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Studies on Ascorbic Acid (Vitamin-C) Content in Different Citrus Fruits and its Degradation During Storage

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Vitamin-C which is chemically L-ascorbic acid is an essential nutrient for human body. Vitamin-C is required for various metabolic functions and its deficiency cause scurvy. Recent studies have clearly indicated antioxidant properties of Vitamin-C and hence its role for human has increased manifold. It is known that citrus fruits are good source of Vitamin-C and because of our fast changing lifestyle packed and stored fruit juice have become very common and they are being administered for therapeutic and nutritional purpose as well. The present investigation has revealed gradual degradation of vitamin-c content present in various citrus fruits even it is stored under refrigeration condition (4-5 0 c). The reduction of Vitamin-C varies from one fruit to other. Hence it is always suggested to consume fresh juice rather than the packed and stored one. V itamin-C or L-ascorbic acid or simply Ascorbate is an essential nutrient for humans and certain other animal species. Vitamin-C is required for a range of essential metabolic reactions in all animals and plants. Deficiency of this vitamin causes the disease scurvy. The biological role of ascorbate is to act as a reducing agent. Vitamin-C is absorbed in the body by both active transport and simple diffusion. Ascorbate concentrations over renal threshold pass freely into the urine and are excreted out. As Vitamin-C is needed for the growth and repair of tissues in all parts of our body and it is used to form an important proteins used to make skin, tendons, ligaments and blood vessels, heal wounds and form scar tissues, repair and maintaining cartilage, bones and teeth. It is one of the many antioxidants, which removes free radicals and stimulate the immune system and prevents and treat cancers. The body is not able to make Vitamin-C on its own, and it does not store Vitamin-C. This Vitamin-C has been a popular remedy for the common cold for many years. Research showed that for most people Vitamin-C supplements or Vitamin-C rich foods do not reduce the risk of getting the common cold. Taking a supplement after a cold starts does not appear to be helpful. Most of the functions of Vitamin-C are related to its property of undergoing reversible oxidation-reduction etc. inter-conversion of ascorbic acid and de-hydroascobic acid.
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VOL. 80, NOS. 9–10 265
Sci. & Cult. 80 (9–10) 265-268 (2014)
Studies on Ascorbic Acid (Vitamin-C)
Content in Different Citrus Fruits and
its Degradation During Storage
Abstract : Vitamin-C which is chemically L-ascorbic acid is
an essential nutrient for human body. Vitamin-C is required
for various metabolic functions and its deficiency cause
scurvy. Recent studies have clearly indicated antioxidant
properties of Vitamin-C and hence its role for human has
increased manifold. It is known that citrus fruits are good
source of Vitamin-C and because of our fast changing life-
style packed and stored fruit juice have become very common
and they are being administered for therapeutic and nutritional
purpose as well. The present investigation has revealed
gradual degradation of vitamin-c content present in various
citrus fruits even it is stored under refrigeration condition (4-
50c). The reduction of Vitamin-C varies from one fruit to other.
Hence it is always suggested to consume fresh juice rather
than the packed and stored one.
Vitamin-C or L-ascorbic acid or simply Ascorbate is
an essential nutrient for humans and certain other
animal species. Vitamin-C is required for a range of
essential metabolic reactions in all animals and plants.
Deficiency of this vitamin causes the disease scurvy. The
biological role of ascorbate is to act as a reducing agent.
Vitamin-C is absorbed in the body by both active transport
and simple diffusion. Ascorbate concentrations over renal
threshold pass freely into the urine and are excreted out.
As Vitamin-C is needed for the growth and repair of tissues
in all parts of our body and it is used to form an important
proteins used to make skin, tendons, ligaments and blood
vessels, heal wounds and form scar tissues, repair and
maintaining cartilage, bones and teeth. It is one of the many
antioxidants, which removes free radicals and stimulate the
immune system and prevents and treat cancers. The body
is not able to make Vitamin- C on its own, and it does not
store Vitamin-C. This Vitamin-C has been a popular remedy
for the common cold for many years. Research showed
that for most people Vitamin-C supplements or Vitamin-C
rich foods do not reduce the risk of getting the common
cold. Taking a supplement after a cold starts does not
appear to be helpful. Most of the functions of Vitamin-C
are related to its property of undergoing reversible
oxidation-reduction etc. inter-conversion of ascorbic acid
and de-hydroascobic acid.
Vitamin-C has received a great deal of attention and
with good reason. Higher blood levels of Vitamin-C may
be the ideal nutrition marker for overall health, say study
Researcher Mark Moyad M.D; MPH of the university. “The
more we study Vitamin–C the better our understanding of
how diverse it is in protecting our health and from CVD,
cancer, stroke, eye health and immunity to living longer”.
Vitamin -C has become the most controversial vitamin in
recent years. This is because of the claims on the use of
the Vitamin-C in mega-doses to cure everything from
common cold to cancer. Vitamin-C is a water soluble
vitamin that is necessary for normal human growth and
development. Leftover amounts of the vitamin leave the
body through the urine. That means we need a continuous
supply of such vitamins in our diet.
Various types of citrus fruits belonging to the family
Rutaceae, and some vegetable are good source of Vitamin-
C. Some common examples of such plants based sources
are Orange, Amla, Mausami, Lemon, Tomato, Strawberry,
etc. Very cheap fruits like Amla and Guava are very rich
sources of Vitamin-C. The fresh Amla juice contains 20
times as much Vitamin-C as Orange juice. A single Amla
fruit is equivalent to one or two oranges in Vitamin-C
content. Heating or drying of fresh fruits or vegetables
usually leads to destruction of most or all of the Vitamin-
C originally present. Amla is however an exception among
fruits not only because of its high Vitamin-C content, but
it also contains substances which partially protect the
vitamin from destruction on heating or drying. Amla juice
which is highly acidic protects Vitamin-C. Use of dried
Amla is an ancient practice and has much nutritional
relevance.
Although some of these fruits and vegetables are part
of normal Indian diet, but the way these fruits are taken is
very important. In Indian context normally cooking styles
and storage processes are not taken care of and this plays
a crucial role in the amount of nutrient available to the
human body.
Research Communication
266 SCIENCE AND CULTURE, SEPTEMBER-OCTOBER, 2014
As we know ascorbic acid that is Vitamin-C is an
essential nutrient for man as he lacks the capacity to
synthesize it like many other animal species. It helps
absorption of dietary iron by keeping it in the reduced form,
i.e. in ferrous form, thus Vitamin-C deficiencies are rarely
seen. Ascorbic acid occurs widely in plants foods,
particularly in fresh fruits and vegetables, especially in
green varieties of all the vegetables.
Ascorbic acid is the most sensitive vitamin in foods,
its stability varying markedly as a function of environmental
conditions such as pH and the concentration of traces metal
ions and oxygen. The nature of the packaging can
significantly affect the stability of ascorbic acid in foods.
The effectiveness of the material as a barrier to moisture
and oxygen as well as the chemical nature of the surface
exposed to the food are important factors. The aerobic and
anaerobic degradation reaction of ascorbic acid in reduced
moisture foods have been shown highly sensitive to water
activity. Ascorbic acid is the most susceptible one to
destruction by atmospheric oxidation. One of the
characteristic properties of this vitamin is its intense
reducing action and hence is oxidized rapidly in air. It is
for this reason that when vegetables become dry and stale
or cut and exposed to air most of the Vitamin-C originally
present is destroyed1. Whenever possible, fresh raw
vegetable should be used for obtaining enough of Vitamin-
C because considerable quantity of Vitamin-C contents of
food is lost during processing, storage and preparation2.
Bruising, peeling, cutting into pieces of different fruits of
leafy vegetables containing ascorbic acid decrease their
ascorbic acid retention3,4. Ascorbic acid decrease gradually
during storage especially at temperature above 00 C5.
Stability of the ascorbic acid also depends upon the
packaging material, exposure to air and storage temperature
condition6.
The present study aims at investigating the Vitamin-
C content of four different citrus fruits (Amla, Lemon, Lime
and Mausami), degradation of ascorbic acid (Vitamin-C)
content of four citrus fruits under the refrigeration condition
after specific time intervals and provide nutritional
education on Vitamin-C.
Materials and Methods : Four citrus fruits were
selected for the present investigation. These are, Amla
(Emblica officinalis), Mausami (Citrus sinesis), Lemon
(Citrus limon) and Lime (Citrus aurantifolia).
Both visual as well as chemical estimation of Vitamin-
C content and its degradation (if any) during storage was
studied.
Juice of citrus fruits was extracted with the help of
common kitchen juicer. Fresh readings were taken. Later
on reading at 24 hours interval was taken for 3 days after
keeping the juice in refrigerator.
Visual estimation : One tablespoon of cornstarch was
added into enough of water and it was stirred well to make
a homogenous mixture. This stock-mixture was used for
further investigation. Nearly 250 ml of water was further
added and the whole mixture was boiled for 5 minutes.
Thereafter 10 drops of starch solution was added to 75 ml
of water. Enough of iodine solution was added to produce
a dark purple blue color. In this way indicator solution
was ready. 5 ml of indicator solution was taken in a test
tube of 15 ml capacity and to it test-material was added
with the help of clean dropper. The dropper was properly
cleaned before its re-use. The test-tubes were held against
bright white background and photographs were taken. The
lighter the solution was, the higher the Vitamin-C content
was established. This is because Vitamin-C is responsible
for discoloring purple blue indicator.
Chemical estimation : For the chemical estimation
of Vitamin-C, following materials and reagents were
required:
Burette, Citrus fruits- Amla, Mausami, Lemon and
Lime, 2,6-dichlorophenol indophenols solution, 52 mg of
sodium salt of the dye and 42 mg of sodium bicarbonate
was dissolved in water to make the final volume to 500
ml. Standard Vitamin-C solution, 10 mg of Vitamin-C in
6% (wlv) meta-phosphoric acid was dissolved to make the
final volume the final volume with meta-phosphoric acid
to 1 litre.
Procedure : Fresh citrus fruits were squeezed to
obtain their respective juices. Juices were strained through
muslin cloth and immediately it was diluted (10 folds) with
6% meta-phosphoric acid. Thereafter 20 ml of standard
Vitamin-C solution was transferred in Erlenmeyer flask, and
it was titrated against the dye solution till the appearance
of a light pink colour was achieved. Then the volume of
the dye used was noted down. Similarly, 20 ml of each
test solution (diluted citrus fruit juice) against the dye
TABLE 1. Vitamin-C content of various citrus fruits.
Sl.No. Name of the citrus fruit Vitamin-C/100ml
1. Amla (Emblica officinalis) 178 mg
2. Mausami (Citrus sinesis) 52 mg
3. Lime (Citrus aurantifolia) 42.5 mg
4. Lemon (Citrus limon) 38.5 mg
VOL. 80, NOS. 9–10 267
solution was titrated and the volume of the dye used was
recorded.
Calculations : Concentration of Vitamin-C standard
solution = 10
gm/ml
Concentration of Vitamin-C in 20 ml of standard
solution= 200 μgm/ml
Suppose
ml of dye was required for the oxidation
of Vitamin-C in 20 ml of the standard solution, then 1 ml
of the dye causes oxidation of 200/


gm of Vitamin-C.
If 20 ml of diluted citrus juices were oxidized by
ml of
dye,
Then,
Amount of Vitamin-C in 20 ml of diluted juice= 200/
x
  
  
 
gm
Vitamin-C in 100 ml of diluted juice= (200/
) x x
(100/20) x (1/1000) = mg
Vitamin-C in 100 ml of undiluted juice= X 10 mg
Results and Discussions : Vitamin-C content in some
common citrus fruits was represented into the Table 1.
Among the studied four citrus fruits namely Amla (Emblica
officinalis), Mausami (Citrus sinesis), Lemon (Citrus limon)
and Lime (Citrus aurantifolia) highest value of Vitamin-C
content was found on Amla (178mg/100ml) followed by
Mausami (52mg/100ml), Lime (42.5mg/100ml) and Lemon
(38.5mg/100ml) respectively.
Different source of Vitamin-C were maintained at
constant temperature condition (under refrigeration at 4-50
C). Table 2 depicts changes in ascorbic acid content of
different sources kept under the refrigeration condition after
24 hours time interval.
From the above mention experiment it was clearly
indicated that ascorbic acid content was gradually decreased
with the time. It was also observed that the maximum
degradation occurred within the 24 hr. of storage. In case
of Amla 8mg/100ml ascorbic acid degraded after 24 hours
followed by 5mg/100ml and 4mg/100ml after 48hr. and
72hr. respectively. In lemon 4.5mg/100ml ascorbic acid was
degraded after 24hr. of storage which was followed by 3mg/
100ml after 48hr. and was constant even after 72 hr. also.
Degradation of Vitamin-C is slightly smaller in Mausami
and Lime after 24 hr. of storage. Only 1.5mg/100ml
Vitamin-C was degraded after 24hr. of storage in Mausami
and 2mg/100ml Vitamin-C was degraded after 24hr. of
storage in Lime.
Conclusion : The results of the analysis showed that
specific time interval significantly affected the stability of
Vitamin-C even stored under the refrigeration condition (4-
50C) and it was observed that there is a significant negative
relation exists between Vitamin-C and time of storage. The
outcome of this work will be highly valuable for the society
because it will throw light on the Vitamin-C content of
commonly used citrus fruits. Most important will be the
data generated regarding degradation of Vitamin-C of
different fruits during the course of storage. Having this
much of data, it will be possible to educate masses
regarding proper storage technology. The results will also
be utilized for proper economic management of nutrient.
This is very-very important aspect because recommendation
of nutrition without taking cost-effectiveness in mind will
fail. The experiment was conducted mainly for the purpose
of revisiting the Vitamin-C content of citrus fruit juices
and to study its degradation during storage. By this
experiment we can compare relative Vitamin-C content and
rank four citrus fruits from highest to lowest concentrations.
It will also help in bringing awareness regarding the factors
responsible for degradation of Vitamin -C during
storage.
AYESHA HASSAN1,
ANJAN KUMAR SINHA2*
AND P K MISHRA2
1Department of Clinical Nutrition and Dietetics,
Vinoba Bhave University,
Hazaribag, 825 301(India)
2Department of Botany,
Vinoba Bhave University,
Hazaribag, 825 301(India)
anjansinha06@gmail.com, malay_mishra@yahoo.com
*Corresponding author, email: anjansinha06@gmail.com
Received : 8 March, 2014
Revised : 15 September, 2014
Table 2: Reduction in Vitamin-C Content in citrus fruits
after storage at 4-5° C temperature
Sl. no. Name of the citrus STORAGE DURATION
fruit
24 HOURS 48 HOURS 72 HOURS
1. AMLA 170±1.1 165±0.9 161±1.2
2. MAUSAMI 49.5±0.9 45±1.1 40±1.6
3. LEMON 33±0.4 30±0.5 30±0.8
4. LIME 40.5±0.9 38.5±1.2 35±1.5
Level of significance- p 0.05
268 SCIENCE AND CULTURE, SEPTEMBER-OCTOBER, 2014
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(2006).
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IOSR Journal of Applied Chemistry (IOSR-JAC). 2(4), 20-24
(2012).
... The obtained findings are entirely agree with those reported by Harding et al. (1940), Sinha et al. (1962), Abdel-Latif (1975) and Cepeda et al. (1993) who mentioned that ascorbic acid content of citrus fruits decreased with the maturity, especially in late Valencia orange. Roongruangsri et al. (2013) and Hassan et al. (2014) reported that the cold stored citrus fruits revealed gradual degradation in vitam. C content under 4-5°C conditions. ...
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Cancer is a global cause of death characterized by uncontrolled proliferation and spread of abnormal cells. Eight extracts composed of the leaf, stem bark, seeds and juice of each of Citrus limon and C. aurantifolia were subjected to in vitro antioxidant assay using DPPH, brine shrimp lethality bioassay (BSL) and cytotoxicity MTT colorimetric assay using human cancer cell lines. Extracts of C. aurantifolia stem bark and leaf had IC50 of 28.2±0.11 and 47.2±0.39 μg/mL, respectively and displayed better radical scavenging activity compared to the other extracts, Ascorbic acid, the reference drug, had an IC50 of 9.2±0.14 μg/mL. Citrus limon stem bark (LC50=10.0±0.33) and C. limon leaf (LC50= 5.0±0.74 μg/mL) extracts were observed to be strongly cytotoxic compared to cyclophosphamide (LC50=98.76±0.15 μg/mL), while the other extracts were either non, weakly, or moderately toxic in BSL assay. Citrus aurantifolia leaf extract (CC50=4.02±2.85 μg/mL, CC50=5.45±2.8 μg/mL) retained a comparable cytotoxicity to cyclophosphamide (CC50=2.23±0.14 μg/ mL, CC50=2.66±0.8 μg/mL) on Rd and Hep-2c human cancer cell lines, respectively. The other extracts exhibited varying degrees of cytotoxicity. This study demonstrated that the extracts of both Citrus species had weak DPPH radical scavenging activity. Citrus aurantifolia leaf extract displayed potent toxicity in BSL assay and on the two human cancer cell lines; Rd and Hep-2c used in the study and were selective to cancer cells than the normal cell, Vero.
  • J Emese
  • P F Nagymate
J. Emese and P.F. Nagymate, British Food Journal. 110(3), 296-309 (2008).
  • M W Davey
  • M Van
  • D Montagn
  • M Inze
  • A Sanmartin
  • N Kanallis
  • Smirnoff
M.W. Davey, M. Van, D. Montagn, M. Inze, A. Sanmartin, A Kanallis and N. Smirnoff. J.f Sci. Food Agric. 80, 825-860 (2000).
  • M A Allen
  • S G Burgess
M.A. Allen and S.G. Burgess. British J. Nutri. 4(2-3), 95-100 (2006).
  • G Maria
  • I A Encarna
  • A A Kade
G. Maria, I.A. Encarna and A.A. Kade. J. Agric. Food Chem. 54, 4284-4296 (2006).
  • O A Oyetade
  • G O Oyeleke
  • B M Adegoke
O.A. Oyetade, G.O. Oyeleke, B.M. Adegoke, and A.O. Akintunde. IOSR Journal of Applied Chemistry (IOSR-JAC). 2(4), 20-24 (2012).