 <1 <1
Apricot (Prunus
armeniaca)
Tinned in syrup,
drained
Rosaceae 24 2 22 <1 <1 <1 1 <1 22 <1
Asparagus (Asparagus
officinalis)
Cooked Asparagaceae 154 2 152 <1 <1 2 <1 149 3
Aubergine (Solanum
melongena)
Raw Solanaceae 9 <1 8 <1 <1 <1 <1 <1 8 <1 <1
Aubergine (Solanum
melongena)
Cooked Solanaceae 8 <1 8 <1 <1 <1 <1 <1 8 <1
Avocado (Persea
americana)
Peeled & stoned Lauraceae 43 9 34 <1 <1 6 <1 <1 24 8
Banana (Musa sp.) Peeled Musaceae 3 2 1 <1 <1 <1 1 <1 <1 <1
Beans, baked
(Phaseolus vulgaris)
Cold Fabaceae 28 5 22 2 3 <1 <1 <1 22 <1 <1
Beans, baked
(Phaseolus vulgaris)
Heated Fabaceae 25 6 19 2 3 <1 <1 <1 19 <1 <1
Beans, Broad beans
(Vicia faba)
Fresh, podded Fabaceae 21 <1 21 <1 <1 <1 <1 <1 20 <1
Beans, Broad beans
(Vicia faba)
Cooked Fabaceae 22 <1 21 <1 <1 <1 <1 <1 21 <1
Beans, Butter beans
(Phaseolus limensis)
Dried Fabaceae 196 51 143 24 21 6 141 2 2
Beans, Butter beans
(Phaseolus limensis)
Cooked from dried Fabaceae 36 13 22 6 5 1 <1 22 <1 <1
Beans, French beans
(Phaseolus vulgaris)
Fabaceae 147 50 94 12 35 2 1 <1 94 <1 3
Beans, French beans
(Phaseolus vulgaris)
Cooked Fabaceae 159 48 109 8 36 2 2 <1 108 <1 2
Beans, Haricot beans
(Phaseolus vulgaris)
Dried Fabaceae 132 21 106 6 14 <1 106 5
G.G.C. Kuhnle et al. / Food Chemistry 116 (2009) 542–554 543
Beans, Haricot beans
(Phaseolus vulgaris)
Cooked from dried Fabaceae 29 6 22 2 4 <1 22 <1 <1
Beans, Kidney beans
(Phaseolus vulgaris)
Dried Fabaceae 172 73 89 15 26 32 88 <1 10
Beans, Kidney beans
(Phaseolus vulgaris)
Cooked from dried Fabaceae 41 14 26 2 6 5 <1 26 <1 <1
Beans, Runner beans
(Phaseolus
coccineus)
Trimmed & strung Fabaceae 201 164 26 64 78 22 <1 <1 26 <1 11
Beans, Runner beans
(Phaseolus
coccineus)
Trimmed, strung &
cooked
Fabaceae 156 132 18 45 70 16 <1 <1 17 7
Beansprouts (Vigna
radiata)
Pre-washed Fabaceae 798 351 86 110 225 16 <1 86 <1 361
Beetroot (Beta vulgaris) Raw, peeled Chenopodiaceae 8 1 7 <1 <1 1 6 1
Beetroot (Beta vulgaris) Cooked Chenopodiaceae 10 <1 10 <1 <1 <1 <1 10 <1
Beetroot (Beta vulgaris) Pickled Chenopodiaceae 5 1 4 <1 <1 <1 <1 4 <1
Beetroot (Beta vulgaris) Precooked Chenopodiaceae 8 1 7 <1 <1 1 <1 7 <1
Blackberries (Rubus sp.) Fresh Rosaceae 57 <1 56 <1 <1 <1 <1 <1 55 1 <1
Blackberries (Rubus sp.) Stewed from fresh Rosaceae 221 <1 220 <1 <1 220 <1
Blackcurrant (Ribes
nigrum)
Fresh Grossulariaceae 109 2 107 <1 <1 2 <1 105 2 <1
Blackcurrant (Ribes
nigrum)
Tinned in juice and
syrup, drained
Grossulariaceae 69 <1 69 <1 <1 65 4 <1
Broccoli (Brassica
oleracea)
Fresh, thick stalks
removed
Brassicaceae 71 <1 71 <1 <1 <1 <1 71 <1
Broccoli (Brassica
oleracea)
Cooked, thick stalks
removed
Brassicaceae 96 3 90 <1 <1 <1 3 <1 89 <1 3
Broccoli, sprouting
(Brassica oleracea)
Tough stalks
removed
Brassicaceae 68 1 66 <1 <1 <1 66 <1 <1
Broccoli, sprouting
(Brassica oleracea)
Cooked from fresh Brassicaceae 41 3 38 2 1 <1 37 <1 <1
Brussel sprouts
(Brassica oleracea)
Brassicaceae 75 <1 74 <1 <1 <1 <1 74 <1 <1
Brussel sprouts
(Brassica oleracea)
Cooked Brassicaceae 59 <1 58 <1 <1 <1 <1 <1 58 <1
Cabbage, green
(Brassica oleracea)
Brassicaceae 11 <1 10 – <1 <1 <1 <1 10 <1 <1
Cabbage, green
(Brassica oleracea)
Cooked Brassicaceae 8 <1 7 <1 <1 <1 <1 7 <1 <1
Cabbage, January King
(Brassica oleracea)
Brassicaceae 14 4 10 <1 3 <1 <1 10 <1
Cabbage, January King
(Brassica oleracea)
Cooked Brassicaceae 6 1 5 <1 <1 <1 <1 4 <1
Cabbage, red (Brassica
oleracea)
Brassicaceae 7 <1 6 <1 <1 <1 <1 <1 5 <1
Cabbage, red (Brassica
oleracea)
Cooked Brassicaceae 4 <1 4 <1 <1 <1 <1 <1 4 <1
Cabbage, Savoy
(Brassica oleracea)
Brassicaceae 30 4 26 <1 2 <1 1 <1 26 <1
Cabbage, Savoy
(Brassica oleracea)
Cooked Brassicaceae 15 3 12 <1 <1 <1 3 <1 12 <1
Cabbage, white
(Brassica oleracea)
Brassicaceae 12 <1 11 <1 <1 <1 <1 <1 11 <1 <1
Cabbage, white
(Brassica oleracea)
Cooked Brassicaceae 8 <1 8 <1 <1 <1 <1 <1 8 <1 <1
Carrots (Daucus carota) Apiaceae 125 4 121 2 1 <1 1 <1 114 7 <1
Carrots (Daucus carota) Cooked Apiaceae 114 3 111 1 2 <1 107 3
Carrots (Daucus carota) Tinned Apiaceae 49 <1 48 <1 <1 <1 <1 <1 47 1 <1
(continued on next page)
544 G.G.C. Kuhnle et al. / Food Chemistry 116 (2009) 542–554
Table 1 (continued)
Food (taxonomic
name)
Preparation Variety Family Phytoestrogens Isoflavones Lignans Daidzein Genistein Glycitein Biochanin A Formononetin Secoisolariciresinol Matairesinol Coumestrol
Cauliflower (Brassica
oleracea)
Brassicaceae 15 <1 14 <1 <1 <1 <1 <1 14 <1
Cauliflower (Brassica
oleracea)
Cooked Brassicaceae 12 <1 11 <1 <1 <1 <1 11 <1
Celeriac (Apium
graveolens)
Peeled Apiaceae 14 2 13 <1 1 <1 <1 <1 10 <1
Celeriac (Apium
graveolens)
Peeled, cooked Apiaceae 36 <1 35 <1 <1 <1 <1 <1 33 <1
Celery (Apium
graveolens)
Apiaceae 7 <1 7 <1 <1 <1 6 <1 <1
Celery (Apium
graveolens)
Cooked Apiaceae 8 <1 8 <1 <1 <1 7 <1 <1
Cherries (Prunus sp.) Stoned Rosaceae 27 20 6 5 2 13 1 <1 6
Cherries (Prunus sp.) Glace Rosaceae 7 4 3 <1 <1 <1 3 <1 1 2 <1
Chestnuts (Castanea
sativa)
Fagaceae 217 2 214 <1 <1 <1 2 <1 201 13 <1
Chestnuts (Castanea
sativa)
Cooked Fagaceae 283 2 280 <1 <1 <1 1 <1 265 15 <1
Chick peas
(Cicer arietinum)
Dried Fabaceae 609 607 2 16 79 9 441 62 <1 2 <1
Chick peas
(Cicer arietinum)
Cooked from dried Fabaceae 420 416 4 4 35 3 351 22 <1 3 <1
Chick peas, as
Houmous
169 135 34 <1 4 <1 127 4 <1 34 <1
Chicory
(Cichorium intybus)
Asteraceae 19 <1 19 <1 <1 <1 18 <1 <1
Chinese leaves
(Brassica rapa)
Brassicaceae 12 1 11 <1 <1 <1 <1 <1 11 <1
Clementine (Citrus
reticulata)
Pith and skin removed Rutaceae 6 <1 5 <1 <1 <1 5 <1 <1
Coconut (Cocos
nucifera)
Fresh Arecaceae 42 10 32 – 4 6 30 2 <1
Coconut (Cocos
nucifera)
Desiccated Arecaceae 26 3 23 <1 2 <1 <1 19 4
Courgette (Cucurbita
pepo)
Cucurbitaceae 35 <1 35 <1 <1 <1 <1 35 <1
Courgette (Cucurbita
pepo)
Cooked Cucurbitaceae 46 3 43 <1 <1 <1 2 <1 43 <1 <1
Cranberries (Vaccinium
sp.)
Ericaceae 93 3 88 2 <1 1 <1 88 <1 <1
Cucumber (Cucumis
sativus)
Cucurbitaceae 12 <1 12 <1 <1 <1 <1 12 <1
Cucumber (Cucumis
sativus)
w/o Skin Cucurbitaceae 13 <1 13 <1 <1 <1 <1 <1 13 <1 <1
Curly kale (Brassica
oleracea)
Cooked Brassicaceae 8 2 6 2 <1 <1 <1 5 1 <1
Dates (Phoenix
dactylifera)
Boxed, stones removed Arecaceae 168 4 163 <1 <1 1 2 161 3 <1
Dates (Phoenix
dactylifera)
Dried Arecaceae 599 14 584 – 7 7 <1 581 3 <1
Dates, medjool
(Phoenix dactylifera)
Stones removed Arecaceae 192 35 157 5 19 6 4 <1 156 2 <1
Fennel (Foeniculum
vulgare)
Apiaceae 72 <1 72 <1 <1 <1 <1 59 12
G.G.C. Kuhnle et al. / Food Chemistry 116 (2009) 542–554 545
Fennel (Foeniculum
vulgare)
Cooked Apiaceae 85 <1 85 <1 <1 <1 <1 <1 72 13
Fig. (Ficus sp.) Moraceae 389 12 376 – 10 2 <1 372 3 <1
Fig. (Ficus sp.) Dried Moraceae 129 14 114 – 12 <1 1 <1 113 1 1
Garlic (Allium sativum) Peeled Alliaceae 99 2 97 <1 2 <1 93 4 <1
Gooseberries (Ribes
uva-crispa)
Grossulariaceae 72 <1 71 <1 <1 <1 <1 <1 71 <1 <1
Gooseberries (Ribes
uva-crispa)
Stewed with sugar Grossulariaceae 121 <1 121 <1 <1 <1 119 2 <1
Grapefruit (Citrus x
paradisi)
Peel, pith and pips
removed
Rutaceae 39 17 21 3 2 11 <1 21 <1 <1
Grapefruit (Citrus x
paradisi)
Tinned in juice Rutaceae 21 13 4 5 8 <1 4 <1 4
Grapes, black (Vitis sp.) Seeds removed Vitaceae 19 5 14 1 4 <1 <1 7 7
Grapes, dried as
Currants (Vitis sp.)
Dried Vitaceae 87 17 70 – 7 10 36 34 <1
Grapes, dried as Raisins
(Vitis sp.)
Dried Vitaceae 88 7 81 – <1 <1 5 1 50 31 <1
Grapes, red, seedless
(Vitis sp.)
Vitaceae 21 6 15 <1 3 <1 2 7 8
Grapes, white, seedless
(Vitis sp.)
Vitaceae 18 <1 17 <1 <1 <1 <1 <1 8 9
Green beans (Phaseolus
vulgaris)
Frozen, sliced Fabaceae 58 19 38 3 16 <1 <1 <1 38 <1
Green beans (Phaseolus
vulgaris)
Frozen, sliced, cooked Fabaceae 46 16 30 3 13 - <1 <1 29 <1 <1
Greengage (Prunus sp.) Stoned Rosaceae 105 2 103 <1 <1 1 <1 <1 102 1 <1
Kiwi (Actinidia delicosa) Skin removed Actinidiaceae 111 <1 111 <1 <1 <1 107 4
Leek (Allium
ampeloprasum)
Alliaceae 66 1 65 <1 <1 <1 1 <1 65 <1
Leek (Allium
ampeloprasum)
Cooked Alliaceae 61 9 52 <1 <1 <1 8 <1 52 <1
Lemon (Citrus x limon) Freshly juiced Rutaceae 4 1 2 <1 <1 <1 <1 <1 2 <1 1
Lemon (Citrus x limon) Peeled Rutaceae 29 4 25 3 <1 <1 25 <1
Lentils, red (Lens
culinaris)
Dried Fabaceae 54 51 3 26 19 6 2 1 <1
Lentils, red (Lens
culinaris)
Cooked Fabaceae 14 13 <1 6 5 2 <1 <1 <1
Lettuce, cos (Lactuca
sativa)
Asteraceae 5 <1 4 <1 <1 <1 <1 <1 4
Lettuce, iceberg
(Lactuca sativa)
Asteraceae 7 <1 7 <1 <1 <1 <1 7 <1 <1
Lettuce, little gem
(Lactuca sativa)
Asteraceae 7 1 6 <1 <1 <1 <1 5 1
Lettuce, round
(Lactuca sativa)
Asteraceae 8 <1 8 <1 <1 <1 <1 7 <1 <1
Lychees
(Litchi chinensis)
Tinned in syrup Sapindaceae 4 3 <1 <1 <1 <1 3 <1 <1 <1 <1
Mandarin (Citrus
reticulata)
Tinned Rutaceae 5 2 2 1 1 <1 <1 2 <1 1
Mangetout (Pisum
sativum)
Cooked Fabaceae 47 38 9 – <1 37 – <1 9 <1
Mango (Magnifera sp.) Skinned & stoned Anacardiaceae 20 1 19 <1 <1 <1 <1 <1 17 1
Mango (Magnifera sp.) Tinned in syrup Anacardiaceae 4 3 <1 <1 3 <1 <1
Marrow (Cucurbita sp.) Cucurbitaceae 9 <1 9 <1 <1 <1 <1 <1 9 <1
Marrow (Cucurbita sp.) Cooked Cucurbitaceae 8 <1 8 <1 8 <1 <1
Melon, cantaloupe
(Cucumis melo ssp.)
Skin & seeds removed Cucurbitaceae 16 <1 16 <1 <1 <1 <1 <1 16 <1
(continued on next page)
546 G.G.C. Kuhnle et al. / Food Chemistry 116 (2009) 542–554
Table 1 (continued)
Food (taxonomic name) Preparation Variety Family Phytoestrogens Isoflavones Lignans Daidzein Genistein Glycitein Biochanin A Formononetin Secoisolariciresinol Matairesinol Coumestrol
Melon, galia (Cucumis
melo ssp.)
Skin & seeds
removed
Cucurbitaceae 11 <1 11 <1 <1 <1 <1 <1 11 <1 <1
Melon, honeydew
(Cucumis melo ssp.)
Skin & seeds
removed
Cucurbitaceae 25 3 22 1 1 <1 <1 <1 21 <1 <1
Melon, water
(Citrullus lanatus)
Skin & seeds
removed
Cucurbitaceae 35 <1 34 <1 <1 <1 <1 <1 34 <1 <1
Mung beans (Vigna
radiata)
Dried Fabaceae 323 32 289 6 15 2 8 <1 289 2
Mung beans (Vigna
radiata)
Cooked from
dried
Fabaceae 50 8 42 <1 4 <1 3 <1 42 <1 <1
Mushrooms (Agaricus
bisporus)
Wiped &
trimmed
Agariaceae n/a n/a <1 Not
determined
52 <1 <1 <1 <1
Mushrooms (Agaricus
bisporus)
Cooked Agariaceae 2 2 <1 <1 <1 <1 1 <1 <1 <1
Mushrooms (Agaricus
bisporus)
Microwaved Agariaceae <1 <1 <1 <1 <1 <1 <1 <1 <1
Nectarine (Prunus
persica)
Stones
removed
Rosaceae 25 1 24 <1 <1 <1 <1 <1 24 <1
Okra (Abelmoschus
esculentus)
Topped & tailed Malvaceae 86 2 84 <1 <1 1 <1 84 <1 <1
Olives, black, pitted
(Olea europaea)
Tinned in brine,
drained in jar of
brine,
Oleaceae 16 2 14 <1 1 <1 <1 5 9 <1
Olives, green (Olea
europaea)
stoned, drained Oleaceae 33 1 32 <1 <1 25 6 <1
Onion rings (Allium
cepa)
Breaded/
battered
Alliaceae 55 44 11 6 28 5 4 <1 8 3 <1
Onions (Allium cepa) Alliaceae 31 <1 31 – <1 <1 <1 <1 30 <1
Onions (Allium cepa) Cooked Alliaceae 21 <1 20 – <1 <1 <1 <1 20 <1 <1
Orange (Citrus
sinensis)
peel & pith
removed
Rutaceae 36 12 21 4 3 5 <1 21 <1 2
Orange (Citrus
sinensis)
Longlife juice Rutaceae 9 <1 4 <1 <1 <1 4 <1 4
Papaya (Carica
papaya)
Peel & seeds
removed
Caricaceae 4 2 2 <1 2 <1 2
Parsley (Petroselium
crispum)
Leaves Apiaceae 197 59 137 - 57 <1 <1 <1 137 <1 <1
Parsnip (Pastinaca
sativa)
Apiaceae 65 5 60 <1 <1 <1 5 36 21
Parsnip (Pastinaca
sativa)
Cooked Apiaceae 66 <1 65 – <1 <1 <1 <1 49 17
Passion Fruit
(Passiflora edulis)
Juice & seeds Passifloraceae 71 43 26 42 <1 <1 <1 <1 26 <1 2
Peach (Prunus perica) Stoned Rosaceae 43 <1 42 <1 <1 <1 <1 <1 42 <1
Peach (Prunus perica) Tinned in
syrup, drained
Rosaceae 2 2 <1 – <1 2 <1 <1
Pear (Pyrus communis) w/o Skin Comice Rosaceae 19 2 17 <1 1 <1 17 <1 <1
Pear (Pyrus communis) w/o Skin Conference Rosaceae 6 <1 5 <1 <1 5 <1
Pear (Pyrus communis) w/o Skin Williams Rosaceae 6 6 <1 <1 <1 5 <1 <1
Pear (Pyrus communis) Tinned, drained Rosaceae 1 <1 <1 <1 <1 <1 <1 <1 <1 <1
Pear (Pyrus communis) w/o skin Comice Rosaceae 8 <1 7 <1 <1 7 <1 <1
Pear (Pyrus communis) w/o Skin Conference Rosaceae 3 <1 3 <1 <1 2 <1
Pear (Pyrus communis) w/o Skin Williams Rosaceae 3 3 <1 <1 2 <1 <1
Peas (Pisum
sativum)
Tinned,
processed,
drained
Fabaceae 2 2 <1 <1 <1 <1 <1 <1 <1 <1
Peas, fresh (Pisum
sativum)
Fabaceae 3 2 1 <1 <1 1 <1 1 <1
G.G.C. Kuhnle et al. / Food Chemistry 116 (2009) 542–554 547
Peas, fresh (Pisum
sativum)
Cooked Fabaceae 1 1 <1 <1 <1 <1 <1 <1 <1 <1
Peas, frozen (Pisum
sativum)
Fabaceae 3 1 2 <1 <1 <1 <1 <1 1 <1 <1
Peas, frozen (Pisum
sativum)
Cooked Fabaceae 2 1 1 <1 <1 <1 <1 <1 <1 <1
Peas, garden (Pisum
sativum)
Tinned, drained Fabaceae 2 1 1 <1 <1 <1 <1 <1 <1 <1
Peas, marrowfat
(Pisum sativum)
Tinned, drained Fabaceae 51 50 <1 <1 3 46 <1 <1 <1 <1
Peas, mushy (Pisum
sativum)
Tinned/frozen Fabaceae 15 14 1 <1 3 10 <1 <1 1
Peas, petit pois (Pisum
sativum)
Frozen Fabaceae 8 6 2 2 1 2 <1 <1 2
Peas, split, dried
(Pisum sativum)
Fabaceae 15 11 4 2 2 7 <1 <1 4 <1
Peas, split, dried
(Pisum sativum)
Cooked Fabaceae 13 12 <1 4 5 3 <1 <1 <1
Peas, sugar snap
(Pisum sativum)
Cooked Fabaceae 44 31 13 – <1 29 <1 <1 13 <1
Peas, whole, dried
(Pisum sativum)
Fabaceae 29 28 <1 7 9 9 3 <1
Peas, whole, dried
(Pisum sativum)
Cooked Fabaceae 10 9 <1 2 3 4 <1 <1 <1
Pepper, green
(Capsicum annuum)
Solanaceae 11 <1 11 <1 <1 11 <1
Pepper, red (Capsicum
annuum)
Solanaceae 16 5 11 <1 <1 4 <1 11 <1
Pepper, yellow
(Capsicum annuum)
Solanaceae 11 6 5 6 <1 5 <1 <1
Pineapple (Ananas
comosus)
Bromelidaceae 38 21 17 21 2 15
Pineapple (Ananas
comosus)
Tinned in juice,
drained
Bromelidaceae 14 2 12 <1 <1 <1 1 <1 <1 11 <1
Plum, red (Prunus
domestica)
Rosaceae 8 2 6 <1 <1 1 <1 <1 6 <1
Plum, Victoria (Prunus
domestica)
Rosaceae 26 2 24 <1 <1 <1 <1 <1 24 <1 <1
Plum, yellow (Prunus
domestica)
Rosaceae 72 2 69 – <1 1 <1 69 <1
Plum, yellow (Prunus
domestica)
Cooked Rosaceae 152 2 150 – <1 <1 <1 <1 150 <1 <1
Pomegranate (Punica
granatum)
Flesh & seeds Lythraceae 304 <1 304 <1 <1 <1 <1 <1 294 9 <1
Potato, chips (Solanum
tuberosum)
From chip–
shop
Solanaceae 11 7 3 <1 2 <1 5 <1 <1 2 <1
Potato, chips (Solanum
tuberosum)
Oven chips Solanaceae 15 11 4 3 2 5 <1 3 1
Potato, crisps
(Solanum
tuberosum)
Solanaceae 22 5 17 3 <1 2 14 2 <1
Potato, for baking
(Solanum
tuberosum)
Boiled Solanaceae 3 2 1 <1 <1 <1 <1 <1 <1 <1 <1
Potato, for baking
(Solanum
tuberosum)
w/o Skin,
boiled
Solanaceae 2 1 <1 <1 <1 <1 <1 <1 <1 <1
Potato, mashed, instant
(Solanum
tuberosum)
Solanaceae 4 2 1 <1 2 <1 <1 1 <1
(continued on next page)
548 G.G.C. Kuhnle et al. / Food Chemistry 116 (2009) 542–554
Table 1 (continued)
Food (taxonomic name) Preparation Variety Family Phytoestrogens Isoflavones Lignans Daidzein Genistein Glycitein Biochanin A Formononetin Secoisolariciresinol Matairesinol Coumestrol
Potato, new (Solanum
tuberosum)
Solanaceae 18 16 2 <1 4 11 <1 1 <1 <1
Potato, new (Solanum
tuberosum)
Boiled Solanaceae 5 2 3 <1 <1 <1 <1 <1 2 <1 <1
Potato, new
(Solanum tuberosum)
w/o Skin Solanaceae 4 2 2 <1 <1 2 <1 1 <1
Potato, new (Solanum
tuberosum)
w/o Skin, boiled Solanaceae 3 3 <1 <1 3 <1 <1
Potato, old (Solanum
tuberosum)
Solanaceae 10 8 2 <1 1 5 <1 <1 <1 2
Potato, old (Solanum
tuberosum)
Baked Solanaceae 3 2 1 <1 1 <1 1 <1
Potato, old (Solanum
tuberosum)
Boiled Solanaceae 1 <1 <1 <1 <1 <1 <1 <1
Potato, old (Solanum
tuberosum)
w/o Skin, baked Solanaceae 3 1 2 <1 <1 <1 <1 <1 1 <1
Potato, red (Solanum
tuberosum)
w/o Skin Solanaceae 10 1 8 <1 <1 <1 <1 <1 8 <1
Potato, red (Solanum
tuberosum)
w/o Skin, boiled Solanaceae 20 5 15 2 3 <1 <1 <1 14 <1 <1
Potato, waffle (Solanum
tuberosum)
Cooked Solanaceae 9 5 4 <1 4 <1 <1 2 1 <1
Prune (Prunus domestica) Dried Rosaceae 363 6 357 1 2 2 <1 <1 352 4 <1
Prune (Prunus domestica) Cooked from dried Rosaceae 108 2 106 <1 <1 <1 <1 105 <1 <1
Prune (Prunus domestica) Semi–dried, ready
to eat
Rosaceae 284 3 281 <1 1 <1 <1 278 3 <1
Prune (Prunus domestica) Tinned in syrup &
juice, not drained
Rosaceae 75 3 72 – 1 <1 <1 <1 68 4 <1
Pumpkin (Cucurbita sp.) Cucurbitaceae 154 <1 154 <1 <1 <1 <1 153 <1 <1
Quince (Cydonia oblonga) Stewed Rosaceae 9 2 7 <1 <1 1 <1 6 <1 <1
Radish (Raphanus sativus) Brassicaceae 3 <1 3 <1 <1 <1 <1 <1 3 <1
Raspberries (Rubus idaeus) Rosaceae 26 2 24 <1 <1 <1 1 <1 24 <1 <1
Raspberries (Rubus idaeus) Tinned in syrup,
drained
Rosaceae 21 5 15 <1 <1 <1 5 <1 15 <1 <1
Redcurrants (Ribes
rubrum)
Grossulariaceae 47 <1 46 <1 <1 <1 45 <1 <1
Rhubarb (Rheum sp.) Polygonaceae 1 1 <1 <1 <1 <1 <1 <1 <1 <1 <1
Rhubarb (Rheum sp.) Cooked Polygonaceae 3 2 <1 <1 <1 <1 <1 <1 <1 <1 <1
Salad cress (Lepidium
sativum)
Brassicaceae 18 3 15 1 1 <1 – <1 15 <1
Satsuma (Citrus unshiu) Rutaceae 24 2 12 2 <1 12 <1 10
Sharon fruit (Diospyros sp.) Ebenaceae 11 6 5 <1 1 <1 4 <1 4 <1 <1
Soya bean (Glycine max) Cooked Fabaceae 17556 17544 11 5730 10664 1144 1 4 11 <1 <1
Soya bean (Glycine max) Frozen, cooked Fabaceae 10687 10621 64 3864 5540 1210 <1 6 64 <1 2
Soya bean, Tofu
(Glycine max)
Microwaved Fabaceae 10619 10609 10 2528 7292 781 3 6 10 <1
Soya flour (Glycine max) Fabaceae 124727 124381 345 54128 62125 8114 <1 14 337 9 <1
Soya mince granules
(Glycine max)
Cooked Fabaceae 20850 20745 101 5747 13770 1225 <1 3 99 2 4
Spinach (Spinacia olearcea) Amaranthaceae 7 2 5 <1 <1 2 <1 5 <1 <1
Spinach (Spinacia olearcea) Cooked Amaranthaceae 4 <1 3 <1 <1 <1 <1 <1 3 <1 <1
Spring green
(Brassica oleracea)
Brassicaceae 56 11 45 <1 <1 <1 10 <1 44 <1
Spring green (Brassica
oleracea)
Cooked Brassicaceae 43 13 30 <1 <1 <1 12 <1 30 <1
Spring onion
(Allium sp.)
Alliaceae 62 9 53 – <1 <1 8 <1 52 <1
G.G.C. Kuhnle et al. / Food Chemistry 116 (2009) 542–554 549
Strawberries (Fragaria
ananassa)
Rosaceae 8 <1 7 <1 – <1 – <1 7 <1 <1
Strawberries (Fragaria
ananassa)
Tinned in syrup,
drained
Rosaceae 40 2 38 – <1 2 <1 38 <1
Sultanas (Vitis sp.) Vitaceae 54 11 44 – <1 <1 8 1 13 31 <1
Swede (Brassica
napobrassica)
Brassicaceae 6 1 5 <1 <1 <1 <1 <1 5 <1
Swede (Brassica
napobrassica)
Cooked Brassicaceae 2 <1 2 <1 <1 <1 <1 <1 2 <1
Sweet potato
(Ipomoea batatas)
Convolvulaceae 259 1 258 <1 <1 <1 <1 136 122 <1
Sweet potato
(Ipomoea batatas)
Cooked Convolvulaceae 251 1 249 <1 <1 <1 <1 118 132 <1
Sweetcorn (Zea mays) Boiled on the cob Poaceae 9 2 7 <1 <1 <1 2 <1 5 2 <1
Sweetcorn (Zea mays) Frozen, tinned,
drained
Poaceae <1 <1 <1 – <1 <1 <1 <1 <1 <1
Sweetcorn (Zea mays) Frozen, tinned,
drained, heated
Poaceae 3 <1 2 – <1 <1 <1 <1 1 <1
Sweetcorn (Zea mays) Kernels from the
cob
Poaceae 2 <1 1 <1 <1 <1 <1 <1 1 <1 <1
Sweetcorn, baby
(Zea mays)
Poaceae 6 <1 5 <1 <1 <1 <1 <1 5 <1 <1
Tomato (Solanum
lycopersicum)
Solanaceae 6 1 4 <1 <1 <1 <1 4 <1
Tomato (Solanum
lycopersicum)
Grilled Solanaceae 7 <1 6 <1 <1 <1 6 <1
Tomato (Solanum
lycopersicum)
Pureed Solanaceae 9 5 5 <1 4 <1 <1 4 <1 <1
Tomato (Solanum
lycopersicum)
Tinned Solanaceae 3 1 2 <1 <1 <1 2 <1 <1
Tomato ketchup 14 7 8 <1 2 <1 4 2 6 <1
Turnip (Brassica rapa) Brassicaceae 12 <1 12 <1 <1 12 <1
Turnip (Brassica rapa) Cooked Brassicaceae 8 <1 8 <1 <1 <1 <1 8 <1
Watercress (Nasturtium
sp.)
Brassicaceae 45 <1 45 <1 <1 <1 <1 <1 45 <1
Vegetable grills Cooked 43 16 27 <1 15 1 23 3 <1
Brown sauce 214 46 168 14 25 4 2 162 7
Fruit cocktail Tinned in syrup,
drained
3 <1 3 <1 <1 <1 – <1 2 <1
Mixed peel 38 32 6 5 19 3 5 <1 <1 5 <1
Pate vegetarian assorted 581 488 92 110 317 42 19 1 87 4 <1
Pate vegetarian chick pea
based (Cicer arietinum)
Fabaceae 1494 1444 49 351 806 111 169 7 36 13 <1
Pate, mushroom (Agaricus
bisporus)
Agariaceae 17 8 8 <1 5 <1 2 <1 4 3 <1
550 G.G.C. Kuhnle et al. / Food Chemistry 116 (2009) 542–554
Wong, Welch, & Bingham, 2001; Kipnis et al., 2003), their use is of-
ten not feasible, particularly in larger studies, and intake has to be
either calculated from dietary information provided by partici-
pants or determined by a combination of biomarkers and dietary
information. Accurate information on the phytoestrogen content
in foods is therefore crucial for the investigation of effects on
health; and to determine population levels for surveillance
purposes.
The main dietary sources of phytoestrogens are plant-based
foods such as fruits and vegetables. In plants, where these com-
pounds occur predominantly as glycosides, they act as antioxi-
dants, screen against light and most importantly act as defensive
agents against predators (Mazur & Adlercreutz, 1998a). The princi-
pal phytoestrogen-classes are isoflavones (found mainly in le-
gumes, e.g. chickpeas and soybean), lignans (e.g. in cereals,
linseed and other fruits and vegetables) and coumestans (e.g. in
young sprouting legumes like clover or alfalfa sprouts) (Committee
on Toxicity of Chemicals in Food, 2003).
Several detailed studies have been conducted to determine the
phytoestrogen content of food previously, amongst others in the
UK (Liggins, Bluck, Coward, & Bingham, 1998a, 1998b; Liggins
et al., 2000; Liggins, Grimwood, & Bingham, 2000; Liggins, Mulli-
gan, Runswick, & Bingham, 2002), Finland (Dwyer et al., 1994; Ma-
zur, 1998; Mazur et al., 1996, 1998b; Valsta et al., 2003), and the
US (US Department of Agriculture, 2002); however, these studies
provide only data for approximately 12% of the UK diet (Mulligan,
Welch, McTaggart, Bhaniani, & Bingham, 2007) and had methodo-
logical limitations (Adlercreutz et al., 1993; Wähälä, Hase, & Adl-
ercreutz, 1995; Wähälä & Rasku, 1997). Previously, we have
developed a sensitive LC/MS/MS method using
13
C
3
-labelled stan-
dards to analyse phytoestrogens in plasma and urine (Grace
et al., 2003). We have adapted this method to be used for food sam-
ples and have measured the phytoestrogen content (isoflavones:
biochanin A, daidzein, formononetin, genistein, glycitein; lignans:
matairesinol, secoisolariciresinol; coumestrol) in more than 240
foods based on fruits and vegetables commonly consumed in the
UK. This is one of the most comprehensive analysis of plant-based
phytoestrogens in the UK and elsewhere.
2. Experimental
2.1. Chemicals
Biochanin A, daidzein, genistein, glycitein, formononetin, seco-
isolariciresinol, matairesinol and coumestrol were purchased from
Plantech (Reading, Berkshire, UK).
13
C
3
-biochanin A
13
C
3
-daidzein,
13
C
3
-genistein,
13
C
3
-glycitein,
13
C
3
-formononetin,
13
C
3
-matairesi-
nol,
13
C
3
-secosiolariciresinol and
13
C
3
-enterolactone were obtained
from Dr. Nigel botting (University of St. Andrews, Fife, UK) (Fryatt
& Botting, 2005; Haajanen & Botting, 2006; Whalley, Bond, & Bot-
ting, 1998; Whalley, Oldfield, & Botting, 2000). b-Glucuronidase
(from Helix pomatia), b-glucosidase (from almonds) and cellulase
(from Trichoderma reesi) were purchased from Sigma (Poole, Dor-
set, UK). Water, methanol, acetic acid and ammonia were pur-
chased from Sigma (Poole, Dorset, UK) and Fisher Scientific
(Loughborough, Leicestershire, UK). To inhibit losses of target com-
pounds by adsorption to glassware, only silanised glassware was
used.
2.2. Sampling
Samples of each food were purchased from at least five different
food outlets (where possible) in Cambridgeshire, UK. If possible,
the foods bought at each outlet were from different manufacturers,
varieties, country of origin and/or batch numbers. Each sample was
weighed, prepared and a representative portion (approximately
35 g dry weight) was taken from each of the five samples. Cooked
food was boiled in water until tender and the water discarded;
more details on preparation are given in Table 1. Tinned foods were
drained unless indicated otherwise; outer leaves were removed
from cabbages; lettuce was analysed as purchased. The samples
were frozen (20 °C), freeze-dried if necessary (BOC Edwards,
Crawley, Sussex, UK) and stored at 20 °C until analysis. For anal-
ysis, samples of each food were pooled (equal amounts), weighed
and processed as described below.
2.3. Analysis
Samples were analysed as described previously (Kuhnle, Dell’A-
quila, Low, Kussmaul & Bingham, 2007). Briefly, approximately
100 mg freeze-dried food was extracted three times with 2.0 ml
10% methanol in sodium acetate (0.1%, pH 5) and deconjugated
with a hydrolysis reagent consisting of purified Helix pomatia juice
(b-glucuronidase), cellulase and b-glucosidase. Deconjugated sam-
ples were then extracted using Strata C-18E SPE cartridges (50 mg/
ml; Phenomenex, Macclesfield, Cheshire, UK), dried, reconstituted
in 40% aqueous methanol and analysed using LC/MS/MS. Analysis
was performed on an LC/MS/MS system consisting of a Jasco HPLC
system (Jasco, Great Dunmow, UK) using a diphenyl column (Varian
Pursuit, 3
l
m, 150 2 mm, Varian, Oxford, Oxfordshire, UK) and a
Waters Quattro Ultima triple quadrupole MS instrument (Waters,
Manchester, UK) fitted with an electrospray ion source in negative
ion mode and a LC/MS/MS system consisting of an Agilent 1100
CapHPLC System (Agilent, Wokingham, Berkshire, UK) and an ABI
4000 QTRAP mass spectrometer (Applied Biosystems, Warrington,
Cheshire, UK) fitted with an electrospray ion source in negative
ion mode. Compounds were quantified using
13
C
3
-labelled internal
standards; Compounds were quantified using
13
C
3
-labelled internal
standards; coumestrol was quantified using
13
C
3
-enterolactone.
The method was validated on both LC/MS/MS systems. The in-
tra-batch CV of this method is between 3% and 14% and the in-
ter-batch between 1% and 6%. As quality control, a sample
consisting of equal amounts of red cabbage, orange and celery
was analysed with each batch. The limit of detection of this meth-
od is 1.5
l
g/100 g dry weight.
2.4. Data analysis
Each sample was prepared in triplicate and analysed twice. Data
are presented as the average of two analyses and is in
l
g/100 g wet
weight. Data was analysed using SPSS 16 (SPSS Inc., Chicago, IL) for
Mac OS X. The data was not normally distributed and therefore
non-parametric tests were used. Differences between plant-fami-
lies were analysed using the Kruskal–Wallis test, the effect of prep-
aration was investigated using Wilcoxon signed rank test. p< 0.05
was considered to be statistically significant.
3. Results
In all foods analysed, with the exception of microwaved mush-
rooms and unheated tinned sweet-corn, phytoestrogens were de-
tected (Table 1). In most foods, the phytoestrogen content was
below 100
l
g/100 g wet weight (median: 20 g/100 g; IQR (inter-
quartile range): 7–66
l
g/100 g) with less isoflavones (median:
2
l
g/100 g; IQR: 1–8
l
g/100 g) than lignans (median: 12
l
g/
100 g; IQR 3–47
l
g/100 g) and a low amount of coumestrol (med-
ian: <1
l
g/100 g). However, 5% of foods analysed contained more
than 400
l
g/100 g phytoestrogens (>134
l
g/100 g isoflavones in
top 5% of foods; >218
l
g/100 g lignans in top 5% of foods), with
the highest content in soya flour (125,000
l
g/100 g) and cooked
soya beans (18,000
l
g/100 g).
G.G.C. Kuhnle et al. / Food Chemistry 116 (2009) 542–554 551
Daidzein, genistein and glycitein were the main isoflavones in
legumes, in particular in soya-based foods such as soya flour, soya
mince granules or tofu. A notable exception was passion fruit
which was the only non-legume with a daidzein content of more
than 40
l
g/100 g. Biochanin A was only found in some foods and
in most foods the content was below 1
l
g/100 g. High contents
of biochanin A were found mainly in chick peas and chick-pea
based foods such as vegetarian pâte and houmous. Similarly, for-
mononetin was found only in a few foods with the highest content
in chick peas and – to a lesser extent – soya. Most types of food
analysed contained lignans, in particular secoisolariciresinol,
which was only absent or in very low concentration in just a few
foods, notably peas and potatoes. The highest amount of secoisol-
ariciresinol was found in dried dates and apricots, but high
amounts were also found in figs, prunes, soya flour and pomegran-
ate. In contrast to secoisolariciresinol, matairesinol was either ab-
sent or present in very low concentrations in most foods. Cooked
sweet potatoes contained the highest amount of matairesinol
(132
l
g/100 g in cooked sweet potatoes). High amounts were also
found in dried grapes (currants, raisins and sultanas), parsnips and
chestnuts. Most foods contained only small amounts of coumestrol
with only 5% containing more than 2
l
g/100 g. Coumestrol was
mainly found in legumes and citrus fruits (Rutaceae) with the
highest content in beansprouts (361
l
g/100 g), raw runner
(11
l
g/100 g) and kidney (10
l
g/100 g) beans.
The phytoestrogen content and composition (proportion of iso-
flavones on total phytoestrogens) varied significantly (p< 0.05,
Kruskal–Wallis test) between foods from different plant families
(Table 2). Legumes (Fabaceae) contained the highest amount of
isoflavones whereas Alliaceae, such as garlic, leek and onion, and
Apiaceae, such as carrots, fennel and parsnips, had the highest con-
tent of lignans. In most foods, lignans were the main class of phy-
toestrogens found, with the exception of legumes which contained
mainly isoflavones.
Sixty-one types of food were analysed cooked and raw (Table 3).
There was a significant difference in phytoestrogen, isoflavone and
lignan content between raw and cooked foods (Wilcoxon signed
rank test, p< 0.05) but no difference in phytoestrogen composition,
suggesting that cooking affects isoflavones and lignans in a similar
way. For most foods, the phytoestrogen content was higher in raw
samples when compared with cooked samples, however, the phy-
toestrogen content increased in some foods, notably in red pota-
toes and celeriac. Peeling decreased the phytoestrogen content in
most foods analysed (Wilcoxon signed rank test, p< 0.05); it also
affected the phytoestrogen composition although not in a uniform
manner. The effect of stewing and drying could only be investi-
gated in a small number of foods but generally resulted in an in-
crease in phytoestrogen content. Tinned food had a lower overall
phytoestrogen content, but the difference was not statistically sig-
nificant (Wilcoxon signed rank test, p< 0.06). Analysis of variance
was conducted, but due to the small numbers of samples analysed,
it was not to investigate the effect of plant family on phytoestrogen
content and composition in more detail.
4. Discussion
Phytoestrogens are formed as secondary metabolites by most
plants and are therefore ubiquitous in plant products (Mazur &
Adlercreutz, 1998a). In this study, we have analysed more than
240 foods based on fruit and vegetables for their phytoestrogen
content to provide a comprehensive database for the assessment
of dietary intake and exposure. The results are expressed per
100 g wet weight to facilitate the use in epidemiological studies
and diet composition databases. Phytoestrogens were found in vir-
tually all foods analysed although the content in most foods was
well below 100
l
g/100 g wet weight with the exception of le-
gumes like soya and some other foods such as dried fruits, figs,
pomegranate, chestnuts and sweet potatoes. Staple foods such as
potatoes contained on average less than 10
l
g/100 g phytoestro-
gens. Other fruits and vegetables commonly consumed in a large
study of free-living adults in the UK (Day et al., 1999), such as ba-
nanas (3
l
g/100 g), raw tomatoes (6
l
g/100 g), apples (12
l
g/
100 g) and cucumbers (12
l
g/100 g), also contained only small
amounts of phytoestrogens.
In contrast to bioanalytical methods for the determination of
compounds in a single matrix such as plasma or urine, the analysis
of food stuff is made difficult by the large variety of different matri-
ces and moreover the lack of true quality controls (Kuhnle, Dell’A-
quila, Low, Kussmaul, & Bingham, 2007). Although most methods
use ‘‘standard foods” as quality control to monitor method perfor-
mance and precision, it is very difficult to monitor accuracy, in par-
ticular since the true content of each compound is not known.
Fortifying samples with neat standards does not provide sufficient
information on recovery and accuracy because most compounds
are present as glycosides and are embedded in the cellular matrix.
To assess relative quality and accuracy of data, they can be com-
pared with data published elsewhere. However, data for compari-
son is only available for a limited number of foods and most
studies focus on different types of phytoestrogens; for example
Horn-Ross et al. (2001) do not include formononetin whereas
Table 2
Phytoestrogen content (in
l
g/100 g wet weight; median and inter-quartile range) and composition (% of total phytoestrogen content) in foods from different plant families. For
this table, only unprocessed foods from families with at least five samples were compared. Percentage of the phytoestrogen content in Solanaceae is too small to provide reliable
information of phytoestrogen composition.
Family nPhytoestrogens Isoflavones Lignans Percentage of isoflavones (%)
Alliaceae 5 62 (31–83) 2 (0–26) 53 (11–81) 2 (2–47%)
Apiaceae 6 69 (7–143) 3 (0–18) 66 (7–125) 6 (2–16%)
Asteraceae 5 7 (5–13) <1 (0–1) 7 (4–13) 2 (1–8%)
Brassicaceae 17 14 (5–51) <1 (0–2) 12 (4–45) 7 (2–7%)
Cucurbitaceae 9 16 (9–35) <1 16 (8–34) 2 (0–4%)
Fabaceae 23 51 (3–201) 28 (2–51) 13 (0–89) 70 (33–92%)
Rosaceae 28 8 (3–37) 2 (0–2) 6 (0–35) 14 (5–46%)
Rutaceae 7 24 (4–36) 2 (0–12) 12 (2–21) 14 (8–34%)
Solanaceae 13 9 (3–11) 2 (0–5) 4 (1–8)
Table 3
Comparison of 61 raw and cooked foods. Phytoestrogen content is given in
l
g/100 g
wet weight (median and inter-quartile range).
Raw Cooked
Total phytoestrogens 29 (9–72)
*
18 (7–52)
Isoflavones 2 (1–11)
*
2 (1–6)
Lignans 14 (5–66)
*
12 (3–39)
%Isoflavones 11% (2–44%) 17% (3–37%)
*
Indicates a significant difference between raw and cooked food (Wilcoxon signed
rank test, p< 0.05).
552 G.G.C. Kuhnle et al. / Food Chemistry 116 (2009) 542–554
Thompson, Boucher, Liu, Cotterchio, and Kreiger (2006) do not in-
clude biochanin A. Furthermore, the phytoestrogen content in
foods depends on a large number of genetic and environmental
factors such as variety, harvest and processing (Eldridge & Kwolek,
1983; Wang & Murphy, 1994), making a comparison difficult. For
soya-based food, fourfold differences between growth location
and varieties have been observed. Previously, we compared the ef-
fect of different sources or countries of origin in nine different
foods and found an average variability of threefold with a coeffi-
cient of variation of more than 30%; however, for some foods the
observed variability was much higher (Kuhnle, Dell’Aquila, Runs-
wick & Bingham, 2009). A comparison of our data with Horn-Ross
et al. (2000), Milder, Arts, Van De Putte, Venema, and Hollman
(2005) and Thompson et al. (2006) using Wilcoxon’s signed rank
test showed no overall significant difference between the phytoes-
trogen and isoflavone content and phytoestrogen composition (as
proportion of isoflavones on total phytoestrogens) found in this
study and the average content found elsewhere. However, lignan
contents were significantly different (p< 0.01) with most values
found in this study being higher, suggesting a better extraction
of these compounds from the sample matrix.
Only limited information is available about the effect of cooking
on the phytoestrogen content of foods. Milder et al. (2005) and
Thompson et al. (2006) investigated the effect for some types of
food and found a decrease in phytoestrogen content. The protocol
for this study was not designed to assess the effect of cooking on
phytoestrogen levels and, although compared with previous stud-
ies, this study includes a larger variety of foods, it was not possible
to control for the effect of family on the probability shown in Table
3that there are losses during cooking. An explanation for this loss
during cooking is the leaching of phytoestrogens into the water
which is later discarded. Although prolonged heating could also re-
sult in the decomposition of phytoestrogen, this effect was not
seen in stewed fruits and it is therefore likely that these com-
pounds are stable during preparation, at least under acidic
conditions.
In summary, this study provides so far the most comprehensive
database of isoflavones, lignans and coumestrol in more than 240
foods based on fruits and vegetables commonly consumed in the
UK. The selection of food was based on consumption data of the
EPIC-Norfolk cohort (Day et al., 1999) and will allow the more
accurate determination of phytoestrogen intake and exposure in
this and other studies and free-living individuals.
Acknowledgements
This work was funded by the UK Food Standards Agency (FSA),
Contract No. T05028 and the Medical Research Council (MRC).
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Lignans encompass a large and complex group of phytochemicals widely distributed throughout terrestrial plant lineages. Lignans play important roles in both plant ecology (interactions with and adaptation to an ever-changing environment) and physiology/ development. As their specialized metabolite nature might suppose, lignans have been related to plant defense against a number of herbivores and microorganisms. For example, their constitutive deposition helps impart durability, longevity, and resistance to the heartwoods of many tree species against wood-rotting fungi, therefore acting as phytoanticipins. Lignans can also act as phytoalexins, being synthesized de novo by plants accumulating quickly at areas of pathogen or herbivore attack. However, the precise roles in planta and the ecological significances of most lignans are still not well established. As for many biologically active compounds originating from plants, lignan exploration has not been restricted solely to the plant research field but has also triggered intensive studies in the fields of human diet and/or health research over the last decades. Some lignans, belonging to the phytoestrogen class are converted, upon ingestion, by human gastrointestinal microbiota into the mammalian lignans enterodiol and enterolactone. The latter display the well-described chemopreventive properties against various tumors (such as breast, colon and prostate cancers) or cardiovascular disorders, whereas some other studies also report their roles in preventing diabetes. Other lignans are already used in pharmacy and medicine such as podophyllotoxin, the natural starting compound for the synthesis of lead anticancer drugs (Etoposide, Teniposide, Etopophos). However, many questions remain concerning i) their bioconversion, pharmacokinetic, and molecular targets, etc. and ii) in simply searching for their perennial and viable sources for human health applications. From a biosynthetic point of view, many lignans are formed by the oxidative coupling of E-coniferyl alcohol moieties. But gene identification, gene regulation or biosynthetic enzyme characterization study are still scarce. For example, lignans can share the same precursor as for lignins, the complex polymers that provide rigidity and support to the vascular plants. The regulation of the genes specifically related to lignan biosynthesis or the precursor partition between lignin and lignans have only rarely been investigated. Such data though could yield not only information about the role of these compounds but also for optimizing their bioproduction using metabolic engineering strategies. Their chemical nature, structural features, physicochemical behavior, and concentrations greatly differ from various plant organs or cultures, food or biological matrices making their extraction, analysis, and purification very challenging. The development of efficient analytical methods dedicated to lignans helps to provide new insight in the natural lignan chemodiversity, evolution throughout the plant kingdom as well as metabolization/detoxification following their absorption/injection in the human body. Moreover, although most in vivo and in vitro data are globally in favor of a chemopreventive effect of lignans, epidemiological studies are sometimes much less conclusive and the mechanism still remains unclear and requires further elucidation. Therefore, the availability of purified lignans at a reasonable cost would allow easier in vivo supplementation experiments and elucidation of mechanisms. All the known in planta biosynthesis roles as well as health benefits of lignans provide new frontiers for scientists from diverse fields of expertise to further study, elucidate or establish biosynthetic pathways, metabolic engineering, analytical methods and health action mechanisms of this important class of phytochemicals. This Research Topic is devoted to the latest new insights, in the form of Reviews and Original Research articles, as well as Communication and Perspective papers covering several aspects of plant lignans including: 1) Biosynthesis: gene expression regulation, gene identification, enzyme characterization, chemical ecology, and pathway evolution, etc. Note that descriptive studies involving omics approaches with no functional insights into plant biology are not considered for review. 2) Phytochemistry/analytical methods: occurrence and diversity in plant lineage, extraction, separation and purification analytical methods, structural elucidation, etc. Note that only studies focusing on the plant standpoint will be considered. 3) Metabolic engineering: plants, in vitro cultures, elicitation, biotransformation, biocatalysts, etc. 4) Biological activity of lignans related to human health: in vitro and in vivo laboratory experiments, epidemiological studies, potential mechanisms and effects on human diseases, toxicology, potential side effects, etc. Note that studies carried out with crude extracts will not be considered for review. Only the use of highly purified, chemically characterized compounds is acceptable. Keywords: Lignans, Metabolic engineering, Analytical methods, Biological activity, Lignans metabolism
... Although some selected types of foodstuff have been analyzed previously, only a few phytoestrogens have been included in the analysis (mostly daidzein, quercetin, genistein and some lignans). Comparable levels were observed with previous studies (Supplementary Table S5) [22,28,46,47] for tomato products (<0.01-14 µg/100 g) and lettuce (not detected-1.63 µg/100 g), except for naringenin in tomato (27.1 µg/100 g [46]). These comparable levels highlight the suitability for the method to quantify phytoestrogens in food. ...
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