CHAPTER 3

ANALYSIS OF FRUITS AND VEGTABLES FOR FLAVONOIDS

97
CHAPTER 3: ANALYSIS OF FRUITS AND VEGTABLES FOR

FLAVONOIDS

3.1 Introduction

Flavonoids are diphenyl compounds with a general structure consisting of two

benzene groups connected by a three-carbon bridge (Kelly et al., 2002) and, to date,

over 4000 naturally occurring flavonoids have been found in nature. Recent interest

in these bioactive components of plants has centred on their potential health benefits

such as antioxidative, antimicrobial, and possibly anticarcinogenic and/or

cardioprotective effects (Hertog et al., 1992a,b; Hertog et al., 1993;

Knekt et al., 1996).

Kuhnau (1976) found that average intake of flavonoids from foods in the United

States (US) to be about 1 g per day (including the consumption of flavonoid dimers

and mono-flavonoids). Flavonoid intakes have been found to range from 4 mg per

day in Finland to 68 mg per day in Japan, where green tea is the major source. In the

UK intakes average 30 mg per day, 82% of which comes from plant beverages,

mostly tea. In Northern Europe and the US, studies repeatedly show that tea and

onions are the most important dietary source, followed by apples (Hertog et al.,

1993; Hertog et al., 1997b; Hollman and Katan, 1999; Knekt et al., 1996).

Advanced analytical techniques and the development of databases that summarise

the flavonoid content of a vast range of fruits and vegetables have resulted in more

accurate estimates of flavonoid consumption being made among different

98
populations. However, obtaining as much data as possible from as wide a range of

fruits and vegetables grown under different conditions in different parts of the world

will help ensure that even more accurate measures of flavonoid intake can be

determined.

Consequently, the aim of this study was to identify the flavonoid content of fresh

fruits and vegetables commonly consumed as part of the Northern Ireland diet.

Analysis was carried out using the HPLC technique developed initially by Hertog et

al. (1992a) and McAnlis (1998) with a number of modifications being made in order

to improve the accuracy of the results.

3.2 Experimental procedures

3.2.1 Preliminary experimental work to improve methodology used for

flavonoid analysis

3.2.1.1 Removal of interfering peaks

Preliminary experimental work using the HPLC method for flavonoid analysis as

employed by McAnlis (1998) showed that following analysis of reagent blanks

containing no flavonoids, two peaks were found to elute at approximately the same

retention times as those of the flavonoids quercetin and kaempferol (Fig. 3.1). Thus,

it was decided to investigate this further in order to ensure that levels of these

flavonoids, if present in fruits or vegetables, could be measured accurately without

interference from other peaks.

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Reagent blanks were subsequently subjected to sonication and filtration only without

hydrolysis. Resultant HPLC chromatograms showed both peaks were absent

in this case. However, when the blanks were subjected to hydrolysis, as would

be the case for samples, both peaks appeared in the resulting chromatograms.

When the antioxidant tert-butylhydroquinene (TBHQ) was subsequently

omitted from the 62.5% aqueous methanol used in the methodology the peaks

were absent following hydrolysis, sonication and filtration. Therefore, it was

concluded that TBHQ was responsible for the presence of the two additional

peaks and consequently it was decided to employ 20 mM sodium

diethylthiocarbamic (DETC) as the antioxidant. Using DETC, the resultant

chromatograms from the reagent blanks following hydrolysis, sonication and

filtration did not contain the additional peaks (Fig. 3.2). The recoveries of pure

flavonoid standards from reagent solutions spiked at a level of 2.5 g/ml were

also checked and found to be unaffected by changing the antioxidant from

TBHQ to DETC. Consequently, it was decided to employ DETC as opposed to

TBHQ in all further experimental work.

100
Figure 3.1 HPLC chromatogram of reagent solution containing 2g/l TBHQ
following hydrolysis, sonication and filtration

Figure 3.2 HPLC chromatogram of reagent solution containing 20 mM DETC

following hydrolysis, sonication and filtration

101
3.2.1.2 Stability of flavonoid standards

Experiments were carried out to determine the stability of the flavonoid stock

standards and of prepared external standards (ESTDs) (Chapter 2; Materials and

Methods) stored at 4C.

Firstly, the stability of the stock standards was checked by preparing 50 ml of a

5 g/ml external standard (ESTD), containing each of the five flavonoids, from the

500 g/ml stock standards of apigenin, kaempferol, luteolin, myricetin and quercetin.

Duplicate 20 l samples were injected onto the HPLC and run under the operating

conditions outlined in Chapter 2 (Material and Methods). Fresh ESTDs were

prepared from the original stock standards after storage of the latter for 0, 1, 2 and 3

months at 4C. The peak height of each standard was measured from the HPLC

chromatograms obtained at each storage period.

Secondly, the stability of ESTDs containing each of the five flavonoids of interest

was determined by preparing 50 ml of a 5 g/ml ESTD from the stock standards at

day 0 and injecting duplicate 20 l samples as described previously onto the HPLC

at intervals of 0 - 4 weeks and following 2 and 3 months storage at 4C.

As can be seen from Table 3.1, all the stock standards proved to be very stable over a

three month storage period at 4C as peak heights did not vary when ESTD, were

prepared and analysed at each time interval.

The stability of the ESTD prepared initially (day 0) and analysed at intervals over a

three month (12 week) storage period was good for apigenin, kaempferol, luteolin

102
and quercetin with some deterioration in stability being observed for myricetin

(Table 3.2). Following 3 weeks of storage myricetin showed a deterioration of

approximately 9%. After 12 weeks, the level of myricetin was approximately 86% of

its original value.

It was therefore concluded that stock standards of the five flavonoids could be used

for up to three months when stored at 4C but that ESTDs should be used within

three weeks of preparation if myricetin was to be included.

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 Table 3.1 Effect of storage at 4C on the stability of stock standards
(500 g/ml) of apigenin, kaempferol, luteolin, myricetin and quercetin
made up fresh as measured by peak height (arbitrary units; AU)

Storage time Apigenin Kaempferol Luteolin Myricetin Quercetin

(months) (AU) (AU) (AU) (AU) (AU)

0 55.4 156.9 135.5 167.7 187.6

1 46.9 154.2 135.4 172.2 190.4

2 51.8 148.7 133.2 171.1 189.6

3 55.2 154.2 135.2 168.0 186.8

All values are the mean of 3 measurements

Table 3.2 Effect of storage at 4C on the stability of external standards (ESTDs)

(5 g/ml) of apigenin, kaempferol, luteolin, myricetin and quercetin

as measured by peak height (arbitrary units; AU)

Storage time Apigenin Kaempferol Luteolin Myricetin Quercetin

(weeks) (AU) (AU) (AU) (AU) (AU)

0 45.4 146.9 135.5 167.6 187.9

1 46.6 140.5 127.7 164.7 186.8

2 47.4 143.4 135.5 165.2 194.4

3 48.0 141.5 135.1 150.8 193.1

4 52.1 148.6 137.5 135.7 185.2

8 51.7 143.9 136.4 136.9 187.9

12 52.6 148.4 137.5 143.8 187.5

All values are the mean of 3 measurements

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3.2.1.3 Determination of limit of detection of flavonoids

This experiment was carried out to determine the limit of detection (LOD) and limit

of quantification (LOQ) of each of the five flavonoids using HPLC. Duplicate 0.5,

0.25, 0.1 and 0.05 g/ml samples of each flavonoid standard were injected onto the

HPLC. The LOD was determined as the lowest concentration at which a peak was

detected corresponding to the expected retention time of each flavonoid and the LOQ

determined as the minimum concentration at which the flavonoid peaks could be

successfully quantified. The LOQ is normally taken to be three times the noise level.

It was found for kaempferol, luteolin, myricetin and quercetin, the absolute LOD was

0.05 g/ml, whilst for apigenin it was 0.1 g/ml. However, the LOQ was found to be

0.1 g/ml for kaempferol, luteolin, myricetin and quercetin and 0.25 g/ml for

apigenin.

3.2.1.4 Determination of effect of storage of final sample extracts for one week

on the stability of flavonoids

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A short experiment was carried out to determine the effect of retaining samples once

hydrolysed on the stability of the five flavonoids under analysis. In order to do

this three samples of green lettuce were treated as described in Chapter 2

(Materials and Methods) and once prepared for analysis, the final extract was

stored for one week at 4C. Following storage they were analysed by HPLC as

described previously. Quercetin was the only flavonoid measured in the

lettuce. A 78% decrease in quercetin concentration was observed between

samples analysed immediately after preparation and from those stored for one

week at refrigeration temperature. The average value for nine samples in each

case was 15.5 g/g for samples analysed immediately and 3.41 g/g for those

analysed post-storage. This experiment highlighted the importance of

analysing samples immediately following preparation which was the case in all

further experimental work described in Chapters 3 and 4.

3.2.2 Analysis of a range of fruits and vegetables for apigenin, kaempferol,

luteolin, myricetin and quercetin

The fresh fruits and vegetables analysed in these studies were selected from those

listed in the Food Frequency Questionnaire (FFQ) taken from the EUREYE study

(Appendix 1). They were purchased from supermarkets in Belfast. The sources of

the fruits and vegetables used in the experimental work are given in Tables 1 to 2,

Appendix 2. The individual products purchased were from the same batch within the

supermarket.

On the day of purchase, where applicable, the edible parts of the fruits and

vegetables were washed, cut finely, weighed, mixed with liquid nitrogen and frozen

106
at -20C. The frozen products were then freeze-dried, re-weighed, ground into a fine

homogenous powder, and stored at -20C pending further analyses as described in

Chapter 2 (Materials and Methods).

Three replicate samples of each product were purchased. In the case of products

such as broccoli, cabbage, cauliflower, cucumber, leeks, onions and peppers, the

products were of a sufficient size and weight to allow for individual items to be used

as single replicates. For products such apricots, Clementine, Mandarin and Satsuma

oranges, and Kiwi fruit where there was insufficient weight from one fruit for

sampling purposes, three fruits were chopped up and a composite sample prepared

from which the required sample weight was taken for analysis. With regard to

grapes, raspberries and strawberries, a punnet of each product was used as a replicate

from which a representative sample was taken for analysis. With frozen peas, one

bag of each product was taken as a replicate from which a homogenous sample was

obtained. Following freeze-drying and homogenisation, triplicate extracts of each

powdered product were analysed thereby giving nine samples for each product.

Two ESTDs were analysed with each HPLC run of fruit and vegetable samples, one

set at the beginning of each run and one at the end. Reagent recoveries and sample

recoveries for each flavonoid were also carried out as described in Chapter 2

(Materials and Methods).

The flavonoids present in the fruit and vegetable samples were identified by

comparing peak retention times to those of flavonoid standards run at the same time

as the actual samples. Spectral analysis as described in Chapter 2 (Materials and

107
Methods) were carried out for further confirmation. All results were expressed as g

flavonoid/g fresh weight of each fruit or vegetable and statistically analysed using

analysis of variance (ANOVA).

3.3 Results

3.3.1 Recoveries

Reagent recoveries ranged from 87% to 111% for quercetin, luteolin, kaempferol and

apigenin while myricetin showed the lowest percentage recovery values ranging

from 30% to 88 % (Tables 1 to 2; Appendix 3).

The percentage spiked sample recoveries ranged from 38% to 99% for quercetin,

luteolin, kaempferol and apigenin while as for the reagent recoveries, myricetin

showed the lower recovery percentage ranging between 0% to 90% (Table 3 to 4;

Appendix 3).

3.3.2 Results of flavonoid analysis of fruit samples

The flavonoids levels measured in the fruits analysed are presented in Table 3.3. The

predominant flavonoid found in the fruits was quercetin, being present in the

majority of products analysed. Apigenin was the next most abundant flavonoid

followed by kaempferol and luteolin. There was no measurable amount of myricetin

detected in the fruits studied.

Plums were found to be the richest source of quercetin containing on average,

78.2 g/g, with red apples and apricots also proving to be abundant in this flavonoid

being measured at levels of 42.2 and 35.9 g/g, respectively. Green apples, red

108
grapes, satsumas and strawberries contained similar amounts of quercetin at

concentrations of 30.0, 23.7, 27.5 and 24.1 g/g, respectively. It was observed that

red apples contained 29% more quercetin than green apples. With regard to oranges,

the satsumas contained quercetin levels approximately five times (27.5 g/g) greater

than those found in Clementine (3.8 g/g) and mandarin (5.3 g/g) oranges and

twice as much as that measured in ordinary eating oranges (13.2 g/g). It was also

noted that red grapes contained approximately three times more quercetin (23.7 g/g)

than white grapes (7.8 g/g). Quercetin was not found in bananas, white or pink

grapefruit.

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Satsuma oranges proved to be the best source of apigenin which was found on

average at a concentration of 76.4 g/g while levels of 66.3 g/g and 63.2 g/g

were measured in pears and ordinary eating oranges, respectively. Pink

grapefruit was found to contain over twice as much apigenin (30.3 g/g) as

white grapefruit

(14.9 g/g). The apigenin contents of Mandarin and Clementine oranges were

within the same range being 23.8 and 18.0 g/g, respectively, and were

significantly lower (p<0.001) than the levels measured in Satsuma oranges and

ordinary eating oranges as previously mentioned.

Kaempferol was found in only four fruits, these being Satsumas, strawberries, pink

and white grapefruit at concentrations of 10.2, 8.7, 6.7 and 3.7 g/g,

respectively.

Small amounts of luteolin were found in white and red grapefruit and melon which

had concentrations of 2.6, 4.5 and 3.7 g/g, respectively.

Chromatograms of raspberries and strawberries exhibited a large peak at the same

retention time as that of myricetin (Fig. 3.3). However, upon examination of

the UV spectrum of this peak it was found to be different from that of

myricetin (Fig. 3.4). Myricetin was not detected in any of the fruits analysed.

110
 Table 3.3 Flavonoid content of selected fruits (values given in logarithmic
transformed means with g/g fresh weight in brackets).

Fruit Apigenin Kaempferol Luteolin Myricetin Quercetin

Log transformation values (g/g fresh weight)

Apple n.d. n.d. n.d. n.d. 1.48

(green) (29.97)
Apple n.d. n.d. n.d. n.d. 1.63
(red) (42.18)
Apricot n.d. n.d. n.d. n.d. 1.56
(35.94)
Banana n.d. n.d. n.d. n.d. n.d.
Clementine 1.26 n.d. n.d. n.d. 0.58
orange (17.98) (3.79)
Grapefruit 1.17 0.56 0.42 n.d. n.d.
(white) (14.90) (3.65) (2.61)
Grapefruit 1.48 0.83 0.66 n.d. n.d.
(pink) (30.34) (6.72) (4.52)
Grape n.d. n.d. n.d. n.d. 1.38
(red) (23.72)
Grape n.d. n.d. n.d. n.d. 0.90
(green) (7.84)
Kiwi n.d. n.d. n.d. n.d. 0.80
(6.36)
Mandarin 1.38 n.d. n.d. n.d. 0.73
orange (23.82) (5.33)
Melon n.d. n.d. 0.57 n.d. 0.75
(3.71) (5.60)

Significance of Effect (Sig.)/Standard Error of the Mean (SEM)

Apigenin Kaempferol Luteolin Quercetin

Sig. SEM Sig. SEM Sig. SEM Sig. SEM

Fruit (F) *** 0.043 *** 0.042 * 0.063 *** 0.030

where: Each value is the mean of 9 measurements

n.d. = below detection limit (<0.5 g/ml); * = p<0.05; *** = p<0.001

111
 Table 3.3 (Continued) Flavonoid content of selected fruits (values given in
logarithmic transformations with g/g fresh weight in
brackets)

Fruit Apigenin Kaempferol Luteolin Myricetin Quercetin

Log transformation values (g/g fresh weight)

Orange 1.80 n.d. n.d. n.d. 1.12

(63.22) (13.17)
Peach n.d. n.d. n.d. n.d. 0.93
(8.52)
Pear 1.82 n.d. n.d. n.d. 1.11
(66.31) (12.94)
Plum n.d. n.d. n.d. n.d. 1.89
(78.20)
Raspberry n.d. n.d. n.d. n.d. 0.85
(7.15)
Satsuma 1.88 1.01 n.d. n.d. 1.44
orange (76.42) (10.18) (27.50)
Strawberry n.d. 0.94 n.d. n.d. 1.38
(8.66) (24.06)

Significance of Effect (Sig.)/Standard Error of the Mean (SEM)

Apigenin Kaempferol Luteolin Quercetin

Sig. SEM Sig. SEM Sig. SEM Sig. SEM

Fruit (F) *** 0.043 *** 0.042 * 0.063 *** 0.030

where: Each value is the mean of 9 measurements

n.d. = below detection limit (<0.5 g/ml); * = p<0.05; *** = p<0.001

112
Figure 3.3 HPLC chromatogram from HPLC analysis of freeze-dried raspberries.
(Peak at retention time of 4.086 min. assumed to be myricetin)

Figure 3.4 UV spectrum of myricetin standard (top spectrum) and peak eluting at
similar retention time in raspberries (bottom spectrum)

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3.3.3 Results of flavonoid analysis of vegetable samples

The flavonoid contents obtained for the range of fresh vegetables analysed are

presented in Table 3.4. The major flavonoid found in the fresh vegetables was

quercetin. This was detected in 22 of the vegetables with kaempferol and luteolin

being the next most abundant flavonoids. Apigenin was measured in only three

vegetables while myricetin was found only in green beans.

The richest source of quercetin was detected in white onions at a concentration of

432.9 g/g which was 31% greater than the concentration of 297.4 g/g found in red

onions. Spinach was also an abundant source of quercetin being found at a level of

406.9 g/g, similar to the amount in white onions. Scallions were found to have a

significantly lower content of quercetin at 18.9 g/g while no quercetin was

measured in leeks. Kale also proved to be a good source of quercetin with an

average concentration of 142.0 g/g (Fig. 3.5). Significantly lower (p<0.001) levels

of quercetin were measured in the other vegetables analysed.

Measurement of quercetin in four types of peppers showed that green peppers

contained the highest amount of this particular flavonoid at a concentration of

13.1 g/g. Red and yellow peppers contained similar levels of 8.2 and 8.6 g/g,

respectively while orange peppers containing the lowest amount at 4.9 g/g.

As noted previously, apigenin was measured in only three of the products studied

with fresh parsley proving to be an excellent source of this flavonoid being present at

a concentration of 2453 g/g (Fig. 3.6). Celery had an apigenin content of

159.2 g/g while the lowest detectable amount was found in watercress at 15.1 g/g.

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 Table 3.4 Flavonoid content of selected vegetables (values given in logarithmic
transformed means with g/g fresh weight in brackets).

Vegetable Apigenin Kaempferol Luteolin Myricetin Quercetin

Log transformation values (g/g fresh weight)

Avocado n.d. n.d. n.d. n.d. n.d.

Bean n.d. 0.84 n.d. 0.72 1.52

(French) (7.00) (5.5) (32.76)
Bean sprouts n.d. n.d. n.d. n.d. 0.89
(7.74)
Beetroot n.d. n.d. n.d. n.d. n.d.
Broccoli n.d. 1.41 n.d. n.d. 1.31
(25.40) (20.34)
Brussels n.d. 2.15 n.d. n.d. 1.17
sprouts (140.8) (14.92)
Cabbage n.d. n.d. n.d. n.d. n.d.
(Red)
Carrot n.d. n.d. n.d. n.d. 0.30
(2.02)
Cauliflower n.d. 0.41 n.d. n.d. 0.44
(2.60) (2.78)
Celery 2.20 n.d. 1.14 n.d. n.d.
(159.2) (13.76)
Cucumber n.d. n.d. n.d. n.d. 0.12
(1.32)
Garlic n.d. n.d. n.d. n.d. 1.05
(11.17)

Significance of Effect (Sig.)/Standard Error of the Mean (SEM)

Apigenin Kaempferol Luteolin Myricetin1 Quercetin

Sig. SEM Sig. SEM Sig. SEM Sig SEM Sig. SEM

Vegetable (V) *** 0.025 *** 0.027 *** 0.048 0.123 *** 0.042

where: Each value is the mean of 9 measurements

n.d. = below detection limit (<0.5 g/ml); *** = p<0.001
1
Only one vegetable contained this flavonoid

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 Table 3.4 (Continued) Flavonoid content of selected vegetables (values given
in logarithmic transformed means with g/g fresh
weight in brackets)

Vegetable Apigenin Kaempferol Luteolin Myricetin Quercetin

Log transformation values (g/g fresh weight)

Kale n.d. 2.78 n.d. n.d. 2.15

(598.3) (142.01)
Leek n.d. 1.71 n.d n.d. n.d.
(50.8)
Lettuce n.d. n.d. n.d. n.d. 1.17
(green) (14.68)
Mushroom n.d. n.d. n.d. n.d. n.d.
Onion n.d. n.d. n.d. n.d. 2.47
(red) (297.4)
Onion n.d. n.d. n.d. n.d. 2.64
(white) (432.9)
Parsley 3.39 n.d. 1.26 n.d. 0.79
(2453) (18.31) (6.18)
Parsnip n.d. n.d. n.d. n.d. 0.66
(4.60)
Peas n.d. n.d. n.d. n.d. 0.82
(frozen) (6.57)
Pepper n.d. n.d. 0.90 n.d. 1.12
(green) (7.92) (13.05)
Pepper n.d. n.d. 0.70 n.d. 0.69
(orange) (4.98) (4.93)
Pepper n.d. n.d. 0.69 n.d. 0.91
(red) (4.91) (8.18)

Significance of Effect (Sig.)/Standard Error of the Mean (SEM)

Apigenin Kaempferol Luteolin Myricetin1 Quercetin

Sig. SEM Sig. SEM Sig. SEM Sig. SEM Sig. SEM

Vegetable (V) *** 0.025 *** 0.027 *** 0.048 - 0.123 *** 0.042

where: Each value is the mean of 9 measurements

n.d. = below detection limit (<0.5 g/ml); *** = p<0.001
1
Only one vegetable contained this flavonoid

116
 Table 3.4 (Continued) Flavonoid content of selected vegetables (values given in
logarithmic transformed means with g/g fresh weight in
brackets)

Vegetable Apigenin Kaempferol Luteolin Myricetin Quercetin

Log transformation values (g/g fresh weight)

Pepper n.d. n.d. 0.80 n.d. 0.93

(yellow) (6.29) (8.58)
Potato n.d. n.d. n.d. n.d. n.d.
Scallion n.d. 1.25 n.d. n.d. 1.28
(18.00) (18.88)
Spinach n.d. n.d. 1.52 n.d. 2.61
(33.46) (406.9)
Watercress 1.18 1.18 1.61 n.d. n.d.
(15.10) (15.20) (40.40)
Sweet corn n.d. n.d. n.d. n.d. n.d.
(Fresh)
Sweet corn n.d. n.d. n.d. n.d. n.d.
(Tinned)
Tomatoes n.d. n.d. n.d. n.d. 0.91
(Vine) (8.21)

Significance of Effect (Sig.)/Standard Error of the Mean (SEM)

Apigenin Kaempferol Luteolin Myricetin1 Quercetin

Sig. SEM Sig. SEM Sig. SEM Sig. SEM Sig. SEM

Vegetable (V) *** 0.025 *** 0.027 *** 0.048 - 0.123 *** 0.042

where: Each value is the mean of 9 measurements

n.d. = below detection limit (<0.5 g/ml); *** = p<0.001
1
Only one vegetable contained this flavonoid

117
Figure 3.5 HPLC chromatogram from HPLC analysis of freeze-dried kale.
(Peak at retention time of 6.151 min. = quercetin; 8.577 min =
kaempferol)

Figure 3.6 HPLC chromatogram from HPLC analysis of freeze-dried parsley.

(Peak at retention time of 6.144 min. = quercetin; 7.123 min =
luteolin; 9.952 min. = apigenin)

118
The richest source of kaempferol proved to be kale with an average concentration of

598.3 g/g being determined. Brussels sprouts were also a good source of this

flavonoid containing 140.8 g/g. Leeks contained twice as much kaempferol

(50.5 g/g) as broccoli (25.4 g/g) while similar concentrations of 18 and 15.2 g/g,

respectively, were found in scallions and watercress. The lowest levels of this

flavonoid were measured in French beans and cauliflower at concentrations of

7.0 and 2.6 g/g, respectively.

Luteolin was most abundant in watercress and spinach at concentrations of 33.5 and

15.2 g/g, respectively. The levels found in celery and parsley were 13.8 g/g and

18.3 g/g, respectively, with the flavonoid also in peppers at levels ranging from

7.9 g/g for green peppers to 4.9 g/g for red peppers.

As noted previously only French beans contained myricetin (Figs. 3.7 and 3.8) at a

low concentration of 5.5 g/g with the major flavonoid in this vegetable being

quercetin measured at a concentration of 32.76 g/g.

Avocado, beetroot, red cabbage, mushroom, potato and sweet-corn (fresh or tinned),

contained no measurable amounts of the flavonoids under investigation. Bean

sprouts, carrot, cucumber, garlic, lettuce, parsnip, frozen peas, and vine tomatoes

contained only quercetin which was found at a low concentration compared to other

vegetables.

119
Figure 3.7 HPLC chromatogram from HPLC analysis of freeze-dried French
beans. (Peak at retention time 4.528 min. = myricetin; 6.076 min =
quercetin; 8.490 min. = kaempferol)

Figure 3.8 UV spectrum of myricetin standard (top spectrum) and myricetin peak
from French beans (bottom spectrum)

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3.4 Discussion

3.4.1. Development of HPLC methodology for measuring flavonoids

The current work is a continuation of studies conducted within the Department of

Food Science, QUB by McAnlis (1998) and further develops the flavonoid

methodology employed which was based on the original method used by

Hertog et al. (1992a). Hertog and co-workers (1992a) used two isocratic mobile

phases, 25% acetonitrile in 0.025 M buffer phosphate and 45% methanol in 0.025 M

buffer phosphate at pH 2.4. Studies by McAnlis (1998) were based on isocratic

elution using, methanol/water/acetic acid in a ratio of 70:29:1 (v/v/v). However,

when the current work commenced it was found that using the method of

McAnlis (1998) resulted in HPLC chromatograms with poor resolution of peaks

from flavonoid standards. This was due to the peaks overlapping as the retention

times of the standards were close together and not separated sufficiently. It was

therefore essential that greater separation of the flavonoid peaks be obtained in order

to allow for more accurate quantification. This was subsequently achieved by

developing the gradient elution method as described in Chapter 2 (Materials and

Methods). Using this method the flavonoid peaks were successfully separated,

thereby allowing for accurate quantification in all subsequent experimental work. In

addition, the use of trifluoroacetic acid in the methodology (Dalluge et al., 1998) as

opposed to acetic acid used by McAnlis (1998) further enhanced the resolution of the

flavonoid peaks and helped eliminate tailing.

Furthermore, the use of DETC as the antioxidant in the current methodology as

opposed to TBHQ as previously employed by Hertog et al. (1992a) and

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McAnlis (1998) further enhanced the reliability of the method. Current studies found

that TBHQ appeared to fragment after hydrolysis in 62.5% aqueous methanol and 6

M HCl yielding two peaks with retention times corresponding to those of quercetin

and kaempferol which could potentially have led to misleading results. Hydrolysis

of DETC under the same conditions did not produce these extra peaks thus it was

used for subsequent analysis of the fruits and vegetables instead of TBHQ.

In the current work, use of a diode array detector (DAD) enabled UV spectra

extracted from the main peaks of the HPLC chromatograms (standards and

unknowns) at 375 nm to be used to further confirm the identity of the flavonoid

compounds in samples as well as comparing retention times of the peaks with those

from standards run at the same time. This proved particularly useful in a number of

instances where there was some doubt as to the authenticity of peaks eluting at the

similar retention times as those of particular flavonoids. For example, in raspberries

and strawberries a large peak was found to elute at a similar retention time to that of

myricetin. As this particular flavonoid can be difficult to detect and measure,

spectral analysis of the peak was carried out. It was found that the spectrum of this

large peak did not match that from the myricetin standard, thus myricetin was not

present in either of these fruits. The ability to use such UV spectra to confirm the

presence or absence of an analyte thus proved to be an effective technique for

ensuring that peaks are not misinterpreted as those of flavonoids and accurate

analytical results can be obtained. Spectral analysis was consequently carried out for

all the samples analysed during this experimental work and that described in

Chapter 4.

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3.4.2 Flavonoid content of fruits and vegetables

3.4.2.1 Fruits

As noted previously, quercetin was the most abundant flavonoid found in all the

fruits studied with plums being the richest fruit source. The levels of quercetin found

in plums by Hertog et al. (1992b) and Justesen et al. (1998) at 9 g/g and 1.5 g/g,

respectively, were significantly lower than the average concentration of 78.2 g/g

found in these studies. This may have been due to different varieties being used or

the stage of maturity of the fruit but as this information was not available from the

literature it is difficult to determine exactly why such differences occur. The plums

used in the current work were Red Beauty and originated in Spain. Early work

carried out by Herrmann (1976) detected small amounts of kaempferol as well as

quercetin in plums. In the current study kaempferol was not detected in the variety

of plum analysed.

Previous work has indicated that red apples contain significantly more flavonoids

than green apples which is in agreement with the findings of this research. For

example, Hertog et al. (1992b) found that Jonas gold apples contained 72 g/g

quercetin while Golden Delicious apples contained 25 g/g. They also showed that

Cox’s Orange Pippin apples contained 41 g/g quercetin. The Golden Cape (green)

apples analysed in this work contained on average 30 g/g quercetin while the Enza-

Royal (red) apples contained 42 g/g quercetin. The results obtained by McAnlis

(1998) for apples also showed that green apples contained significantly less quercetin

than red apples although it was noted that the levels measured were significantly

higher than those reported in this work and by other workers.

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Red grapes were also found to be richer in quercetin than green grapes with levels of

approximately 23.7 and 7.8 g/g, respectively. The quercetin concentrations of

12 g/g reported by Hertog et al. (1992b) for white grapes were similar to those

obtained in this work for green grapes with levels in black grapes being 15 g/g.

Oszmianski and Lee (1990) found that red grapes contained 35.4 g/g quercetin with

a similar level of 37 g/g being found in blue grapes by Justesen et al. (1998) and a

lower concentration of 2 g/g being measured in green grapes. Franke et al. (2004)

found an average concentration of 6 g/g quercetin in red seedless grapes as well as

measuring luteloin at an average level of 33 g/g. This is contrary to the findings of

other workers and the findings of this study where luteolin was not detected in either

red or green grapes.

It was also noted from this research that pink grapefruit had approximately twice the

concentration of apigenin (30.3 g/g) and kaempferol (6.7 g/g) than white

grapefruit (14.9 and 3.7 g/g, respectively). Previous work by Justesen et al. (1998)

showed that grapefruit pulp, not specifying whether it was white or pink, contained 5

g/g quercetin and 4 g/g kaempferol. Work undertaken by Franke et al. (2004)

found that ruby red grapefruit contained 3 g/g quercetin, 14 g/g apigenin and <0.1

g/g kaempferol.

It can be observed from these results that Clementine and Mandarin oranges

contained similar quantities of quercetin (3.79 and 5.33 g/g, respectively) but that

higher amounts were found in ordinary eating oranges (13.17 g/g) and even higher

concentrations in Satsuma oranges (27.5 g/g). Considerably higher quantities of

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apigenin were found in the ordinary eating oranges and Satsuma oranges (63.2 and

76.4 g/g, respectively) than in Clementine and Mandarin oranges (18.0 and

23.8 g/g, respectively). Results reported by Franke et al. (2004) for two types of

orange, which were most likely ordinary eating oranges, demonstrated the presence

of quercetin at levels of 4 - 6 g/g but with apigenin at concentrations of <1 g/g.

They also found luteolin present at concentrations of 14 - 15 g/g which is contrary

to the findings of this work. Work reported by Justesen et al. (1998) did not find any

of these particular flavonoids either in orange pulp or orange juice. The presence of

myricetin and quercetin has been also reported in raw orange juice by Hertog et al.

(1993).

Concentrations of quercetin measured in apricots (35.9 g/g) were slightly lower

than those of 25 and 26 g/g, respectively, reported by Hertog et al. (1992b) and

Justesen et al. (1998). The same workers found levels of 7.9 and 6.5 g/g,

respectively, for kaempferol and quercetin while the present study revealed a similar

level of 8.7 g/g for kaempferol but a higher concentration of 24.1 g/g for

quercetin. Häkkinen et al. (1999) also found both kaempferol and quercetin in

strawberries at concentrations of 12 and 8.6 g/g, respectively, as did Franke et al.

(2004) at levels of 6 and 9 g/g, respectively. The latter workers also found frozen

strawberries to contain 13 and 9 g/g of both flavonoids. The difference in quercetin

content between the present work (24.1 g/g) and the values obtained by others

workers, may be due in part to a different variety of strawberry being analysed or

seasonal variation.

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The average concentration of quercetin in raspberries measured in this study

(7.2 g/g) was similar to that of 5 g/g obtained by Justesen et al. (1998). However,

research carried out by Häkkinen et al. (1999) found that red raspberries contained a

significantly higher concentration at 29 g/g. Franke et al. (2004) measured

concentrations of 11 g/g for quercetin in frozen raspberries. As for strawberries,

such differences in concentrations may be seasonal or due to variety.

In the current study green pears contained high levels of apigenin (66.3 g/g) and

moderate amounts of quercetin (12.9 g/g). Justesen et al. (1998) found pear peel to

contain 45 g/g quercetin while values for quercetin obtained by Hertog et al.

(1992b) ranged from 3.3 g/g to 10 g/g which are comparable to the values

obtained in this work. In the available literature (Hertog et al., 1992b) no

measureable flavonoids were found for peaches which is in contrast to this work

where a small amount of quercetin (8.5 g/g) was quantified. A low concentration of

quercetin was also found in Kiwi fruit (6.4 g/g) and in melon (5.6 g/g), which also

contained a small amount of luteolin (3.7 g/g).

3.4.2.2 Vegetables

As for fruits, quercetin was found to be the predominant flavonoid in the majority for

vegetables analysed, with onions, spinach and kale containing the highest

concentrations and the lowest amounts being found in cauliflower and parsnips. The

current study found green leafy vegetables including celery, kale, leek, green lettuce,

parsley, scallions, spinach and watercress to be main flavonoid sources with regard to

vegetables, which is similar to the findings of other workers such as Herrmann

(1976), Hertog et al. (1992 a,b), McAnlis (1998) and Justesen et al. (1998).

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Hertog et al. (1992b) found that onions (not specified if red or white) contained

quercetin levels ranging from 284 to 347 g/g while Justesen et al. (1998) reported

quercetin concentrations of 340 g/g for yellow onion and 450 g/g for red onions.

Crozier et al. (1997) reported concentrations of 201 g/g quercetin in red onion with

levels for white onions ranging from 185 - 634 g/g. Chu et al. (2000) quantified

quercetin in the outer leaves of onions to be 258.9 g/g with the inner leaves

containing 26.1 g/g. They also found a kaempferol content of 26.1 g/g in the outer

leaves with 0.56 g/g present in the inner leaves. Kaemperferol was not found in

onions in the current study. McAnlis (1998) reported the presence of apigenin and

kaempferol in onions which is contrary to the findings of this work and that of the

Chu et al. (2000) mentioned above.

The concentration of quercetin in spinach found in the current work (406.9 g/g)

proved to be significantly higher than the level of 19.6 g/g reported by Chu et al.

(2000). These workers also measured low amounts of myricetin (0.4 g/g) and

kaempferol (0.6 g/g) with no luteolin being found which is contrary to the current

findings where neither myricetin or kaempferol were detected and a luteolin content

of 33.5 g/g was measured.

Kale proved to be an excellent source of kaempferol (598 g/g) and quercertin

(142 g/g). Justesen et al. (1998) found kale to contain 470 g/g kaempferol and

120 g/g quercetin, which is within the range of values obtained in this work whilst

Hertog et al. (1992b) obtained lower concentrations of 110 g/g for quercetin and

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211 g/g kaempferol. Leeks were also a source of kaempferol which was found at an

average concentration of 50.8 g/g in this study compared with levels of

11 - 56 g/g reported by Hertog et al. (1992b) and 31 g/g by Justesen et al. (1998).

The current work also showed Brussels sprouts to be a good source of kaempferol,

the concentration of 140.8 g/g reported being significantly higher than that of 7.4

g/g obtained by Hertog et al. (1992b) and 9 g/g reported by Justesen et al. (1998).

Broccoli contained both kaempferol (25.4 g/g) and quercetin (20.34 g/g). Hertog

et al. (1992b) also quantified both these flavonoids at levels of 30 and 72 g/g,

respectively, while Justesen et al. (1998) found them to be present at concentrations

of 37 g/g and 60 g/g, respectively.

The other product shown to be a rich source of flavonoids was parsley. This

commonly used herb was found to contain 2453 g/g apigenin with small amounts of

luteolin (18.3 g/g) and quercetin (6.2 g/g). There appears to be no other literature

reporting the presence of apigenin in parsley with others measuring only kaempferol,

myricetin and quercetin at average concentrations of 4.4, 80.8 and

3.3 g/g (Justesen and Knuthsen, 2001; Justesen et al., 1998; Lugasi and Hovari,

2000; USDA, 2003). Watercress, another herb, was found to contain the three

flavonoids, apigenin (15.1 g/g), kaempferol (15.2 g/g) and luteolin (40.4 g/g).

Justesen and Knuthsen (2001) also found kaempferol at a concentration of 10 g/g in

watercress as well as quercetin at a level of 40 g/g but did not report the presence of

apigenin or luteolin. Apigenin was also abundant in the celery used in the present

work at a concentration of 159.2 g/g with luteolin also being present at a level of

13.76 g/g. Hertog et al. (1992a) found celery to contain a significant concentration

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of apigenin (1787 g/g dry weight) with luteolin also being present

(358 g/g dry weight). Later work by Crozier et al. (1997) found both flavonoids to

be present in different varieties of celery stalks ranging from 49 - 104 g/g (fresh

weight) for apigenin and 36 - 40 g/g (fresh weight) for luteolin.

All four types of peppers analysed (green, orange, red and yellow) contained both

luteolin and quercetin at relatively low levels ranging between 4.9 - 7.9 g/g for

luteolin and 4.9 - 13.1 g/g for quercetin. Work carried out on green, red and yellow

peppers by Justesen et al. (1998) reported that only green peppers contained

quercetin at a concentration of 5 g/g. Lugasi and Hovari (2000) found that green

peppers contained both luteolin and quercetin at levels of 6.9 and 6.5 g/g,

respectively, which is in agreement with the quantities reported in this research.

All the other vegetables analysed in this work contained either none of the flavonoids

being studied or they were present in low amounts. The latter included bean sprouts

(quercetin; 7.7 g/g), carrots (quercetin; 2.0 g/g), cauliflower (kaempferol, 2.6

g/g; quercetin; 2.8 g/g), cucumber (quercetin; 1.3 g/g), garlic (quercetin; 11.2

g/g), lettuce (quercetin; 14.7 g/g), parsnips (quercetin; 4.6 g/g), scallions

(kaempferol; 18.0 g/g; quercetin; 18.9 g/g), and vine tomatoes (8.2 g/g).

As well as containing kaempferol (7.0 g/g) and quercetin (32.8 g/g), French beans

was the only vegetable in which myricetin (5.5 g/g) was measured. This is not

surprising, however, as this flavonoid appears to be difficult to detect and quantify.

Hertog et al. (1992b) reported that a stock standard solution of myricetin degraded

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up to 10% after one month storage at 4C. Results from the current study have

supported previous findings with a 16% deterioration in myricetin being observed in

the external standard stored for a 3 month period at 4C with approximately 9% of

the deterioration occurring during the first 3 weeks (Section 3.2.1.2).

The problems with the detection and measurement of myricetin were also reflected in

results obtained from both the reagent and spiked sample recoveries reported in

Section 3.3.1. These findings were in agreement with those reported by Justesen et

al. (1998) who obtained only a 30% recovery of myricetin standard. They suggested

that this low recovery could be explained by degradation in the acid hydrolysis

process. The paper by Justesen et al. (1998) also pointed to the work carried out by

Hertog et al. (1992b) in relation to the instability of myricetin and also to research

reported by Shepherd and Ibe (1995) who remarked on the interference of other

compounds with myricetin. Justesen et al. (1998) did not observe the interference

reported by Shepherd and Ibe (1995) and thought that this may be explained by the

50 min. gradient used in their chromatographic system, allowing the very polar

compounds to elute prior to myricetin. They did, however, agree that myricetin is

less stable than other flavonoids.

A summary of the total flavonoid content of the fruits and vegetables studied is

presented in Table 3.5. It clearly shows the variation in flavonoid

concentrations found in these plant foods and highlights the

importance of eating as wide a range of fruits and vegetables as

possible in order to obtain the optimum intake of these natural

antioxidants.

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Figure 3.5 Total flavonoid content (g/g fresh weight) of fruit and vegetables
analysed

Fruit Total flavonoids Vegetable Total flavonoids

(g/g fresh weight) (g/g fresh weight)

Apple (green) 30.0 Avocado n.d.

Apple (red) 42.2 Bean (French) 45.3
Apricot 35.9 Bean sprouts 7.7
Banana n.d. Beetroot n.d.
Clementine orange 21.8 Broccoli 45.7
Grapefruit (white) 21.2 Brussels sprouts 155.7
Grapefruit (pink) 41.6 Cabbage (Red) n.d.
Grape (red) 23.7 Carrot 2.0
Grape (green) 7.8 Cauliflower 5.4
Kiwi fruit 6.4 Celery 173
Mandarin orange 29.2 Cucumber 1.3
Melon 9.3 Garlic 11.2
Orange (Ordinary) 76.4 Kale 740
Peach 8.5 Leek 50.8
Pear 79.3 Lettuce (green) 14.7
Plum 78.2 Mushroom n.d.
Raspberry 7.2 Onion (red) 297
Satsuma orange 114 Onion (white) 433
Strawberry 32.7 Parsley 2478
Parsnip 4.6
Peas (frozen) 6.6
Pepper (green) 21.0
Pepper (orange) 9.9
Pepper (red) 13.7
Pepper (yellow) 14.9
Potato n.d.
Scallion 37
Spinach 441
Sweet corn (Fresh) n.d.
Sweet corn (Tinned) n.d.
Tomatoes (Vine) 8.2
Watercress 70.7

n.d. = not detected

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