TOTAL PHENOLICS AND TOTAL FLAVONOIDS IN BULGARIAN FRUITS AND VEGETABLES

D. Marinova, F. Ribarova, M. Atanassova
Journal of the University of Chemical Technology and Metallurgy, 40, 3, 2005, 255-260
TOTAL PHENOLICS AND TOTAL FLAVONOIDS
IN BULGARIAN FRUITS AND VEGETABLES
D. Marinova, F. Ribarova*, M. Atanassova
National Center of Public Health Protection,
Received 06 June 2005
Department of Food Chemistry, Sofia 1431, Bulgaria
Accepted 21 July 2005
*E-mail: f.ribarova@nchmen.government.bg
ABSTRACT
The increasing interest in powerful biological activity of plant phenolics and flavonoids outlined the necessity of
determining their contents in Bulgarian fruits and vegetables.
The study comprised 42 food products ? 20 fruit and 22 vegetable species.
The total phenolic content was determined by using the Folin-Ciocalteu assay. The content of total flavonoids was
measured also spectrophotometrically by using the aluminum chloride colorimetric assay.
The results of fruits showed the highest total phenolic content in blueberries (670.9 mg gallic acid equivalents
(GAE)/100 g), dogwood berries (432.0 mg GAE/100 g) and sour cherry (429.5 mg GAE/100 g). The greatest total flavonoid
content was revealed in blueberries (190.3 mg catehin equivalents (CE)/100 g). The lowest total phenolics and total
flavonoids were established in peaches (50.9 mg GAE/100 g and 15.0 mg CE/100 g, respectively).
The results of vegetables showed the greatest value of phenolics in green peppers (246.7 mg GAE/100 g) and red
peppers (173.2 mg GAE/100 g). Significant difference was found between total phenolic content in red and spring onions
at almost equal total flavonoid values.
The present paper shows particular specificity not only in the total phenolic and flavonoid content, but also in their
ratio and distribution in the different parts of the studied fruits and vegetables.
Keywords: phenolics, flavonoids, fruits, vegetables.
INTRODUCTION
nols. The group of simple phenols contains also the so-
called ?phenolic acids? or phenols with carboxyl group
Phenolics are ubiquitous secondary metabolites
underlying the specificity of their function. Polyphenols
in plants. They comprise a large group of biologically
contain at least two phenol rings. Flavonoids, a subject of
active ingredients (above 8000 compounds) ? from simple
comprehensive studies in recent years, belong to this group.
phenol molecules to polymeric structures with molecu-
More than 4000 flavonoids have been identified in dif-
lar mass above 30000 Da [1]. On the basis of the number
ferent higher and lower plant species [2].
of phenol subunits, the modern classification forms two
The presence of phenolic compounds in diet was
basic groups of phenolics ? simple phenols and polyphe-
long regarded as a negative feature because they were
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Journal of the University of Chemical Technology and Metallurgy, 40, 3, 2005
doubted to decrease the availability of nutrients, lead-
in hermetically sealed packages and stored at 4ºC until
ing to lower nutritional value of the product [3]. After
the time of analysis. Lyophilization is applied aiming
the identification of the ?French paradox? and reveal-
to uniform the storage conditions and submit the prod-
ing that the moderate consumption of red wine (rich in
ucts for analysis in similar form.
polyphenols) decreases cardiovascular morbidity rate
Reagents: Gallic acid, (+)-catechin and Folin?
among French people, particular emphasis was put on
Ciocalteu?s phenol reagent were purchased from Sigma
studying phenolic compounds as food ingredients. A
Chem. Co. All other chemicals were of analytical grade.
number of data showed that the presence of phenolics
in foods is particularly important for their oxidative sta-
Procedures
bility and anti-microbial protection [4, 5].
Phenolics possess a wide spectrum of biochemi-
Sample preparation
cal activities such as antioxidant, antimutagenic,
anticarcinogenic, as well as ability to modify the gene
A ground freeze-dried sample of 0.5 g was
expression [6, 7]. Numerous epidemiological studies
weighted and phenolic and flavonoid compounds were
confirm significant relationship between the high di-
extracted with 50 ml 80 % aqueous methanol on an
etary intake of flavonoids and the reduction of cardio-
ultrasonic bath for 20 min. An aliquot (2 ml) of the
vascular and carcinogenic risk [8]. The formulation of
extracts was ultracentrifugated for 5 min at 14000 rpm.
preventive and healthy nutrition requires information
about phenolic and flavonoid composition in plant foods.
Total phenolic assay
Based on numerous evidence on the strong bio-
logical activity of phenolic compounds and on the scar-
The total phenolic content of fruits and vegetables
city of data for their content in foods the aim of current
were determined by using the Folin?Ciocalteu assay [9].
study was focused on determination of total phenolic
An aliquot (1 ml) of extracts or standard solution of
and total flavonoid content in Bulgarian fruits and veg-
gallic acid (20, 40, 60, 80 and 100 mg/l) was added to
etables.
25 ml volumetric flask, containing 9 ml of distilled
deionised water (dd H O). A reagent blank using dd
2
EXPERIMENTAL
H O was prepared. One millilitre of Folin?Ciocalteu?s
2
phenol reagent was added to the mixture and shaken.
Materials
After 5 min, 10 ml of 7 % Na CO solution was added
2
3
to the mixture. The solution was diluted to volume (25
The study covered 42 food products - 20 fruit
ml) with dd H O and mixed. After incubation for 90
and 22 vegetable species. Randomized market sampling
2
min at room temperature, the absorbance against pre-
was applied. The average sample consisted of represen-
pared reagent blank was determined at 750 nm with an
tative amounts of three individual samples from respec-
UV-Vis Spectrophotometer Lambda 5. Total phenolic
tively different region of origin, in similar stage of bio-
content of fruits and vegetables was expressed as mg
logical development and ripeness. Only samples com-
gallic acid equivalents (GAE)/100 g fresh weight. All
plying with the requirements of good manufacturing
samples were analysed in duplicates.
practice were considered. The sampling lasted one year
according to the seasonality of harvesting for individual
Total flavonoid assay
species. All samples data are stated in the sampling pro-
tocol. After removing the non-edible parts the samples
Total flavonoid content was measured by the alu-
were cut into small pieces and frozen under liquid ni-
minum chloride colorimetric assay [10]. An aliquot (1
trogen. The samples were freeze-dried after one week.
ml) of extracts or standard solution of catechin (20, 40,
The water content in lyophilized fruits and vegetables
60, 80 and 100 mg/l) was added to 10 ml volumetric
was not more than 3.5 %, providing for safety storage
flask containing 4 ml of dd H O. To the flask was added
2
conditions. The freeze-dried samples were vaccumized
0.3 ml 5 % NaNO . After 5 min, 0.3 ml 10 % AlCl was
2
3
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D. Marinova, F. Ribarova, M. Atanassova
added. At 6th min, 2 ml 1 M NaOH was added and the
1.2 mg GAE/100 g fresh mass; recovery: 97% and re-
total volume was made up to 10 ml with dd H O. The
producibility (RSD): 2.7 %. The determined analytical
2
solution was mixed well and the absorbance was mea-
parameters of the total flavonoids method was as fol-
sured against prepared reagent blank at 510 nm. Total
lows: limit of detection: 0.6 mg CE/100 g fresh mass;
flavonoid content of fruits and vegetables was expressed
limit of determination: 1.8 mg CE/100 g fresh mass;
as mg catechin equivalents (CE)/100 g fresh mass.
recovery: 96 % and reproducibility (RSD): 3.7 %. These
Samples were analysed in duplicates.
results proved the viability of the both used methods to
determine phenolic and flavonoid compounds in fruit
RESULTS AND DISCUSSION
and vegetable extracts.
The results for total phenolic and total flavonoid
The determined analytical parameters of the to-
content and the ratio total flavonoids/phenolics in the
tal phenolics method was as follows: limit of detection:
studied fruits are presented in Table 1. The data clearly
0.4 mg GAE/100 g fresh mass; limit of determination:
outline the richest phenolics sources ? blueberries
(670.9 mg GAE/100 g),
Table 1. Content of total phenolics and total flavonoids in fruits.
dogwood berries (432.0
mg GAE/100 g) and sour
Fruit Latin
name Total
Total flavonoids Flavonoids/
cherry (429.5 mg GAE/
phenolics
mg CE/100g
Phenolics
100 g). Blueberries have
mg GAE/100g
fresh mass
also the highest flavonoid
fresh mass
content (190.3 mg CE/100
Pear (unpeeled)
Pyris communis
124.7
69.9
0.56
g) with ratio of 0.32. This
Pear (peeled)
Pyris communis
91.0
48.5
0.53
result enables us to sup-
Apple, yellow
Malus pumila

port the proverb popular
(unpeeled)
99.7
34.8 0.35
in
northern countries
Apple, yellow
Malus pumila

?When there are blueber-
(peeled)
75.8
20.9
0.28
Apple, red
Malus pumila

ries, we shall not need a
(unpeeled)
125.4
48.6 0.39
doctor?.

In dogwood ber-
Apple, red
Malus pumila

ries the total flavonoids
(peeled)
104.3
32.7 0.31
have

a smaller share of
Plum
Prunus domestica
303.6
136.2
0.45
total phenolics, while in
Apple, green
Malus pumila

sour cherries the ratio is
(unpeeled)
118.1
40.4 0.34
0.32,

even slightly greater
Apple, green
Malus pumila

than in blueberries. The
(peeled)
97.5
17.3 0.18
discussion of this differ-
Peach
Prunus persica
50.9
15.0
0.3
ence would have a higher
Blackberry
Rubus coesins
355.3
55.5
0.16
degree of uncertainty, due
Raspberry
Rubus ideaus
178.6
26.6
0.15
to the small number of
Strawberry
Fragaria vesca
244.1
69.7
0.29
tests. It is clear that phe-
Sweet cherry
Prunus avium
78.8
19.6
0.25
nolic acids prevail in dog-
Prunus cerasus

wood berries, which ex-
Sour cherry
vulgaris
429.5
138.6 0.32
Blueberry
Vaccinium myrtilus
plains their sour astringent
670.9
190.3
0.28
taste.
Fig
Ficus carica
59.0
20.2
0.34
The second group
White grape
Vitis vinifera
184.1
36.5
0.2
with high total phenolic
Black grape
Vitis vinifera
213.3
77.1
0.36
content comprises black-
Dogwood berry
Cornus mas
432.0
91.4
0.21
berries (355.3 mg GAE/
Results are presented as mean value of duplicates.
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Journal of the University of Chemical Technology and Metallurgy, 40, 3, 2005
100 g), plums (303.6 mg GAE/100 g) and strawber-
flavonoids are found in the greatest ratio in plums
ries (244.1 mg GAE/100 g). In this group the total
(0.45). We could suppose that this is due to the rich
abundance
of
Table 2. Content of total phenolics and total flavonoids in vegetables.
anthocyanidines in combi-
nation with the other fla-


Total phenolics Total flavonoids Flavonoids/
vonoids. The low ratio
Vegetable
Latin name
mg GAE /100g
mg CE /100g
Phenolics
(0.16) in blackberries is
fresh mass
fresh mass
probably a result of the
Carrot
Daucus carota
96.0
26.7
0.28
Apium

rich spectrum of phenolic
Celery (leaves)
graveolens
113.0
46.4 0.41
acids.

In strawberries the
Petroselium

total flavonoids contribute
Parsley
sativum
188.0
27.2
0.14
one third of the total phe-
Hibisons

nolics, with the ratio simi-
Okra
eculentus
153.7
49.1 0.32
lar to that of blueberries.

Tomato
S. lycopersicum
76.9
12.8
0.17
Pears analysis pro-
Capsicum

Red pepper
anuum
173.2
13.7 0.08
vides
very interesting re-
Capsicum

sults. We found the highest
Green pepper
anuum
246.7
27.4 0.11
percentage

rate of total fla-
Salad
Lactuca sativa
116.2
76.5
0.66
vonoids vs. total phenolics
Lactuca sativa

compared to all other stud-
Lettuce
capitata
124.5
97.2 0.78
ied fruits. This ratio is the
Brassica

oleracae var.

same for both unpeeled and
Kohlrabi
caulorapa
44.9
8.9 0.19
peeled
pears. The unpeeled
Brassica

pears showed significantly
oleracae var.

greater content of total phe-
Red cabbage
botritis
139.3
23.7
0.17
Brassica

nolics and total flavonoids.
oleracae var.

The results for unpeeled and
Brussels sprout
Gemmifera
161.5
33.1 0.20
peeled

apples are quite simi-
Brassica

lar. Our data suggest richer
oleracae var.

Broccoli
Italica
101.7
18.8 0.18
presence

of phenolics in the
Raphanus

skin of the fruits. The com-
sativus, var.

parative assessment between
Radish
Radicula
160.0
48.5
0.30
red and yellow apples favors
Red onion
Allium cepa
154.1
18.7
0.12
the red ones because of the
Spring onion
Allium cepa
120.0
16.0
0.13
greater content of phenolics.
Spring onion


(leaves)
Allium cepa
81.0
11.7
0.14
The differences in total fla-
Spring onion
Allium cepa

vonoid content, following
(stem)
36.5
2.5 0.07
the same

pattern, are quite
Leek (leaves)
Allium porrum
35.7
3.9
0.11
smaller.
Leek (stem)
Allium porrum
27.7
2.6
0.09
The analysis of both
Phaseolus

grapes species ? white and
Green bean
vulgaris
35.5
4.1
0.12
black logically revealed
Phaseolus

higher content of total phe-
Yellow bean
vulgaris
55.7
8.2 0.15
nolics and total flavonoids
Results are presented as mean value of duplicates.
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D. Marinova, F. Ribarova, M. Atanassova
in red grapes, associated with the higher content of
mg CE/100 g). Different content was also detected in
anthocyanidines.
the different parts of the plant. The green part of the
Peaches show particularly low content of total
spring onion has more total phenolics compared to the
phenolics and total flavonoids (50.9 mg GAE/100 g and
white part, while the total flavonoids are more concen-
15.0 mg CE/100 g, respectively) as well as figs (59.0 mg
trated in the green part.
GAE/100 g and 20.2 mg CE/100 g, respectively) at al-
The analytical data for leeks follow the same
most equal ratios ? respectively 0.3 and 0.34.
pattern concerning the stem and the leaves. The results
The variation of phenolic compounds content
for yellow and green beans are also similar. It is clear
in the fruits depends on many factors. It is known that
that the change in the colouring of the plant is a process
it decreases in the process of fruits development. Thus,
associated with redistribution of phenolics and fla-
for example, in white-coloured fruits it decreases con-
vonoids.
stantly with the progress of the ripening, while in red-
The results from the analysis of vegetables show
coloured varieties it increases during the last ripening
specific total phenolic and flavonoid content and spe-
stage due to the maximal accumulation of
cific distribution in the particular representatives, es-
anthocyanines and flavonols. Although we applied a
pecially for peppers and onions that are traditional for
very strict selection of the samples in equal ripening
stage we support the opinion that this criterion out-
Bulgarian diet.
lines data variations to the greatest extent.
The general assessment of the analytical results
CONCLUSIONS
for fruits definitely shows individual specificity of each
The presented data for total phenolic and total
studied sample and a rich diverse spectrum of phenolic
compounds differing from the flavonoids group.
flavonoid content are a basis for assessment of the pre-
Table 2 presents the analytical data for total
ventive role of fruits and vegetables against free radicals
effect and will enrich the national food composition
phenolic and total flavonoid content of the studied
database.
vegetables. The highest total phenolic content was
They are a necessary step towards in-depth stud-
found in green peppers (246.7 mg GAE/100g), fol-
ies on the spectrum of multiple phenolics and flavonoids
lowed by parsley (188.0 mg GAE/100g) and red pep-
representatives.
pers (173.2 mg GAE/100g). The ratio total flavonoids/
phenolics is comparatively low (about 0.1). The re-
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