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RESEARCH ARTICLE
Abstract: The ascorbic acid and total phenolic content to antioxidant activity of fresh juices of four
ripe and unripe citrus fruits namely Citrus limon, C. reticulata, C. sinensis and C. aurantium were
compared. The fruits were collected from local market and the pulp and seed free juices were
collected. The pH and total acidity were determined. Ascorbic acid content and total phenolic content
of fresh fruit juices were determined by volumetric and Folin-Ciocalteu reagent method respectively.
Antioxidant activity of fruit juices was determined by two in vitro assays namely DPPH free radical
scavenging assay and Ferric reducing assay. The pH was lesser in unripe fruit juices. Acidity, ascorbic
acid and total phenolic contents were high in unripe fruit juices. Ascorbic acid and total phenolic
content was high in C. aurantium and C. sinensis respectively. In DPPH assay, C. limon, C. reticulata
and C. sinensis exhibited stronger scavenging potential when compared to C. aurantium. Ferric
reducing potential was higher in C. sinensis followed by others. Overall, unripe fruit juices have
displayed stronger antioxidant activity when compared to ripe fruit juices. In this study, the antioxidant
activity of fruit juices was shown to be directly related to the content of ascorbic acid and total phenolics
except in case of C. aurantium. The lower antioxidant activity of ripe fruit juices could be due to the
possible reduction in the ascorbic acid and total phenolic content during ripening.
Keywords: Citrus, Ascorbic acid, Antioxidant activity, Total phenolic, Folin-Ciocalteu method
Introduction
Studies have revealed that increased consumption of grains, fruits and vegetables is
associated with reduced risk of diseases. This may be attributed to the presence of natural
antioxidants such as vitamin C, tocopherols, carotenoids, polyphenolics and flavonoids
which prevent free radical damage1. The plant phenolics are commonly present in fruits,
vegetables, leaves, nuts, seeds, barks, roots, etc. The antioxidant property of phenolics is
mainly due to their redox properties. They act as reducing agents, hydrogen donors, singlet
oxygen quenchers and metal chelators2,3.
304 Chem Sci Trans., 2012, 1(2), 303-310
Vitamin C (Ascorbic acid) is the most important vitamin in fruits and vegetables.
Except human and other primates, most of the phylogenetically higher animals can
synthesize vitamin C (L-ascorbate). More than 90% of the vitamin C in human diets is
supplied by fruits and vegetables (including potatoes). Vitamin C is defined as the
generic term for all compounds exhibiting the biological activity of L-ascorbic acid.
Ascorbic acid is the principal biologically active form but L-dehydroascorbic acid, an
oxidation product, also exhibits biological activity. Vitamin C is required for the
prevention of scurvy and maintenance of healthy skin, gums and blood vessels. It
functions in collagen formation, absorption of inorganic iron, reduction of plasma
cholesterol level, inhibition of nitrosoamine formation, enhancement of the immune
system, and reaction with singlet oxygen and other free radicals. As an antioxidant, it
reportedly reduces the risk of arteriosclerosis, cardiovascular diseases and some forms
of cancer4,5.
The consumption of fruit juices is beneficial and the health effects of fruits are
ascribed, in part to ascorbic acid, a natural antioxidant which may inhibit the
development of major clinical conditions including cardiovascular diseases and cancer.
Many fruit juices also contain phenolic compounds and carotenoids which have
antioxidant potential and intake of such fruits is beneficial6. Citrus species (Rutaceae),
the most popular fruits, originated in South-East Asia and then gradually spread to
different parts of the world. These fruits contain a variety of sugars, citric acid, ascorbic
acid, carotenoids, minerals, essential oils, etc and play an important role in human
nutrition as excellent source of antioxidants (ascorbic acid, carotenoids and phenolic
compounds). These constituents are considered to be essential components of functional
foods. Many of these substances prevent damage to cell membrane and other structures
by neutralizing free radicals. Ascorbic acid is the most important antioxidant in citrus
fruit juices and it protects the organism from oxidative stress7. The objective of the
present study was to relate the content of ascorbic acid and total phenolics to the
antioxidant potential of four unripe and ripe Citrus fruits namely Citrus limon,
C. reticulata, C. sinensis and C. aurantium.
Experimental
The ripe and unripe Citrus fruits namely Citrus limon, C. reticulata, C. sinensis and
C. aurantium were purchased from the local market of Shivamogga city, Karnataka.
Preparation of juice
The fruits were washed thoroughly in water. The juices were extracted by cutting the fruits
in half and carefully squeezing to extract juices. The collected juices were filtered through
4-fold muslin cloth and the pulp free juice was collected in clean containers.
Determination of total acidity and pH
Total acidity of the juices was determined by titration method. 10% fruit juices were
prepared by taking 10 mL juice and making up to 100 mL using distilled water in a
volumetric flask. 10 mL each of the juices were titrated against 0.1N NaOH (Standardized
using standard Oxalic acid) using Phenolphthalein indicator. The end point was noted (the
colour changed from colorless to pale pink). Total acidity was calculated in terms of citric
acid using formula, Acidity (g/100 mL) = Normality of the juice x Equivalent weight of
citric acid. The pH of 10% juices was determined using pH meter8.
Chem Sci Trans., 2012, 1(2), 303-310 305
In the presence of a substance capable of donating an hydrogen atom, its free radical
nature is lost and hence the reduction in DPPH radical was determined by the decrease in its
absorbance at 517 nm2. A dose dependent scavenging of free radical was observed and the
unripe fruit juices have demonstrated strong scavenging activity when compared to ripe fruit
juices. Among citrus fruits, C. sinensis, C. limon and C. reticulata have shown almost
similar scavenging activity when compared to C. aurantium (Table 4).
Table 4. DPPH scavenging activity of citrus fruit juices
% Scavenging activity
Concentration,
C. limon C. reticulata C. sinensis C. aurantium
in µL
Ripe Unripe Ripe Unripe Ripe Unripe Ripe Unripe
100 95.0 98.8 96.6 98.8 97.5 98.7 88.8 89.5
50 92.5 97.7 95.5 97.7 96.7 97.7 80.6 84.3
25 90.0 95.5 77.7 96.6 93.7 96.2 71.1 76.2
12.5 86.25 80.0 68.8 61.1 90.0 95.0 57.7 60.5
Ferric reducing activity of citrus fruit juices
In order to determine reducing potential of fruit juices, we have employed ferric reducing
assay. In this, the absorbance of the reaction mixture at 700 nm was found to increase with
the increase in concentration of fruit juices which indicates reducing potential of juices. The
highest reducing potential was shown by unripe fruits. Reducing activity was more in
C. sinensis followed by C. reticulata, C. limon and C. aurantium (Table 5).
Table 5. Ferric reducing activity of citrus fruit juices
Absorbance at 700nm
Concentration,
C. limon C. reticulata C. sinensis C. aurantium
in µL
Ripe Unripe Ripe Unripe Ripe Unripe Ripe Unripe
100 0.58 0.76 1.06 1.77 1.73 1.92 0.52 0.65
50 0.52 0.68 0.98 1.02 1.02 1.19 0.46 0.58
25 0.28 0.44 0.70 0.96 0.54 0.74 0.26 0.43
12.5 0.18 0.31 0.49 0.72 0.47 0.35 0.15 0.30
The genus citrus belongs to the family rutaceae and comprises of 40 species
distributed in India, China, Malaysia, Sri Lanka and Australia. Citrus is one of the most
important fruits being consumed fresh or as juice due to high nutritional value and flavor.
Citrus fruits have been shown to possess anti-inflammatory, antioxidant, antitumor and
antifungal activities11. Flavonoids are most important natural phenolics. These possess broad
spectrum chemical and biological properties including radical scavenging properties.
Hesperidin is a member of flavonone group of flavonoids. It is commonly found in Citrus
species. It is reported to possess antioxidant, anticarcinogenic, antihypotensive and
antimicrobial properties11,12.
Ascorbic acid is highly bioavailable and is consequently the most important water
soluble antioxidant vitamin in cells, effectively scavenging reactive oxygen species (ROS).
When relating the antioxidant activities of fruit juices to health and disease risk, it is
important to consider the contribution of ascorbic acid in addition to that of phenolic
compounds with antioxidant activity6. Traditional methods for ascorbic acid assessment
involve titration with an oxidant solution: dichlorophenol indophenol (DCPIP), potassium
iodate or bromate. Chromatographic methods, particularly HPLC with electrochemical
detection has turned out to be a selective and sensitive method for ascorbic acid assessment
308 Chem Sci Trans., 2012, 1(2), 303-310
reduce the DPPH radial (purple colour) to a yellow coloured compound, diphenylpicryl-
hydrazine. The extent of colour change depends on hydrogen donating ability of the
antioxidants. It has been documented that ascorbic acid, tocopherol, cysteine, glutathione,
gallic acid and few other compounds can reduce and decolourise DPPH radical16,17. Radical
scavenging activities of citrus juices have been investigated. In a study by Zvaigzne et al.7, it
was found that the highest radical scavenging activity was shown by freshly squeezed
orange juice and reconstituted grapefruit juice. Ali et al.18 showed DPPH radical scavenging
and ferric reducing antioxidant potential of fruits including few citrus fruits. The citrus fruit
extracts have shown scavenging and reducing potential. In this study, a dose dependent
scavenging of DPPH free radical by fruit juices was observed. Least scavenging activity was
observed in C. aurantium.
In the reducing power assay, the presence of antioxidants (reductants) in the sample
would result in the reducing of Fe3+ to Fe2+ by donating an electron. The amount of Fe2+
complex formed by the reduction of Fe3+ can be monitored by measuring the formation of
colour complex at 700 nm. Increase in absorbance indicates an increase in reductive ability.
The reducing capacity of compound may serve as a significant indicator of its potential
antioxidant activity. This would have the effect of converting free radicals to more stable
products and thus terminating free radical initiated chain reactions16,17. In this study, the
juices have been shown to cause reduction in a dose dependent manner. Juices of
C. aurantium showed least reducing potential when compared to other fruit juices.
Vitamin C is not used as a co-enzyme but is required for the continued activity of
enzyme prolyl hydroxylase which synthesizes 4-hydroxyproline, an amino acid that is
required in collagen. The enzyme prolyl hydroxylase has iron at its active site. The ferrous
state of iron is necessary of the activity of prolyl hydroxylase. By reducing iron atom to its
ferrous state, Vitamin C maintains the enzyme in active form. Ascorbate is oxidized to
dehydroascorbic acid. Thus, ascorbate acts as a specific antioxidant by reducing ferric ions4,19.
In the case of all fruit juices selected, unripe fruits showed higher antioxidant activity in
both DPPH scavenging assay and ferric reducing assay than the ripe fruits. This is due to the
changes that occur during the process of fruit ripening. Unripe fruits have high amount of
ascorbic acid, phenolics, starch, chlorophyll, pectins, acids and organics. During ripening, a
phytoharmone ethylene is released which activates the transcription genes for the synthesis
of various enzymes which degrade the phytocontituents and involve in ripening process. The
metabolism speeds up and also a number of free radicals are generated. Antioxidants like
ascorbic acid, phenolics, etc. come for the rescue and detoxify the free radicals generated
into harmless reduced substances. Thus, there is a reduction in the ascorbic acid, phenolic
and other antioxidant contents in the fruits while ripening as a result of which the
antioxidant activity of ripe fruit juices is comparatively less than that of unripe fruit juices20.
Acknowledgement
Authors express sincere thanks to Principal, SRNMN College of Applied Sciences and NES,
Shivamogga for providing all facilities to conduct work.
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