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Chem Sci Trans.

, 2012, 1(2), 303-310 Chemical Science Transactions


DOI:10.7598/cst2012.182 ISSN/E-ISSN: 2278-3458/2278-3318
Ascorbic Acid, Total Phenol Content and Antioxidant Activity
of Fresh Juices of Four Ripe and Unripe Citrus Fruits
C. REKHA, G. POORNIMA, M. MANASA, V. ABHIPSA, J. PAVITHRA DEVI,
H T. VIJAY KUMAR

and T R. PRASHITH KEKUDA
*

Department of Biochemistry, SRNMN College of Applied Sciences, Balraj Urs Road,
Shivamogga-577201, Karnataka, India
*
Department of Microbiology, SRNMN College of Applied Sciences, Balraj Urs Road,
Shivamogga-577201, Karnataka, India
p.kekuda@gmail.com
Received 16 May 2012 / Accepted 4 June 2012
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 damage
1
. 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 chelators
2,3
.
RESEARCH ARTICLE
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 cancer
4,5
.
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 beneficial
6
. 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 stress
7
. 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 meter
8
.
Chem Sci Trans., 2012, 1(2), 303-310 305
Estimation of Ascorbic acid content
Ascorbic acid content in fruit juices was estimated by volumetric method
9
. 5 mL of
standard ascorbic acid (100 g/mL) was taken in a conical flask containing 10 mL 4%
oxalic acid and was titrated against the 2,6-dichlorophenol indophenols dye. The
appearance and persistence of pink colour was taken as end point. The amount of dye
consumed (V
1
mL) is equivalent to the amount of ascorbic acid. 5 mL of sample
(prepared by taking 5g of juice in 100 mL 4% oxalic acid) was taken in a conical flask
having 10 mL of 4% oxalic acid and titrated against the dye (V
2
mL). The amount of
ascorbic acid was calculated using the formula, Ascorbic acid (mg/100 g) = (0.5
mg/V
1
mL) (V
2
/15 mL) (100 mL/Wt. of sample) 100.
Estimation of total phenolic content
The phenolic content in the fruit juices was estimated by Folin-Ciocalteu method
9
. 0.5 mL of
each fruit juice was mixed with 2.5 mL of distilled water. To this, 0.5 mL of F-C reagent
(1:1) was added and incubated for 3 minutes. To each tube, 2 mL of 20% sodium carbonate
was added and the tubes were kept in boiling water bath for 1 minute. Tubes were cooled
and the absorbance of reaction mixture was read at 650 nm. A standard curve was plotted
using different concentrations of Gallic acid (standard, 0-1000 g/mL). Total phenolic
content was estimated as g Gallic acid equivalents (GAE)/mL of fruit juice.
Antioxidant activity of fruit juices by DPPH free radical scavenging assay
The radical scavenging ability of fruit juices was tested on the basis of the radical
scavenging effect on the DPPH free radical. The fruit juices (12.5 L to 100 L/mL) were
prepared in methanol. In clean and labeled test tubes, 2 mL of DPPH solution (0.002% in
methanol) was mixed with 2 mL of different concentrations of fruit juices separately. The
tubes were incubated at room temperature in dark for 30 minutes and the optical density was
measured at 517 nm using UV-Vis Spectrophotometer. The absorbance of the DPPH control
was also noted. The scavenging activity of the juices was calculated using the formula:
Scavenging activity (%) = [(A B) / A] x 100, where A is absorbance of DPPH and B is
absorbance of DPPH and fruit juice combination
10
.
Antioxidant activity of fruit juices by ferric reducing assay
Fruit juices (12.5 L to 100 L/mL) in 1 mL of methanol were mixed in separate tubes with
2.5 mL of phosphate buffer (200 mM, pH 6.6) and 2.5 mL of 1% potassium ferricyanide.
The tubes were placed in boiling water bath for 20 minutes at 50
o
C, cooled rapidly and
mixed with 2.5 mL of 10% trichloroacetic acid and 0.5 mL of 0.1% ferric chloride. The
amount of iron (II)-ferricyanide complex formed was determined by measuring the
formation of Perls Prussian blue at 700 nm after 10 minutes. The increase in absorbance of
the reaction mixtures indicates increased reducing power
10
.
Results and Discussion
Total acidity and pH of fruit juices
The pH of citrus fruit juices (10%) ranged from 3.50 to 4.33 and was found to be lesser in
case of unripe fruits when compared to ripe fruits. (pH of C. aurantium < C. limon
< C. reticulata < C. sinensis). The total acidity was found to be higher in C. limon juice
followed by C. aurantium, C. sinensis and C. reticulata. In all fruit juices, the total acidity
was higher in unripe fruits when compared to ripe fruits (Table 1).
306 Chem Sci Trans., 2012, 1(2), 303-310
Table 1. pH and total acidity of citrus fruit juices

Ascorbic acid content of citrus fruit juices
The ascorbic acid content in the fruit juices, as estimated by volumetric method, ranged from
6.34 to 26.01 g/100 mL (Table 2). Unripe fruits contained high ascorbic acid when
compared to ripe fruits. Among fruits, high ascorbic acid content was observed in
C. aurantium followed by C. sinensis, C. limon and C. reticulata.
Table 2. Ascorbic acid content of citrus fruit juices
Citrus fruit Ascorbic acid, g/100 mL
C. limon
Ripe 10.60
Unripe 12.70
C. reticulata
Ripe 06.34
Unripe 07.41
C. sinensis
Ripe 17.40
Unripe 19.04
C. aurantium
Ripe 24.90
Unripe 26.01
Total phenolic content of citrus fruit juices
F-C method was employed to estimate phenolic content. The total phenolic content ranged
from 532 to 960 g GAE/mL of fruit juice. High phenolic content was observed in unripe
fruits. Among Citrus fruits, high phenolic content was observed in C. sinensis followed by
C. reticulata, C. limon and C. aurantium (Table 3).
Table 3. Total phenolic content of citrus fruit juices
Citrus fruit TPC (g GAE/mL)
C. limon
Ripe 600
Unripe 760
C. reticulata
Ripe 800
Unripe 850
C. sinensis
Ripe 820
Unripe 960
C. aurantium
Ripe 532
Unripe 620
DPPH free radical scavenging activity of citrus fruit juices
Free radical scavenging activity of juices of citrus fruits was determined by DPPH free
radical scavenging assay. DPPH is a stable free radical having maximum absorption at
517 nm that accepts an electron or hydrogen atom to become a stable diamagnetic molecule.
Citrus fruit pH Total acidity, g/100 mL
Ripe
Unripe
4.0 3.328
3.6 3.404
Ripe
Unripe
4.28 0.563
4.20 0.588
Ripe
Unripe
4.33 1.024
4.03 1.075
Ripe
Unripe
3.9 2.816
3.5 2.920
Chem Sci Trans., 2012, 1(2), 303-310 307
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 nm
2
. 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
Concentration,
in L
% Scavenging activity
C. limon C. reticulata C. sinensis C. aurantium
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
Concentration,
in L
Absorbance at 700nm
C. limon C. reticulata C. sinensis C. aurantium
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 activities
11
. 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 properties
11,12
.
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 activity
6
. 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
in foodstuffs and biological fluids. Fluorimetric methods and UV-VIS absorbance-based
determinations were also used for ascorbic acid estimation
13
. In this study, the ascorbic acid
was estimated by volumetric method. Ascorbic acid reduces the 2,6-dichlorophenol
indophenol dye to a colourless leuco-base. The ascorbic acid gets oxidized to
dehydroascorbic acid. Though the dye is blue coloured compound, the end product is the
appearance of pink colour. The dye is pink coloured in acid medium. Oxalic acid is used as
the titrating medium
9
. All fruit juices, tested in this study, have been found to contain
ascorbic acid. Antioxidant activity was found to be higher in fruit juices containing high
ascorbic acid (except in case of C. aurantium) and this is in accordance with the result
obtained by Gardner et al.
6
and Zvaigzne et al.
7
. Gardner et al.
6
showed that vitamin C is the
main antioxidant in orange juice but not the major antioxidant contributor in apple juice.
The candidate phenolic antioxidants in foods include flavonoids, anthocyanins,
catechins, chalcones and hydroxybenzoic and hydroxycinnamic acids. Many of these are
found to be present in fruit juices
6
. In this study, the total phenolic content of the citrus fruit
juices was estimated by Folin-Ciocalteu method. Phenols react with an oxidizing agent
phosphomolybdate in F-C reagent under alkaline conditions and result in the formation of
blue coloured complex, the molybdenum blue which is measured at 650 nm colorimetrically
9
.
In our study, the antioxidant activity of fruit juices was shown to be influenced by the total
phenolic content also. Fruit juices containing high phenolic contents have been found to
exert high antioxidant potential. The study of Ghafar et al.
11
has shown a direct relation
between antioxidant activity of citrus species and phenolic contents.
A well maintained balance exists between production of free radicals and antioxidant
defense mechanisms in a healthy individual. In pathophysiological conditions, this balance
shifts towards overproduction of free radicals or lowering of antioxidant defenses results in
oxidative stress. Oxidative stress is excess formation and/or incomplete removal of highly
reactive molecules such as ROS, including superoxide radical, hydroxyl radical etc. as well as
nonradical species such as hydrogen peroxide. Free radicals are implicated in the etiology of
several degenerative diseases such as stroke, coronary artery diseases, rheumatoid arthritis,
diabetes and cancer. These radicals are capable of damaging essential biomolecules such as
proteins, DNA and lipids. In case of aerobic organisms, cell membrane is the major target for
ROS where lipid peroxidation is induced. Membrane structure and function are affected and
some oxidation products, such as malondialdehyde, are produced which react with
biomolecules and exert cytotoxic and genotoxic effects. Endogenous antioxidants such as
vitamin C, vitamin E, etc act as primary defense systems. However, in diseased states, there is
an extra requirement for antioxidants from exogenous sources. Cells have several defense
mechanisms, for prevention of adverse effects of ROS, which includes antioxidative enzymes
(such as superoxide dismutase, catalase and glutathione peroxidase) and small molecules such
as vitamin C and E. Substances termed antioxidants can influence the oxidation process
through simple or complex mechanisms including prevention of chain termination, binding to
transitional metal ion catalyst, decomposition of peroxides, prevention of continued hydrogen
abstraction and radical scavenging. They reduce oxidative damage and prevent or alleviate
chronic diseases
14-16
. In recent years, there is an increasing interest in finding antioxidants from
natural origin. The most effective antioxidants are flavonoids and other phenolic compounds
present in plants particularly in herbs, seeds and fruits
17
.
DPPH free radical scavenging method is a widely used method to evaluate the free
radical scavenging ability of various samples. The DPPH radical is stable organic free
radical with absorption maximum band around 515 to 528 nm. In this assay, the antioxidants
Chem Sci Trans., 2012, 1(2), 303-310 309
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 radical
16,17
. 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 Fe
3+
to Fe
2+
by donating an electron. The amount of Fe
2+
complex formed by the reduction of Fe
3+
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 reactions
16,17
. 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 ions
4,19
.
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 juices
20
.
Acknowledgement
Authors express sincere thanks to Principal, SRNMN College of Applied Sciences and NES,
Shivamogga for providing all facilities to conduct work.
References
1. Choi Y, Jeong H S and Lee J, Food Chem., 2007, 103, 130-138.
2. Kaviarasan S, Naik G H, Gangabagirathi R, Anuradha C V and Priyadarshini K I,
Food Chem., 2007, 103, 31-37.
3. Yen G C, Duh P D and Su H J, Food Chem., 2005, 89, 379-385.
310 Chem Sci Trans., 2012, 1(2), 303-310
4. Sarkar N, Srivastava P K and Dubey V K, Curr Nutri Food Sci., 2009, 5, 53-55.
5. Lee S K and Kader A A, Postharvest Biology Technology, 2000, 20(3), 207220.
6. Gardner P T, White T A C, McPhail D B and Duthie G G, Food Chem., 2000, 68,
471-474.
7. Zvaigzne G, Karklina D, Seglina D and Krasnova I, Chemine Technologija, 2009,
3(52), 56-61.
8. Khosa M A, Chatha S A S, Hussain A I, Zea K M, Riaz H and Aslam K, J Chem Soc
Pak., 2011, 33(2), 188-192.
9. Thimmaiah S K, Standard Methods of Biochemical Analysis, Kalyani Publishers,
Noida, 1999.
10. Kekuda P T R, Shobha K S and Onkarappa R, J Pharm Res., 2010, 3(1), 26-29.
11. Ghafar M F A, Prasad K N, Weng K K and Ismail A, Afr J Biotechnol., 2010, 9(3),
326-330.
12. Garg A, Garg S, Zaneveld L J and Singla A K, Phytotherapy Research, 2001, 15(8),
655-669.
13. Pisoschi A M, Pop A, Negulescu G P and Pisoschi A, Molecules, 2011, 16, 1349-1365.
14. Harish R and Shivanandappa T, Food Chem., 2006, 95(2), 180-185.
15. Chatterjee S, Poduval T B, Tilak J C and Devasagayam T P A, Clinica Chimica Acta,
2005, 352, 155-163.
16. Ardestani A and Yazdanparast R, Food Chem., 2007, 104, 21-29
17. Chung Y, Chien C, Teng K and Chou S, Food Chem., 2006, 97, 418-425.
18. Ali M Y, Nayan V, Chanu K V, Ralte L and Devi L I, World J Agricultural Sci.,
2011, 7(3), 327-332.
19. Berg J M, Tymoczko J L and Stryer L, Biochemistry, W.H.Freeman and Company,
New York, 2002.
20. Jacob-Wilk D, Holland D, Goldschmidt E E, Riov J and Eval Y, The Plant J., 1999,
20(6), 653-661.

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