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Anthocyanins
inbloodoranges:
Gofllositionand
liologioalaotiuity
Paolo Rapisarda, Santina Elisabetta Bellomo and Francesco Intrigliolo
IstitutoSperimentale
per l'Agrumicoltura,CorsoSavoia190,I-95024Acireale(CT), Italy
Abstract
Qualitative and quantitative aspect of anthocyanins occurring in blood orangesfruit
and juice are described.
Anthocyanins content ín fruit juice of the three most importctnt cultivars of blood
oranges, determined at the same degree of maturation, always varies according to the
following order: Moro > Sanguinello ) Tarocco. Marked dffirence in anthocyanin level
among the single Tarocco clones are also noted.
The effect ofextraction technology,processing and storage on anthocyaninspresents
in blood orangejuice is documented.
Anthocyqnins extraction-from by-product of blood orangesprocessing may be used as
Conespondence/Reprintrequest: Paolo Rapisarda, Istihrto Sperimentaleperl'Agrumicolhfa,
Acireale (CT), Italy
Corso Savoia 190, I-95024
Paolo RaPisarda ef a/.
2L8
substances'
food colorants or health-promoting
vitro and in vivo
such as
-in
Biological proprietiàs of blold oranges anthocyanins
described'
also
are
properties
antioxidant activity and phaimacological
Introduction
are the most cultivated
Tarocco, Moro and Sanguinello blood (pigmente{) 9ra-ng.es
The main characteristic
(L.iOsUe"kl
yóitru,
sinensis
in ltalV.
varieties of sweet orurrg",
is the presence of hydrosoluble
which distinguish"s tfrJs" vuri"ti"s from bloni o*tg"t
pigmentsinthefleshu.'o,o*"ti-"sintherind,belongingtotheanthocyaninclass.other
peculiaraspectsu,".t'"-igioulflu.,,o*,thehigh"o"t""lofvitaminC,flavanonesand
of these substancesvaries greatly in
hydroxycinnamic acids. The"amount and compoJition
physiological or environmental
genetic,
of
relation to a nrmber of factors which may bè
origin.
climatic conditions play a very
Anthocyanins are mainly cultivar-specifc but also
of the
oJ these pigments' In particular the development
important role in the b;Ctiesis
(cool
night
and
day
mnge between
red colour in the flesh is favoured by the temperature
nights and warm days) and by the intensity of light'
fruit. However, the rapid increase in
Blood orange, ur. ouai io"uuy consumed ís fresh
and the *.eateÎ attentionby colìsumersto
the consumptionof oru"g" ú"" in ítary and in Ernope
up the
taste of frestrly squeezedorangejuice, have opened
more healthy products *'"-djd,h"
elceflgnt
their
by
characterised
are
fnése
products.
iuices
market to various ,"0 o*oéj"ui*s
élour, sharp sweet -taste) and valued for
sensorial properties (rnterise"aroma,brilliant red
mncefúrationsof antioxidantstrbstances
the high vitamin c content and the p€senc€ of higfr
for health'
fr'lo V"it rnl" acids)whiih arevery important
(anthocyanins,luuu"*r,
Qualitative and quantitative
aspect
^Anthocyanins
represent one of the
of water-soluble
groups
most important
plant kingdom'
the
in
present
pigments
responsible for
main
the
i*ottg
ut"
îÈ'"y
in flowers
colours
blue
pink
and
ied,
the
and fruiis. Their chemical structure is
2-PhenlbetuoPYnlium
bY
formed
(flarylium) sàlt variously substituted. with
ùydróxylic or methoxylic groups (Fig'- 1)
and usually glycosilated in position 3 but
also in 5- and less frequently in other
positionsof the A or B rings.
The more frequentlY found sugar is
glucose but also galactose, rhamnose and
àrabinosecan occur. Other kinds ofsugars
as well as biosides and triosides are more
rarely present. In many cases, the sugar
residues are acylated by acetic, malonic or
Anthocyanidins
-
Cyanidin
Delphinidin
Pelargonidin
Peonidin
Petunidin
Malvidin
-Rt
OHH
OH
HH
OCH3
ocH3
OCH3
Rr-
OH
H
oH
OCH3
Fig. 1 - Structure of the most common
anthocyanidins
Anthocyanins in blood oranges
2t9
hydroxycinnamic acids (p-coumaric, ferulic, sinapic and caffeic). The first
observations on the pigments in blood oranges go back to the early years of the
twentieth century. Wheldale (1) stated that anthocyanins are present in the red'blood oranges'. Matlack (2) observed through the
fleshed varieties of so called
microscope that crushed red juice sacs showed spherites or needle crystals of
anthocyanins and performed samplings to confirm the chemical nature of these
crystals. In a review, Carrante (3) reported that the level of pigmentation of fruits
was linked to genetic factors, to climatic conditions and the intensity of light. In
addition, other factors such as mineral fertilisation and rootstocks play an important
role. The first attempts to isolate and quantify anthocyanins from the juices of
different varieties of blood oranges_were conducted by Patanè (4) who by
colorimetric methods, found an anthocyanin content of 0.1% in Sanguinello and
1.5% in Moro orange juices. Chandler (5) was the first to identify the cyanidin-3glucoside as the predominant anthocyanin in the Moro variety; traces of a second
pigment, provisionally identified as delphinidin-3-glucoside, were also found.
The application of.new chromatographic and spectroscopic techniques such as
'H-NMR
have allowed to identify the complete pattern of the
HPLC, HPLC-MS and
anthocyanins present in the three varieties of Moro, Tarocco and Sanguinello
oranges (6, 7, 8). Two anthocyanins were predominant: cyanidin-3-glucoside [A]
and cyanidin-3-(6"-malonyl)-glucoside [B] while other eight anthocyanins were
identified, some of which acylated with hydroxycinnamic acids (Fig. 2). The
anthocyanin content in blood orange represents an important index of quality both
for the fresh fruit as well as the processing industry. In fact, it is always used as a
criterion of economic evaluation of the product, since the colour is one of main
factors in consumer choice (9).
In the past the method of determining anthocyaninsin blood orange juices made
reference to those methods adopted for other fruit species as well as strawberries,
cranberries,etc. (10, ll, l2). Currently five spectrophotometricmethods are utilised
for determining anthocyaninsin blood orange juices. Three ofthem are described in
the literature (13, 14, l5), the remaining two are currently used in the Italian juice
processing factories (16). Methods are based on the measurementof absorbanceat
l,.r* of juice after dilution with an acidic solvent, but the procedure to obtain an
adjusted value of absorbance and to calibrate concentration of anthocyanins are
typical of each method.
Recently, Rapisarda et al. (17) have carried out an investigation in a series of
blood orange juices following various currently used methods, including HPLC
analysis as a reference point, in order to investigate the causes of the different
anthocyanin content, obtained for the same sample of juice. Spectrophotometric
methods utilising aqueous ethanol as a solvent furnished a higher anthocyanin
content than that determined by HPLC (13, 15, l6). Discrepancieswere ascribed to
the use of impure standards andlor unsuitable calibration lines. The most consistent
results with the HPLC finding were obtained by a method utilising water as a
solvent (pH differential method) and cyanidin-3-glucoside as standard (14). Finally,
the actual concentration of anthocyanins in the examined blood oranges juice was
remarkably lower than currently determined by procedures used in the methods
cited.
Paolo Rapisarda e/ a/.
220
OHH
Cyanidin-3-glucoside
Cyanidin-3-(6"-malonyl)-glucoside
tAl
tBl
Pigment
|
2
J
f
)
6
7
I
9
10
Retention time
[dentification
(min)
19.2
Delphinidin-3,5-diglucoside
zt,+
Cyanidin-3,5-diglucoside
24.4
Delphinidin-3-glucoside
26.8
Peonidin-3,5-diglucoside
30.2
Cyanidin-3-glucoside
Cyanidin-3-(6"-malonyl)-glucoside
46.6
Cyanidin-3-(ferulyl)- glucoside
51.6
Cyanidin-3-(coumaryl,ferulyl)-glucoside
56.9
58.3
Cyanidin-3-(sinapyl)-glucoside
Peonidin-3-(coumaryl)-glucoside
59.7
Fig. 2- HPLC of anthocyanins from Moro, Tarocco and Sanguinello oranges Modifled fiom
Maccarone et al. (7)
Anthocyanins in fresh fruits
Anthocyanins are mainly located in the skins of the fruits such as in apples, pears,
grapes, etc. In other casesthey are found in the skin and flesh (strawberries, peaches).
They accumulate in the vacuoles of epidermal and subepidermaltissue (18). In any case
their concentration gradually decreaseson passing from the outer layers to the more
inner layersof the fruits.
Blood oranges are distinct from other fruits speciesbecausealmost the total amount
of the anthocyaninsis found in the flesh and only in some varieties (Moro, Sanguinello,
'Vitale') is it to be found in
Sanguignodoppio) or in some clones of Tarocco ('Rosso',
221
Anthocyanins in blood oranges
the rind. The external coloration of blood
oranges is however very much influenced
"ì*{.
$0i
by climatic conditions and by light. In
ff$i:
fact, cold and dry winters can induce a
**i
n 3$$l
greater external pigmentation in the fruit.
X]
28i'
S
qi
Anthocyanin content in the fruit juice of
q r$,i'
the three most important cultivars of blood
s 1ù*i.
oranges, determined at the same degree of
F *i'
ai
maturation (total soluble solid, TSS / total
acidity, TA ratio of 7.5 and 10.0), always
r€.{
varies according to the following order:
3$rli'
Moro > Sanguinello> Tarocco (Fig. 3).
*, wa
the
internal
in
Differences
E rmi
pigmentation of fruit have also been
È r*i'
observed between old line (OL) and
? tE*.i'
{i
nucellar line (NL) in the same cultivar. In
{ m*i'
particular, the anthocyanin level was
* s*i''':
l :./
higher in Moro NL and Sanguinello NL
tl'f- :
than in the corresponding OL. The reverse
was observed for Tarocco (19). These
results have been confirmed in another
study in which the juice of 14 different Fig. 3- Comparison between anthocyanin level
cultivars and clones of Moro. Tarocco and in fruit juices of the three pigmented cultivars at
Sanguinello were analysed (20). The two TSS/TA values in NL and OL clones. From
highest anthocyanin content was observed Rapisardaand Giuffrida (19)
'm 27', Tarocco
in Moro NL, Moro
'Rosso' and Sanguinello oMoscato'
clones. Besides, anthocyanin content was
&
positively correlated with the chromaticity
6w
value a/b ratio determined in the juice
vffi
€e
with a Minolta CR-200 Chromameter.
X,*
Finally, in these works it has emergedthat
sq 1f*
anthocyanin formation is closely linked to
4
the degree of fruit ripening. Results of a
linear regtession between anthocyanin
i-:*i."41
content and TSS/TA ratio found in OL
in
Fig.
4.
Moro
are
reported
and NL
Studies on blood oranges h1y" Fig. 4 - Simple linear regressionbetween
continued in order to evaluate fruit quality TSS/TA and total anthocyanins. From
of commercial products. Five years of RapisardsandGiuffrida(I9)
analytic data on the fruit of Tarocco,
Moro and Sanguinello sampled in the market have allowed to individuate the ranges
within which the anthocyaninsand other quality parametersvary (Table 1).
Among the three pigmented varieties, the Tarocco orange is the most widespread
cultivar in Italy. The successof this cultivar in domestic and international markets is
owing to its exceptional organoleptic characteristics,its large size, the balanced levels of
J ì
Paolo Rapisarda e/ a/.
222
iaUie f - Qualiw parametervaluesrangeof blood orangessqrnp&{jl the rnarkglTotal Anthocyanins
TSS/TA Vitamin C
TA
TSS
(%\
(%)
(mellooml-)
Tarocco
min.
max.
med.
8.4
12.5
10.5
0.7
1.5
1.2
6.7
14.2
9.0
50.1
79.6
64.6
8.7
69.2
23.5
Moro
min.
max.
med,
8.4
12.5
10.5
1.1
t.7
1.4
6.1
9.4
7.5
41.4
62.8
49.8
56.8
186.2
I 19.1
min.
Sanguinello ma:(.
med.
8.2
12.8
10.6
0.9
1.5
1.1
6.8
12.0
9.2
48.4
62.5
55.5
ts.2
6t2
32.5
@c&)-
This
sugar and acids, the high vitamin C content and the presence of_anthocyanins.
number
a
modify
commonly
which
mutations
to
spontaneous
ru. is readily subject
Different clones
"oIti
of morphological and physiol'ogical characteristics of the plant (21)'
(22).
have been seÉcted and àescribed, a number of which have become widespread
at
addressed
At the moment researchinto genetic improvement of the cv. Tarocco is
with
seasons,
obtaining new selections characterisedby fruit with early or late ripening
of
ftgtr t"ót content and limited total acidity but, more importantly, with high levels
price
since
higher
a
earn
generally
fruit
uritho"y"unirrr.In fact, better-coloured Tarocco
they are demanded both for the fresh fruit market and processing ildustry' Furthermore,
proven
higler anthocyanin content positively influences consumer choice for their
(see
later).
vivo
and
in
in
vitro
bolh
antioxidant activity
'vitale" 'Tringale"
Fruit quality of five Tarocco selections respectively called
.Sciara', .da[o; and 'Rosso' were studied recently (23). Results performed at different
difference
dates, showed that anthocyanin levels increased during ripening with marked
among the single clones (Fig. 5).
In particular, in February the
{*^**
'Rosso' and 'sciara' clone fruits were
with
pigmented,
most
the
à
anthocyanin values in the juice
E
&$i I
in
while
between 60 and 80 mg/L
g
anthocYanin
the highest
March
r
'Tringale' and
content was found in
o oi :l
E
'sciara' clones with values of 110
i &
ii
'*ll
'*'l
--il
mglL.
The clone with the lowest
anthocyanin levels, in all samplings,
'Gallo' which reached a
was the
value of 40 mgll' onlY in March. In
addition, it has emerged from these
'Tringale' and 'Sciara'
data that only
clones provide juice with an ideal
?oll Fi;.XÌ
";d_*sd
,nx
,
:
r
'Vltale'
$
'Trln8ale'
FCg
Apft
l\ÀAg
i:l S{!a|a' il
'Gallo'
€l
ì
'Rîùso'
Fig. 5 - Change in anthocyanins values of different
Taiocco clones during ripening. From Rapisarda and
Russo(23)
Aathocyaninsin bloodoranges
coloration for processing, given that the
s.tandard level of anthocyanins in
coÍìmercial red juice is 90-100 mg/L.
Formation of anthocyanins in blood
oranges also continues after harvesting
when the fruits are stored at low
temperatures (24). Tarocco fruits of
different clones ('Gallo', 'Vitale',
'Scirè' and 'Meli') stored
at 8oC for 4
months, have shown a marked increase
in the anthocyanin concentration in the
flesh(Fig.6).
In the
223
gp
2fi
3rm
I
.Fro
$'r
I
{5s
,/
(r;'t'-..t;:
....'r"'l'^
. : ) a"'. . - |
;;;:i;;:'
0
01234
]lÍ€of€ba€6(Ítorúr)
+vrtde'
:
-+-'fi,bll' ...;...,Q|d - -r .,sdrè
'Vitale'
clone
the
- Change in anthocyanins values of
anthocyanins increase in the first two Fig. 6
different Tarocco clones fruits during storage at
months to then level off in the following
goc.
months, while in the other clones the
increase is progressive and occurs
2W
throughout the period of storage.
't80
Concomitant increases in the levels of
l r r4o
*
b
phenylalanine ammonia-lyase (PAL),
È t2o
the first enrpe
involved in the
.E 10o
oao
biosynthesis of the anthocyanins,
{.o
occurred during the first month of
le o
20
storage in all clones; after this time a
o
clear decreasewas observed(Fig. 7).
îma of stoilgc {mnth)
Further comparative studies made
-*-'ilrl'
+\,ibb
...r..,Gc[o' . .r .'SdrÈ'
using Tarocco and Moro oranges stored
at
8oC
have
higblighted
that
- Change in PAL activity of different
anthocyanin content rose by 500% in Fig. 7
Tarocco clones during storageat 8oC.
Tarocco whereas values for Moro only
increased bv 20oA. In both varieties the
highest production ofanthocyanins occurs after 40 days storage(25).
The factors determining anthocyanin biosynthesis in blood oranges are still unclear.
In a recent work, two fragments of genes coding the chalcone syntase (CHS) and
anthocyanidin syntase (ANS), two en4,mes which act at the beginning and end of the
anthocyanin biosynthetic pathway, were amplified and sequenced.The sequenceswere
very similar respectively to CHS of Citnts, Juglans nigra and Casuaria glauca and to
ANS of Malus and Petunia. In addition, a high expression of CHS was observed in the
flesh ofblood orangesand very little was found in blond oranges(26).
Anthocyaninsin Juice
Since the 1960's the citrus processing industry in Italy has been using blood orange
fruits for processing (27). At presenttwo types ofblood orangejuices are produced: 'not
from concentrated' (l.tpc) and 'concentrated' orange juice, the latter primarily
designated for the production of'reconstituted from concenkate' orange juice (RFC).
The quality of NFC and RFC blood orangejuice has widely been studied. Di Giacomo et
Paolo Raoisarda et al
al. (28) have analysed RFC juices
produced in two different regions of
southem Italy (Sicily and Calabria).
The results from the Sicilian juices
had a decisively higher anthocyanin
content than the Calabrian ones
(Table 2).
Another investigation carried out
on freshly squeezedand NFC orange
juices gave indications on the ranges
of the principle
of
variation
quality
parameters of
of these juices
(29). The influence of extraction
qualitative
on
technology
characteristics of blood orange juice
was studied by Arcoleo et al. (30).
Lastly, Di Mauro et al. (31)
reported the average trend of the
anthocyanin content, observed over a
Table 2 - Anthocyaninvalues (mgil) in
orange juices from two different regions
of Italv
'Calabria''
'Sicilv'
min.
max.
8.57
69.21
69.21
150,25
med.
24.74
t02.73
Modified from Di Giacomoet al. (28)
2m
180
1@
eilo
g 120
É,m
*ao
l'l 4 soo
.
..'í
'ff
...
. -
--l.k- .
.'.'\.
.
tt'
four year period, in the months of
n
production of blood orangejuice (Fig.
0
the
right
possible
to
note
that
8). It is
ilbr.
period in which the juice reaches an
íFntt
optimum coloration for the market
(90-100 mgll) is from February to
Fig. 8 - Monthly anthocyaninscontentin Sicilian
the whole of April.
orangejuices. Modified from Di Mauro et al.
blood
Since anthocyanins in blood
t
3
1
)
orangcs are very sensitive to
discoloration, much researchhas been
undertaken with the aim of understandingthe causesof colour degradation of red juice.
In particular, the effect of pH and temperature (32), of the addition of SO2 (33) and 5hydroxymethylfurfural (34), and treatment with enzymatic systems have been evaluated
(35).
The stability of the red colour of blood orange juice has somewhatbeen improved
by pasteurizationwith microwaves and addition of tartaric acid and gluthatione as mildly
acidic and antioxidant agents, respectively. Highest stability was obtained through the
formation of complexes between anthocyanins and phenolic compounds, such as rutin
and caffeic acid (Fig. 9) (36).In fact, it is well known that phenolic compounds form
intermolecular complex with anthocyanins (copigmentation) by hydrogen bonding and
charge transfer (37, 38, 39). This complex is also regarded as one of the significant
factors of anthocyanin stabilisation in vivo condition (40).
'tannic acid', a mixture of
The addition of different concentrations of commercial
polyphenols with about T\Yopenta-meta-digalloyl-glucose,retarded loss ofanthocyanins
in the blood orange juice (41). In particular the half-lives were 50, 70 and 90 days in
presenceof 0. I , 0.2 and 0.3% of additive, respectively, in comparison with the 40 days
ofthe referenceiuice.
Anthocyaninsin bloodoranges
225
Recent studies on degradation of
anthocyanins of freshly squeezed and RFC
orange juices during storage, have shown that
reaction
between
anthocyanins
and
intermediates of degradation of sugar and
ascorbic acid are the main cause of formation
of brown-red polymers in juice (42).
The effects of storage time and
temperature on the quality of NFC juice was
investigated by Trifirò et al. (43). They found
that anthocyanin in the juice stored at 18 and
30 oC degraded more rapidly than in the
sample stored at 3"C. However, all parameters
describing red colour (anthocyanins, Gardner
colour values) degradeaccording to I't order
kinetics.
Thermal treatments of blood orange juice
cause a significant modification in distribution
Fig. 9 - (A) anthocyanin-rutincomplex; of original constituents and a partial
(B) anthocyanin-caffeicacid complex. transformation of some of them. In parlicular
alteration of the sensory profile by loss of
From Maccaroneet al. (36)
aroma components(44) and generation of offflavour (45) occur during storage of the processedjuice. Also a change of colour was
observed and this modification was ascribed to degradation of anthocyanins.
Experimental results carried out by Arena et aL @6) have demonstrated, instead, that
alteration ofthe original colour ofblood orangejuice after thermal processing is not due
to the degradation ofanthocyanins but to the different distribution ofcarotenoids (yellow
pigments) between serum and pulps and to the modification of pulp particle size.
Only a slight decrease(13%o)in the anthocyanins level was observed passing from
freshly squeezedto RFC orangejuices (47).
The stability of blood orange anthocyaninshas also been studied in model systems.
Degradation profiles at different values of pH and in the presence of glucose,
ascorbic acid, 3,4-dihydroxybenzoic acid, caffeic acid, asparagine, quercetin-3rutinoside, cysteine and a number of their mixtures, have revealedthat the degradation of
the anthocyanins is primarily due to ascorbic acid which interacts with these pigments
forming non-coloured products. Furthermore, the presence of caffeic acid and cysteine
has improved the stability of the solutions of cyanidin-3-glucoside containing ascorbic
acid (48). Other studies aimed at determining the effects of phenolic copigments on the
flavylium/carbinol equilibrium of anthocyanins, point out that the copigment works by
reducing the equilibrium constant of the flavylium/carbinol system,thus recoveringpart
ofthe latent colour stored in colourless carbinol (39).
The effects of light on anthocyanins are usually deleterious (49). Maccarone et al.
(50) reported that, in acidic aqueous solution, 3,S-diglucosidesare more stable than the
3-monoglucosidesunder UV and visible light. In addition, photolysis of cyanidin yields
3,4-dihydroxyberuoic acid and 2,4,6,-trihydroxybenzaldehyde. These results indicate
that the photodegradation products derive from chalcone species present at the
equilibrium of anthocyaninsin acidic aqueoussolution (Fig. 10).
226
Paolo Raoisarda et a/.
The stability ofblood oranges
anthocyanins in carbonated and
ethanolic beverage was adversely
influenced by storage temperature
and exposureto daylight (51).
HrO
-
rr
Anthocyanins from byproduct
Extraction of anthocyanins
from pulp wash liquors or juice
and the use ofthis extract as a food
health-promoting
colorant
or
substances have recently been
studied (52,53\.
OH
îIl l
oH
l't ,\Y""
HoìA/'\42
l(
A
| .
ll
\l
I
I
\4o,
Chalcone
Útatewltow)
oHr
îro
'ov+oH
î*t
"4"
l( A Jl
Y
OH
2,4,6,-trilrydroxybenzaldehyde
l\
l\ B /l
Yr*
OH
3,4-dihydroxybenzoic
acid
Fig. 10 - Equilibrium of cyanidin in aqueousacid
solution and degradationproduct afterphotolysis
The process described by
Calvarano et al. (53) entails the
extraction of anthocyanins from
the pulp with an ethanol-water
mixture containing 20À of citic
acid, removal of the alcohol by
distillation and ultrafiltration of the
aqueous phase. Pigments afe,
successively, adsorbed on nonionic resin, and eluted by ethanolwater at loA of citric acid. After
distillation of the alcohol and
spray-drying
of the aqueous
solution, a powder product rich in
anthocyaninsis obtained (Fig. I 1).
production
the
The
of
anthocyanin extracts from the byproducts
of
blood
oranges
processing (prrlp, peel, secondpressure juice) to be used in the
food or pharmaceutical industries
opens new economic prospects for
processing
in
ltaly,
citrus
these new
that
considering
products may be sold at high
prices.
Biological activity
with potential
Citrus fruits are recognisedas importantsourcesof phytochemicals
health benefits. In addition to the vitamin C. these include flavonoids,limonoids,
carotenoids
andphenolicacids(54).
in bloodoranges
Anthocyanins
227
Citrus flavonoids have been
reported to have several biological
effects such as antioxidant (55),
anticarcenogenic and antitumor (56,
57, 58), antiviral (59) and arfiiinflammatory activities (60, 6l).
Water
+
Bthanol
2o/ocitic
acid
Fig. 11 - Schemefor production ofblood oranges
anthocyanins extract.
Four types of flavonoids are to be
found in citrus fruits. These are
flavanones, flavones, flavonols and
anthocyanins, with the latter present
only in blood oranges(62).
Anthocyanins have also been
associated with potentially beneficial
effects on various diseases such as
(63),
fragility
diabetic
capillar
retinopathy (64), human platelet
aggregation (65). Many of these
biological properties of anthocyanins
have been correlated with their
antioxidant activity. Recent studies
have shown that cyanidin-3-glucoside,
the most abundant anthocyanin of
blood oranges, had the highest
antioxidant activitv between the 14
tested(66). In addition,cyanidinwas found
and anthocyanidins
coÍrmon anthocyanins
to havean antioxidantactivity 4.5 timeshigherthanvitaminC (67) while, in the rat liver
and cyanidinexhibitedstrongeractivity than
microsomalsystem,cyanidin-3-glucoside
tocopherol(68).
Fruits and vegetablesare the
dietary
sources
of
main
antioxidant substances such as
C,
carotenoids,
vitamin
flavonoids and other phenolic
compounds. After processing,
these compounds are distributed
between the juice and byproducts.
When blood oranges are
processed, almost the total
amount of anthocyanin and some
part
of
flavanones,
and
hydroxycinnamic
acids
vitamin C are found in the juice.
The presence ofthe anthocyanins
and high levels of vitamin C,
flavonoids and hydroxycinnamic
-&
g
&
.9,
d
f{
0
!fllturo
$TsÈo*(E
rdèru
w
: r$$*$hfns{nnrÀel n v*bflsià lató
juwrulu
Fig. 12 - Total antioxidantactivity (TAA) estimated
methodu.
usingthe ferrylmyoglobin/ABTS
"(Rice Evans C. and Miller, N. J. 1994. Methods
et al. (69)
Enzymol.,234,279).ModifiedfromRapisarda
Paolo Rapisarda et a/.
228
acids gives blood orangejuices a greater antioxidant activity with respect to the orange
juice
lom the blond v-arieties.In particular, Rapisarda et al. (69) have found that the
-antioxidant
activity, assessedby means of different in vitro tests, of blood varieties
Moro, Tarocco and Sanguinello orange juice, was higher than the Valencia late and
. Furthermore, the antioxidant effrciency of
Washlngon navel blond varieties.
blood oianges juice appearsto be widely influenced by the anthocyanin level' In fact,
orange juici with higÀè. anthocyanin content was a better antioxidant than those with
lowei anthocyanin iontent (Fig. l2). Total antioxidant activity (TAA) was also
juice
determined inNFC and RFC blood orange juice, coming from the same batch of
before and after the process of concentration respectively, as well as during storage at
two different temperatures (2" and 20"C) (70). The results have highlighted that TAA
remains unchanged after the concentration process ofjuice and during storage at the two
temperaturescited.
In vitro antioxidant activity and in vivo photoprotective effect of a red (blood)
extract (ROE) containing 3.1% of anthocyanins, 2'070À of
orange juice
(caffeic, ferulic,p-coumaric, sinapic acids),8.1% of flavanones
acids
hydróxycinnamic
giycosides (narirutin and esperidin) and 5.0oÀ of ascorbic acid have been studied by
Bonina et al. (71).
The results obtained in the in vitro tests demonstrated the strong antioxidant
properties of ROE, with a clear relationship between ROE scavengerefficiency and its
lottt"ttt in antioxidant components. During in vivo experiments, ROE proved to
efficiently protect against photooxidative skin damage when topically appliedimmediatelyafter skin exposuie to UVB radiation. Interestingly, the protective effect of
ROE appeared greater than that elicited by another natural antioxidant (tocopherol)
.o*-only employed in cosmetic formulations. The same ROE was used in in vitro lests
in order io uri.ri the protective effect on two human skin-derived cells lines (NCTC
2544 keralinocytes and IIFFF2 fibroblast) subjected to chemical stress such as ironinduced lipid peroxidation. The results showed thal a significant inhibition of
malondialdehyaè GrlO,! was observed in cells treated with ROE (72). Another
interesting reiearch conducted on a group ofapparently healthy smokers has shown that
the supplÉmentationof diet with ROE significantly augmented serum levels of thiolic
groups
thereby reducing the oxidative stressin all the volunteers'
Therefore ROE mày be considered as a useful antioxidant for smokers (73). Finally,
juice have been studied in in
pharmacological aspectsof anthocyaninsof Moro orange
introduction of Moro orange
the
that
have
shown
(74).
They
vivo model by Saija eI al.
juice into the-diet-can modulate the permeability of the blood vessel wall and induce a
protective effect on gastric mucosa. In addition, it seemsto elicit an immunostimulatory
effect.
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