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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.33 n.2 Santiago  2000

http://dx.doi.org/10.4067/S0716-97602000000200007 

Antioxidant flavonols from fruits, vegetables and
beverages: measurements and bioavailability

ALAN CROZIER,1* JENNIFER BURNS,1,3 AZLINA A AZIZ,1,2 AMANDA J STEWART,1
HELENA S RABIASZ,1 GARETH I JENKINS,1 CHRISTINE A EDWARDS2 AND MICHAEL
EJ LEAN3

1 Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Bower Building, University of Glasgow, Glasgow, G12 8QQ, UK
2 University of Glasgow Department of Human Nutrition, Yorkhill Hospitals, Glasgow G3 8SJ, UK
3 University of Glasgow Department of Human Nutrition, Queen Elizabeth Building, Royal Infirmary, Glasgow G31 2ER, UK

ABSTRACT

Flavonols are polyphenolic secondary plant metabolites that are present in varying levels in commonly consumed fruits, vegetables and beverages. Flavonols have long held an interest for nutritionists, which has increased following a Dutch study in the early 1990’s showing that dietary intake of flavonols was inversely correlated with the incidence of coronary heart disease. The main factors that have hindered workers in the field of flavonol research are (i) the accurate measurement of these compounds in foods and biological samples, and (ii) a dearth of information on their absorption and metabolism. This review aims to highlight the work of the authors in attempting to clarify the situation. The sensitive and selective HPLC procedure to identify and quantify common flavonols and their sugar conjugates is described. In addition, the results of an on-going screening program into the flavonol content of common produce and beverages are presented. The bioavailability of dietary flavonols is discussed with reference to an intervention study with onions, as well as pilot studies with tea, red wine and cherry tomatoes. It is concluded that flavonols are absorbable and accumulate in plasma and that consuming high flavonol-containing varieties of fruits and vegetables and particular types of beverages could increase their circulatory levels.

Key terms: bioavailability, dietary antioxidants, flavones, flavonols, HPLC analysis

INTRODUCTION

Flavonoids are polyphenolic secondary metabolites widely dispersed throughout the plant kingdom and found in substantial levels in commonly consumed fruits, vegetables and beverages. The flavonoid family is divided into a number of sub-groupings; the six main classes are flavonols, flavones, flavan-3-ols, isoflavones, flavanones and anthocyanidins (Fig. 1). Nutritionists became interested in flavonoids in the 1930’s when it was shown that flavonoids from citrus fruits decreased capillary permeability and had vitamin C sparing properties. Rusznyák and Szent-Györgi (1936) suggested that flavonoids be known as vitamin P or vitamin C2. However, by the 1950’s the vitamin claim had been abandoned due to a lack of substantive evidence.


FIGURE 1. Structures of the six main classes of flavonoids.

In the 1990’s flavonoids were once again becoming fashionable, principally due to a combination of increased concern surrounding high levels of coronary heart disease and a burgeoning interest in its prevention by dietary components. Hertog et al. (1993a) reported on a study of 805 elderly men in Zutphen, the Netherlands which showed that flavonol/flavone intake was inversely associated with both mortality from CHD (p = 0.015) and with the incidence of myocardial infarction (p = 0.08). The protection offered by these flavonoids is believed to be due to their antioxidant activity. The aromatic rings of the flavonoid molecule allow the donation and acceptance of electrons from free radical species (Kanner et al., 1994). In addition to quenching free radicals, flavonoids are able to regenerate the traditional antioxidant vitamins, vitamin C and vitamin E (Vinson et al., 1995).

Flavonols and flavones are synthesized in plant tissues from a branch of the phenylpropanoid pathway (Fig. 2). The major flavonol aglycones found in produce are quercetin, myricetin, kaempferol and isorhamnetin, while a more limited number of fruits and vegetables contain the structurally-related flavones, apigenin and luteolin (Fig. 3). In plant tissues, flavonols and flavones are found conjugated to sugars, primarily glucose, rhamnose and rutinose (Herrmann, 1988). Most conjugation occurs at the 3 position of the B ring, although it can also occur frequently at the 7 and 4' positions.


FIGURE 2. The phenylropanoid pathway by which plants synthesize a wide range of secondary metabolites. Chalcone synthase (CHS) is the first step in the branch of the pathway that produces the flavonoids including isoflavones, flavones, flavonols and anthocyanins. PAL, phenylalanine amminia lyase; CA4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; IFS, isoflavone synthase; FN3H, flavanone 3-hydroxylase; FS, flavone synthase; F3¢H, flavone 3¢-hydroxylase; FLS, flavonol synthase; FL3¢H, flavonol 3¢-hydroxylase; FL3¢M, flavonol 3¢-methylase; FL5¢H, flavonol 5¢-hydroxylase.

Quantitative analysis of flavonols and flavones

There are a large number of flavonol and flavone conjugates and, as reference compounds are rarely available, this makes it difficult to determine their concentrations in plant produce. Hertog et al. (1992b) overcame this problem by subjecting samples to acid hydrolysis, which cleaved the sugar moieties releasing the flavonols and flavones as aglycones which could be readily analyzed by reversed-phase high performance liquid chromatography (HPLC). This procedure was used to determine the flavonol/flavone content of a range of common fruits, vegetables and beverages purchased in a Dutch supermarket on four occasions over a 12-month period (Hertog et al., 1992a, 1993b). From detailed dietary records obtained over a 5-year period, these values were then used to estimate the intake of flavonols and flavones by the subjects in the Zutphen study (Hertog et al., 1993a). This quasi-quantitative method conveniently avoided the problems associated with the analysis of the wide variety of flavonol and flavone conjugates that exist in nature. The protocol developed by Hertog et al. (1992b) was adapted and optimized by Crozier et al. (1997a) for the analysis of flavonols and flavones in fruits, vegetables and beverages (Crozier et al., 1997b).

FIGURE 3. Structures of the main dietary flavonols and flavones.

In HPLC method development the most important variable, after solvent choice, is the column. The ability of a number of different columns to separate nine flavones and flavonols was investigated (Crozier et al., 1997a). Although each column was packed with a C18 reversed phased support, their chemistries were different. Large variations in the performances of the columns were observed using theoretical plates and peak tailing as the performance parameters. However, good separation of the flavonols was obtained using a C18 Genesis column (Jones Chromatography, Mid Glamorgan, UK) eluted with a 20–40% gradient of acetonitrile in water adjusted to pH 2.5 with trifluroacetic acid. High efficiency separations were also obtained with C18 Symmetry and C18 Novapak supports (Waters, Milford, MA, USA) (Fig. 4). Initially, the only detector was an absorbance monitor operating at 365 nm but subsequently a second detection system was also employed. Some flavonols, by virtue of their chemical structure can be chelated with aluminum nitrate to form fluorescent complexes. By introducing a post-column derivatization step, involving the addition of methanolic aluminum nitrate after the column eluant had passed through the absorbance monitor, it became possible to use a fluorimeter to detect quercetin, myricetin, morin, kaempferol and isorhamnetin (Figs. 5A and B). This second detector system not only increased selectivity but also lowered the limits of detection ca. 100-fold to <50 pg (Hollman et al., 1996; Aziz et al., 1998).

FIGURE 4. Gradient reversed phase HPLC analysis of free and conjugated flavonoids. Trace A: 250 x 4.6 mm i.d. 5 µm RP-C18 Lichrosphere column eluted with a 20 min gradient of 15-35% acetonitrile in water adjusted to pH 2.5 with trifluoroacetic acid. Trace B: 100 x 8 mm i.d. 4 µm C18 Nova-Pak column eluted with a 20 min gradient of 20-40% acetonitrile in water adjusted to pH 2.5 with trifluoroacetic acid. Trace C: 150 x 3.9 mm i.d. 5 µm C18 Symmetry column eluted with a 20 min gradient of 15-35% acetonitrile in water adjusted to pH 2.5 with trifluoroacetic acid. Trace D: 150 x 4.6 mm i.d. 4 µm C18 Genesis column eluted with a 20 min gradient of 20-40% acetonitrile in water adjusted to pH 2.5 with trifluoroacetic acid. Flow rate: 1 mL min-1. Detector: absorbency monitor operating at 365 nm. Sample: (1) rutin, (2) quercetin-3-glucoside, (3) quercitrin, (4) myricetin, (5) luteolin, (6) quercetin, (7) apigenin, (8) kaempferol and (9) isorhamnetin.

Flavonols and flavones in fruits and vegetables

Using these methods a selection of fruits and vegetables were analyzed for their flavonol and flavone content (Crozier et al., 1997b). Different varieties of tomatoes, lettuce, onions and celery were purchased at various dates throughout the year. Traditional size Scottish tomatoes, large Dutch beef tomatoes, as well as Spanish and English cherry tomatoes were examined. Cherry tomatoes were found to have a markedly higher flavonol content than either the traditional or beef varieties, with the Spanish-grown cherry tomatoes having a slightly higher flavonol content than their English counterparts (Table I).

Table I.

Quercetin content of selected produce

Produce

Quercetin Content *

Tomatoes

 

Scottish tomatoes var. Spectra

7.3 ± 0.9

Dutch Beef tomatoes var. Trust

2.2 ± 0.3

Spanish cherry tomatoes var. Paloma

55 ± 2.9

English cherry tomatoes var. Favorita

41 ± 1.8

Lettuce

 

Iceberg, whole

2.2 ± 0.1

Round, whole

11 ± 0.5

Lollo Rosso outer leaves

911 ± 22

inner leaves

450 ± 17

Onions

 

Red

1337 ± 31

Yellow

1359 ± 65

White

10 ± 2.3

Peas

 

Mangetout

145 ± 6.5

Sugar snap peas

98 ± 2.3

* Data expressed as m g total quercetin g-1 fresh weight ± SEM, n=3.

Sizeable variations in flavonol content were also observed with lettuce (Table I). The commonly consumed small "round" lettuce contained only 11 µg g-1 fresh weight of quercetin, and the levels in "iceberg" lettuce were even lower. In contrast, the outer leaves of "Lollo Rosso," a red variety of lettuce, contained 911 µg g-1. The red color of this lettuce is due to high levels of anthocyanins, which like quercetin, are products of the phenylpropanoid pathway (Fig. 2). As one end-product of the pathway has been elevated, it may well be that other related compounds, including the flavonols, are also found in higher concentrations.

Onions were also found to be a rich source of flavonols, particularly quercetin conjugates, although once again variety was an important consideration. While yellow and red onions contained approximately 1350 µg g-1 fresh weight of total flavonols, white onions contained only 10 µg g-1 (Table I). Other high-flavonol vegetables include sugar snap peas and mange-tout, which were found to contain 98 and 145 µg quercetin g-1 respectively (Table II). Celery contains flavones rather than flavonols and widely varying levels of luteolin and apigenin were detected in different samples (Table II).

Table II
Flavone content of celery

Produce

Luteolin*

Apigenin*

White celery stalks

   

var. Greensleeves

40

90

var. Ista

36

104

Green celery hearts

   

var. Victoria

35

191

White celery stalks

   

var. Celebrity

6.6

17

* Data expressed as m g g-1 fresh weight

Flavonols in tea and red wines

Apart from fruits and vegetables, some beverages, particularly tea and red wine, can also be important sources of flavonols (Hertog et al., 1993b). Green and black tea contain a large range of flavonoids. While green tea contains predominately flavan-3-ols, fermented black tea has mainly condensation and polymeric forms of these compounds (Kuhr and Engelhardt, 1991). Both green and black tea infusions contain conjugated quercetin, kaempferol and myricetin, with the total flavonol content ranging from 21.0 to 32.8 mg L-1 (Table III). Tea is an important contributor to the total dietary intake of flavonols in Japan and the UK.

Table III
Total Flavonol Content of selected beverages

Beverage

Flavonol Content *

Loose black tea

 

Safeway

21.0 ± 0.3

Typhoo

39.1 ± 0.9

Loose green tea

 

Japanese

32.8 ± 0.4

Red Wines

 

Cabernet Sauvigon, Chile

58.4 ± 4.0

Pinot Noir, California

30.2 ± 0.6

Merlot, Chile

25.2 ± 1.2

Beaujolais, France

9.9 ± 0.9

White Wines

 

Reisling, Australia

1.7 ± 0.2

Bordeaux Blanc, France

n.d.

Dry White, South Africa

n.d.

* Data expressed as mg L-1 total flavonols ± SEM, n=3. Teas contain predominately conjugated myricetin, quercetin and kaempferol, while red wines contain varying ratios of free and conjugated flavonols particularly myricetin, quercetin, kaempferol and isorhamnetin. n.d., not detected.

The phenolic components in red wines have been implicated as the active agents responsible for the French Paradox, the low incidence of coronary heart disease in southern France despite the high saturated fat intake (Renaud and de Logeril, 1992). Red wines, in contrast to white wines, contain significant amounts of free and conjugated grape skin-derived flavonols, principally quercetin and myricetin (Table III). There is marked variation in the levels of these compounds in different wines, and it is believed that this is a consequence of the variety of grape used, the climate, cultural conditions and the vinification technique employed (McDonald et al., 1998).

Bioavailability of flavonols

Onions. Onions have been used as the flavonol source in a number of bioavailability studies with human volunteers (Hollman et al., 1995; Aziz et al., 1998). Common yellow onions have a consistently high level of flavonol with the outer layers containing 56% of the total flavonol content compared with 30% and 14% in the middle and inner segments. Thus, higher flavonol intakes can be achieved by eating only the outer layers rather than the whole onion. Onions can be made palatable by light frying which does not reduce the flavonol levels to any extent (Crozier et al., 1997b). A further advantage in using onions is that their endogenous flavonol conjugates have been identified (Tsushida and Suzuki, 1995). The main components are quercetin-3,4'-diglucoside and quercetin-4'-glucoside with smaller amounts of isorhamnetin-4'-glucoside (Figs. 5 and 6) and traces of other quercetin conjugates. Quercetin-4'-glucoside and isorhamnetin-4'-glucoside can be chelated with aluminum nitrate and are analyzed in unhydrolyzed samples by HPLC using fluorescence detection as well as an absorbency monitor (Figs. 5C and D). The enhanced selectivity and sensitivity of this analytical procedure facilitates the detection of these flavonol conjugates in plasma (Fig. 5E and F) and urine after the consumption of onions.

FIGURE 5. Gradient reverse phase HPLC analysis of flavonols. Column: 150 x 3.0 mm i.d 4-µm Genesis C18 cartridge column with a 10 x 4.0 mm 4-µm Genesis C18 guard cartridge. Mobile phase: 25 min gradient of 15-40% acetonitrile in water containing 0.1 % trifluoroacetic acid. Flow rate: 0.5 mLl min-1. Detector: absorbency monitor operating at 365 nm and, after on-line post-column reaction with methanolic aluminum nitrate, a fluorimeter operating at excitation 420 nm and emission 485 nm. Samples: (A) 150 ng of (1) quercetin-3,4'-diglucoside, (2) quercetin-3-glucoside, (3) quercetin-4'-glucoside, (4) isorhamnetin-4'-glucoside, (5) morin, (6) quercetin, (7) kaempferol and (8) isorhamnetin with detection at A365 nm; (B) as in A, but with post-column derivatization and fluorescence detection (excitation 420 nm emission 485 nm); (C) aliquot of an unhydrolyzed extract of lightly fried onions, with detection at A 365 nm; (D) as C but with post-column derivatization and fluorescence detection; (E) unhydrolyzed 12 µL aliquot of plasma collected immediately prior to the consumption of 300 g of lightly fried onions, with post-column derivatization and fluorescence detection; (F) as E but plasma collected 1.5 h after eating fried onions. Numbers indicate peaks that co-chromatograph with standards listed for sample A.

FIGURE 6. Structures of the main flavonol conjugates in onions.

(Aziz et al., 1998) have reported on a detailed investigation of the absorption and excretion of onion-derived flavonols. Five subjects, who had been on a low flavonol diet for three days, consumed 300 g of lightly fried onion. Blood was collected at 0 min, 0.5, 1.0, 1.5, 2, 3, 4, 5 and 24 h after supplementation. The subjects also collected their urine for 24 h. The time course profiles of the appearance of isorhamnetin-4'-glucoside and quercetin-4'-glucoside in plasma are presented in Figure 7. As the amounts of flavonols in the onions consumed varied from person to person, the flavonol levels in the plasma are expressed as a percentage of the amount ingested. The plasma flavonol profiles show some variation among the five subjects (Fig. 7). Volunteers 2 and 3 exhibited similar profiles with a rapid increase in flavonol levels after onion consumption followed by a swift decline in their concentration. On the other hand, the flavonols in subjects 1, 4 and 5 appeared to have a second peak concentration later in the time course. In addition they had a slower decline in flavonol content than subjects 2 and 3 (Fig. 7) with maximum levels, defined as a proportion of intake, of 10.7 ± 2.6% and 0.13 ± 0.03% respectively. The mean values for the key features of flavonol accumulation in plasma are presented in Table IV. Quercetin-4'-glucoside peak plasma concentration was 1.3 ± 0.2 h after ingestion of the onions while a value of 1.8 ± 0.7 h was obtained for isorhamnetin-4'-glucoside. Isorhamnetin-4'-glucoside and quercetin-4'-glucoside were also detected in urine at levels approximating 17.4 ± 8.3% and 0.2 ± 0.1% of intake (Table V).

Table IV

Mean values ± standard error of key features of flavonol conjugate accumulation in plasma following the consumption of 300 g of lightly fried onions by five subjects.

Flavonol

Intake

Peak plasma
concentration

Time of peak plasma concentration

Peak plasma concentration as a proportion of intake*

Quercetin-4'-glucoside

102 ± 22 mg
(197 ± 43 m moles)

45 ± 11 ng mL-1
(0.09 ± 0.02 m M)

1.3 ± 0.2 h

0.13 ± 0.03%

Isorhamnetin-4'-glucoside

10.5 ± 1.5 mg
(21 ± 3 m moles)

370 ± 91 ng mL-1
(0.75 ± 0.18 m M)

1.8 ± 0.7 h

10.7 ± 2.6%

* Calculated on the basis of 3000 mL plasma/person

Table V

Mean values ± standard error for the excretion of flavonol conjugates in urine following the consumption of 300g of lightly fried onions by five subjects.

Flavonol

Intake

Excretion period

Total excreted as a proportion of intake

   

0-6 h

6-12 h

12-24 h

Quercetin-4'-glucoside

102 ± 22 mg
(197 ± 43 m moles)

100 ± 27 m g
(0.1 ± 0.05 m moles)

65 ± 43 m g
(0.1 ± 0.08 m moles)

4.8 ± 2.6 m g
(0.01 ± 0.005 m moles)

0.2 ± 0.1%

Isorhamnetin-4'-glucoside

10.5 ± 1.5 mg
(21 ± 3 m moles)

1175 ± 482 m g
2.4 ± 1.0 m moles)

620 ± 374 m g
(1.2 ± 0.7 m moles)

23 ± 15 m g
(0.05 ± 0.03 m moles)

17.4 ± 8.3%

FIGURE 7. Concentration of quercetin-4'-glucoside and isorhamnetin-4'-glucoside in plasma collected from five human volunteers after the ingestion of 300 g of lightly fried onions. Data expressed as percentage of the intake based on flavonol content of onions ± S.E. (n = 5) and calculated on the basis of 3000 mL of plasma per person.

While quercetin-4'-glucoside is found in onions in a ten-fold excess to isorhamnetin-4'-glucoside, the opposite is found in plasma and urine (see Tables IV and V). Preferential absorption and accumulation of the isorhamnetin conjugate could explain this. However, as quercetin is metabolized to isorhamnetin in rats (Manach et al., 1996), an alternative possibility is that quercetin-4'-glucoside is absorbed and converted to isorhamnetin-4'-glucoside by a 3'-O-methylation reaction. It is of interest that the main flavonol in onions, quercetin-3,4'-diglucoside, was not detected in body fluids. However, this does not necessarily mean that the quercetin-3,4'-diglucoside is not absorbed. The diglucoside does not chelate with aluminum nitrate and form a fluorescent complex (see Fig. 5C and D) and, as a consequence, it was not analyzed by HPLC with the same sensitivity and selectivity as quercetin-4'-glucoside and isorhamnetin-4'-glucoside.

The study by Aziz et al. (1998) showed for the first time that the onion flavonol glucosides, quercetin-4'-glucoside and isorhamnetin-4'-glucoside accumulate in the bloodstream and are excreted in urine seemingly without undergoing structural modification. However, it is important to appreciate that the homeostasis of flavonol pools is almost certainly in a state of flux due to the combined effects of transport through the gut wall into the bloodstream and removal by sequestration, metabolism and excretion. Further comprehensive studies are required using isotopically labeled substrates to monitor the underlying physiological and metabolic events associated with these processes.

Tea, red wine and cherry tomatoes. Pilot studies on the bioavailability of tea, red wine and cherry tomato flavonols have shown comparable absorption profiles. Using the same protocol as used with onions, 400 mL black tea, 300 mL red wine and 250 g Spanish cherry tomatoes were consumed and the accumulation of quercetin conjugates in plasma was followed (Fig. 8). In this study the nature of the quercetin conjugates was unknown, and the quercetin in plasma was determined both before and after acid hydrolysis. Peak absorption levels of conjugated quercetin occurred between 60 and 90 min in each instance, with a maximum absorption in the range of 1% of flavonol consumption (Burns and Stewart, unpublished data). Further work using radio-labeled compounds is required before any firm conclusions can be drawn as to the specific mechanisms of flavonol absorption, metabolism and accumulation.

FIGURE 8. Concentration of conjugated quercetin in plasma collected from individual human volunteers after the ingestion of red wine, black tea and cherry tomatoes. Data expressed as percentage of the flavonol intake calculated on the basis of 3000 mL of plasma per person.

SUMMARY

The results presented in this review are of potential importance in view of recent epidemiological studies indicating that flavonol intake is associated with a reduced risk of cancer and coronary heart disease and stroke. It is estimated that at least 20% of coronary heart disease is attributable to diet, and dietary factors are considered responsible for 40-60% of cancer incidence and 35% of cancer deaths (National Research Council, 1989). Fruits and vegetables have consistently been found to be protective against coronary heart disease (Hertog et al., 1993a; 1995) and against a variety of cancers (NRC, 1989; WHO, 1990). The quantitative roles of antioxidants are not known precisely in relation to their health benefits, nor are the specific contributions of carotenoids, tocopherols and ascorbic acid. However, evidence obtained with an in vitro oxidation model for heart disease has demonstrated that several plant flavonols, such as quercetin, myricetin and rutin, are more powerful antioxidants than the traditional vitamins (Vinson et al., 1995). The health influences of flavonols have yet to be fully established. However, they have been shown to function in a way similar to antioxidant vitamins, to protect against lipoprotein oxidation in vitro (Negre-Salvayre and Salvayre, 1992) and to have anti-platelet and anti-thrombotic actions (Gryglewski et al., 1987; Cook and Samman, 1996). There are, therefore, grounds for encouraging the consumption of foods rich in flavonols. It is evident with tomatoes, lettuce and onions, and in all probability with other produce, that there are very large varietal differences in flavonol content (Table II). Identification and incorporation of flavonol-rich foods into the diet is clearly one means by which the intake of flavonols derived from fruits and vegetables could be increased markedly.

The relative contribution of vegetables to total flavonol intake will depend to a large degree on the consumption of other rich dietary sources of flavonols, like tea and wines (see Hertog et al., 1993b). Tea is considered one of the main dietary sources of flavonols for adults in the UK, but its limited use by younger people is declining in favor of carbonated drinks and coffee, which are relatively low in flavonols. Although the levels vary among different types of red wines (Table IV) (McDonald et al., 1998), red wines are a major source of flavonols in countries such as Italy and France where there is relatively little consumption of tea (Hertog et al., 1995). In contrast, red wine is consumed by only a minority of the UK population. It is likely that the processes leading to coronary heart disease and cancers are initiated many years before the diseases manifest themselves. If flavonol intake is important in children, who do not consume tea or wine in any quantity, then the varieties of fruits and vegetables they consume could be critical for future health.

Recent years have seen an exponential increase in research on the absorption and metabolism of flavonoids, particularly flavonols. Evidence suggests that flavonol conjugates are absorbed to a greater extent than the parent aglycone. However radio-labeled pure compounds are required before any firm conclusions can be drawn. It may be that the active antioxidant compounds are not the dietary flavonols per se, but their metabolites.

If it is accepted that higher intakes of flavonols from foods are associated with long-term health benefits, then the data presented in this paper offer possible avenues for horticultural approaches toward health promotion by identifying and selecting varieties rich in flavonols by optimizing growth and storage. It should be noted that as accurate measurement of flavonols in foods is relatively inexpensive and not particularly time consuming, it thereby offers a novel method for product quality assurances.

ACKNOWLEDGEMENTS

J.B. was funded by a BBSRC CASE award from Safeway Stores plc. A.A.A. was supported by a postgraduate studentship from the University of Malaya, Malaysia, and A.J.S. was funded by a BBSRC CASE award from Scotland’s Tomatoes plc. H.S.R. was funded by Safeway Stores plc.

Received: September 9, 1999. Accepted: January 28, 2000

Corresponding Author: Alan Croizer. Telephone: 44-141-330-4613 . Fax: 44-141-330-5394. e-mail: A.Crozier@bio.gla.ac.uk

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