DOI: 10.21276/sjavs.2016.3.6.

Scholars Journal of Agriculture and Veterinary Sciences e-ISSN 2348–1854

Sch J Agric Vet Sci 2016; 3(6):416-423 p-ISSN 2348–8883
©Scholars Academic and Scientific Publishers (SAS Publishers)
(An International Publisher for Academic and Scientific Resources)

Effects of Cooking on Content of Vitamin C in Green Leafy Vegetables

Rama Ramesh Singh*, Dr. Anupma Harshal
Department of Biotechnology, K.C. College, Churchgate, Mumbai- 400020, India

*Corresponding Author
Name: Rama Ramesh Singh
Email: ramasingh0794@gmail.com

Abstract: This study was designed to determine the effect of processing on the Vitamin C content of five Indian green
leafy vegetables namely, Spinach (Spinacia oleracea), Methi (Trigonella foenum-graecum), Lal maath (Amaranthus
cruentus), Chauli (Thotakur avepudu) and Bhathua (Chenopodium album). The Vitamin C content of the lemon sample
was also estimated using DNPH colorimetric assay, DCPIP, Iodometry and N-Bromosuccinimide titration methods.
Among the methods used for Vitamin C estimation of lemon, N-Bromosuccinimide method was found to be most
accurate and sensitive. Hence, it was further used to estimate the Vitamin C content in the above mentioned vegetables.
Processing methods employed were boiling, blanching and microwave heating the vegetables for particular time interval.
The vegetable extracts were subjected to 0.01% N-Bromosuccinimide titration to estimate their Vitamin C content before
and after the treatment. Results revealed that maximum reduction in Vitamin C activity of vegetable samples were
observed with microwave heating followed by boiling and blanching. Vitamin C content of boiled vegetable samples
showed 40-50% reduction in their vitamin C activity, microwave heated samples resulted into 50-75% reduction and
blanching lead to minimum reduction i.e. 20-35% as compared to unprocessed vegetable samples. Vitamin C is easily
destroyed by excessive heat and water as well as exposure to air. For retention of Vitamin C in cooked foods, it is
recommended that foods containing Vitamin C be cooked as fast as possible with less heat and small amount of water.
Keywords: Vitamin C, Lemon, Green leafy vegetables, Boiling, Blanching, Microwave heating

INTRODUCTION Population studies have shown that individuals with

Vegetables and fruits are valuable components high intakes of Vitamin C have lower risk of chronic
of the daily diet contributing carbohydrate in form of diseases including, heart disease, cancer, eye diseases
dietary fibre, vitamins and minerals to the body [1]. and neurodegenerative conditions [6]. Diet supplies
Most tropical countries diet contains a diversity of leafy more than 90% of the Vitamin C in human by fruits and
vegetables such as spinach, amaranth, lettuce. The main vegetables [7]. Except human and other primates, most
protective action of vegetables has been attributed to the of the higher animals can synthesize Vitamin C (L-
antioxidants present in them. The potential cancer ascorbate). Humans lack the enzyme (L-gluconolactone
inducing oxidative damage might be prevented or oxidase, GLO) which is needed to convert glucose to
limited by dietary antioxidants found in vegetables [2]. Vitamin C [8]. Ascorbic acid is widely distributed in
Researchers had found that vegetables lead to the fresh fruits and vegetables like orange, lemons,
diminution of several diseases like: cardiovascular, grapefruit, watermelon, papaya, tomatoes, broccoli,
neurological and carcinogenic destruction of different green and red peppers [9]. Vitamin C cannot be
body parts [3]. synthesized through body cells nor does stores it. It is
therefore important to include plenty of Vitamin C-
Vitamins are essential to maintain normal containing foods in daily diet [10].
metabolic processes and homeostasis. The amount of
specific vitamin required by an individual varies Vitamin C plays significant functions in the
considerably [4].Vitamin C (Ascorbic Acid) is a water- body. The biochemical functions of Vitamin C includes:
soluble antioxidant. It was first isolated in 1928 by the Stimulation of certain enzymes, collagen biosynthesis,
Hungarian biochemist and Nobel Prize winner Szent- hormonal activation, antioxidant, detoxification of
Gyorgyi. Vitamin C is available in reduced form (L- histamine, phagocytic functions of leukocytes,
ascorbic acid) and oxidized form (L-dehydroascorbic formation of nitrosamine and proline hydroxylation
acid). It is an unstable, easily oxidized acid and can be amongst others. Vitamin C has been associated with
destroyed by oxygen, alkali and high temperature [5]. reduction of incidence of cancer, blood pressure,
Great interest has been seen in the clinical roles of immunity and drug metabolism and urinary
Vitamin C because of evidence that oxidative damage is hydroxyproline excretion, tissue regeneration
a root cause and mostly associated with many diseases. [11]. Study published in Anticancer Research in 1992

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reported that use of high-dose antioxidants in microwave and conventional cooking have only slight
conjunction with chemotherapy and radiation prolonged difference on vitamin retention in foods [19].
survival in patients with small-cell lung cancer [12].
The recommended dietary allowance (RDA) for The recent scientific literature indicates that
Vitamin C for non smoking adult male and female was beyond protecting against scurvy, Vitamin C
60 mg/day [6]. The new higher Recommended Dietary contributes to many aspects of human health regulating
Allowance (RDA) for Vitamin C is 75mg for women many metabolic processes. The data indicates that the
and 90mg for men because it acts as an antioxidant as Vitamin C requirement of smokers is higher by at least
well as for protection from deficiency [13]. Since 60mg per day than that of non-smokers due to increased
smokers suffer increased oxidative stress there metabolism. Important functions such as, immune
recommended dietary allowance is increased by response, pulmonary function and iron absorption are
35mg/day. related to Vitamin C intakes in diet. Daily Vitamin C
intake of at least 150-200mg per day is required for
Most vegetables are commonly cooked before these functions. Vitamin C also play critical roles in the
consumption. In general, vegetables are cooked at home prevention of CHD, cancer and cataract [20].
on the basis of convenience and taste preference of
consumer rather than retention of nutrient and health- Studies from the UK and North America have
promoting compounds. Study shows that cooking reported Vitamin C deficiency in around 1 in 5 men and
methods affect the contents of nutrient and health- 1 in 9 women in low income groups [21]. The
promoting compounds such as Vitamin C, carotenoids, prevalence of Vitamin C deficiency is highest among
polyphenols and glucosinolates in broccoli [14]. It is Indians and people of South Asian origin compared to
known that cooking of vegetables induces significant other races except the Mexican population. Lower
changes in chemical composition which influences the intake of fresh fruits and vegetables and over-cooking
concentration and bioavailability of bioactive of food by South Asians might be the reason for higher
compounds. However, both positive and negative prevalence of Vitamin C deficiency in these
effects have been reported depending upon differences populations. Also this deficiency of Vitamin C may be
in process conditions and morphological and nutritional the one of the reason for higher rates of cardiovascular
characteristics of vegetable species. Physical properties disease and cancer among South Asians individuals in
are greatly affected by heat treatments to vegetables. Pakistani, Indian, Malay and Chinese populations
Although consumption of fresh unprocessed plant food compared to most Western populations [22]. The
is widely advocated to obtain maximum nutrition but research done on prevalence of Vitamin C deficiency in
evidence is emerging that in vivo bioavailability of India clearly highlights that serum ascorbic acid
many protective compounds present in vegetables is deficiency is associated with elevated markers of
enhanced when they are cooked. Along with positive chronic disease in this population of young adults which
attributes of boiling vegetables before consumption, may have long-term adverse health consequences [23].
consumers are also deprived of important nutrients as The age, sex and season standardized prevalence of
the nutritional properties of the vegetables are greatly Vitamin C deficiency was 73.9% in 2668 people in
affected after cooking [15]. The degree of vitamin loss north India and 45.7% in 2970 from south India. Only
is influenced by various factors including: type of food, 10.8% in the north and 25.9% in the south met the
variety of vegetables, method of cutting, duration and criteria for adequate levels of Vitamin C. Deficiency
processing of cooking [16]. Ascorbic acid is sensitive to was more prevalent in men with increasing age, users of
heat and oxygen and is a water soluble vitamin, tobacco and biomass fuels with poor nutrition and with
therefore leaching into the cooking water may occur lower intakes of dietary Vitamin C [21].
during processing resulting in the potential loss during
industrial processing or domestic cooking. In addition, The purpose of this study is to investigate the
leaves of the vegetables may absorb a large amount of effects of different cooking processes like boiling,
cooking water and this can lead to dilution and further blanching and microwave cooking on Vitamin C
reduction in the level of vitamins in the cooked product content of green leafy vegetables namely spinach,
[17]. Research has demonstrated that thawing of methi, lal maath, bhathua and chauli. Also to find the
vegetables before cooking is useless and leads to more most suited and accurate method to determine the
Vitamin C loss. Therefore, frozen vegetables must not Vitamin C content in vegetables among the various
be thawed before cooking. To prevent Vitamin C from chemical methods available.
destruction, use of double based stainless steel pan,
minimum amount of water and cooking of frozen MATERIAL AND METHODS
vegetables are recommended [18]. Microwave cooking Estimation of Vitamin C by various methods using
has gained considerable importance as an energy- lemon as sample
saving, convenient, and time-saving cooking method. A Various methods were examined to accurately
large amount of data is available on the effects of determine the Vitamin C content in lemon and using
microwaves on vitamins. It is concluded that Vitamin C tablet as a standard. Methods used are as
follows:

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1) Iodometry [24] [volume of dye required to titrate test/Volume of dye

Vitamin C can be determined in food by use of required to titrate standard] * conc. of std * Dilution
an oxidation-reduction reaction. The redox reaction is factor
preferable to an acid-base titration because a number of
other species in juice can act as acids but relatively few D) N-Bromosuccinimide [27]
interfere with the oxidation of ascorbic acid by iodine. Stock solution of ascorbic acid was prepared
The solubility of iodine is increased by complexation freshly by dissolving 0.05g in 100cm3 of 0.5% oxalic
with iodide to form tri iodide. Tri iodide then oxidizes acid solution. Serial dilutions were made from the stock
Vitamin C to dehydroascorbic acid: solution (50mg ascorbic acid/100cm3) to give the
working solutions of 40mg, 30mg, 20mg and 10mg of
C6H8O6 + I3 - + H2O C6H6O6 + 3I- + 2H+ (2) ascorbic acid in 100cm3 of 0.5% oxalic acid solution.
10cm3 each of the standard ascorbic acid solutions were
The endpoint is indicated by the reaction of transferred to a standard flask and the volume made up
iodine with starch suspension which produces a blue- to 100cm3 with de-ionized water. 10cm3 of this solution
black product. Take 25ml of standard Vitamin C was then titrated against 0.01% N-bromosuccinimide
solution (1mg/ml) into 150ml conical flask. Add 10 solution according to the method earlier reported. All
drops of 1% starch solution and titrate it against iodine titrations were performed in triplicate and the results
solution till end point is reached, take 3 readings. used to construct a calibration curve for ascorbic acid.
Repeat the titration taking 25ml of lemon juice and
calculate Vitamin C gm% of lemon. Titration of lemon sample to determine ascorbic acid
content
2) DNPH method [25] Take approximately 20gm of lemon and
Extract 5gm of sample in 25-50ml 4% oxalic squeeze to obtain sufficient amount of juice. Take 10 ml
acid as described. Transfer an aliquot (10ml) to a of lemon juice and titrate against 0.01% N-
conical flask and add bromine water drop wise with bromosuccinimide. Note the reading and estimate the
constant mixing. The enolic hydrogen atoms in ascorbic amount of Vitamin C present in 10gm of sample using
acid are removed by bromine. When the extract turns standard ascorbic acid curve obtained above.
orange-yellow due to excess bromine, expel it by
blowing in air. Make up to a known volume (25 or B) Estimation of vitamin C in vegetables using N-
50ml) with 4% oxalic acid solution. Prepare stock Bromosuccinimide titration method
solution of Vitamin C using tablet of 100mg/ml. Take Sample collection and preparation
aliquots in the range of 0.2-1ml, make up the volume Vegetables were selected on the basis of
with distilled water to 3ml. Add 1ml of 2% DNPH availability and their popular consumption. Fresh
solution and 2ml of 10% thiourea. Incubate in waterbath vegetables were purchased from the local market and
for 1hr at 60o C. The dehydroascorbic acid is then were used on the same day. These were washed
reacted with 2, 4-dinitrophenyl hydrazine to form thoroughly with water, leafs of the vegetables were
osazone and dissolved in sulphuric acid to give an grinded using a pestle and mortor and a buffer as
orange-red color solution which is measured at 540nm. specified by the individual assay methods. Filtrate was
Take 1ml of the processed lemon juice sample as collected after crushing and used for Vitamin C assay.
unknown. Calculate the ascorbic acid content in the The samples used for analysis of Vitamin C were
sample using the standard graph. Spinach, Methi, Lal maath, Chauli and Bhathua.

C) DCPIP (Dichlorophenoliodophenol) titration [26] Processing treatments

Standard: 5ml of standard solution was 5g of vegetable samples were taken and
pipetted out into conical flask, 10ml of 4% oxalic acid subjected to boiling (10mins), blanching (2mins) and
was added and titrated against the dye. The end point microwave heating (10mins). Then the vegetables were
appeared as pink color which persists for few minutes. crushed in a mortor and pestle along with 0.5% oxalic
The amount of dye consumed is equivalent to the acid, it was filtered with muslin cloth and the volume
amount of ascorbic acid. was made upto 100ml using measuring cylinder. The
filtrate obtained was refiltered using filter paper for
Test: 5ml of test sample (lemon juice) was obtaining more clear solution. The treated samples were
pipetted out into a 100ml of conical flask. 10ml of 4% extracted in 0.5% oxalic acid and volume of the filtrate
oxalic acid was added and titrated against the dye. The was made up to 100ml, 10ml of the sample solution was
endpoint appeared as pink color which persists for titrated against 0.01% N-bromosuccinimide. The
sometimes and reading was noted. content of Vitamin C was determined before and after
the extracts were exposed to blanching, heating and
Calculations: The amount of ascorbic acid present in the microwave radiation was determined using standard
given sample is calibration curve of ascorbic acid.

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RESULTS

Table1: Estimation of Vitamin C by various methods using lemon as sample

Amount of
Weight of Mean Value
Volume of Juice Vitamin C/10g
Method Lemon sample (mg/10gm) of
extracted (ml) of Lemon
taken (g) lemon sample
sample (mg)
75.63 40 7.616
Iodometric Titration 09.44 45 5.35 7.62
99.22 45 9.88
6.510 2.9 46.39
DNPH Method 6.11 2.5 41.90 42.99
5.07 2 40.68
24.13 12 42.0
DCPIP Dye Titration
29.66 17 52.3 48.86
Method
28.92 14 52.3
20.3 11 60.3
By N-Bromosuccinimide
21.6 13 59.4 61.47
Method
19.8 11 64.7

From the above methods used to determine the colorimetric assay followed by DCPIP dye titration
Vitamin C content in lemon juice sample, Iodometry method. N- bromosuccinimide titration method gave
method is least sensitive followed by DNPH best results to estimate Vitamin C content.

Fig-1: Comparison of the results obtained by various method for estimation of Vitamin C in lemon juice sample

Fig-2: DNPH method: standard and sample tubes

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Table 2: Vitamin C content in vegetables before and after processing

Sampl Vitamin C content (mg%) after processing
e Before (raw) Mea Boiling Mea Blanching Mea Microwave heating Mea
n n n n
Spina 55.3 67.3 61.2 61.3 17.5 35.5 35.6 29.5 34.8 61.2 49.0 48.3 14.1 12.4 16.0 14.2
ch 7 1 1 0 6 6 6 7 1 2 6 4 3 9 2
Methi 66.0 59.4 65.2 63.5 32.5 30.0 31.4 31.3 38.8 39.4 36.6 38.2 27.8 29.2 23.3 26.7
9 6 7 2 2 7 8 9
Lal 223. 215. 219. 219. 136. 102. 140. 126. 148. 147. 181. 158. 57.8 60.0 61.5 59.7
maath 4 96 56 64 8 8 6 73 2 4 0 86 8 9
Bhath 217. 146. 151. 171. 118. 94.9 56.1 89.7 133. 110. 89.1 115. 100. 81.3 79.4 87.1
ua 28 32 43 67 25 5 1 7 78 5 2 94 7 5 5
Chauli 46.8 54.9 48.6 50.1 22.8 27.0 29.6 26.5 33.2 44.8 34.6 37.5 13.2 20.7 22.0 18.6
3 1 9 2 0 8 6 5 2 7

The above table shows that Vitamin C content processes including boiling, blanching and microwave
of various green leafy vegetables considered for study heating. The amount of vitamin C was calculated in mg
when they are unprocessed and also the amount of of Vitamin C present in 100gm of the vegetable sample.
Vitamin C available after subjecting to various cooking

Fig-3: Vitamin C content in vegetables before and after processing

Table 3: Comparative values of Vitamin C after various treatment

% reduction in the content of vitamin C
Sample Microwave
Boiling Blanching
heating
Spinach 61.29 52.42% 22.03% 76.57%
Methi 63.56 50.72% 39.61% 57.65%
Chauli 50.11 46.59% 25.39% 62.10%
Lal maath 219.64 42.33% 33.75% 72.77%
Bhathua 172.62 48.30% 35.18% 48.89%

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Fig-4: Percentage reduction in content of Vitamin C after subjecting to various cooking methods

Fig-5: DCPIP Titration

DISCUSSION often used as an estimation for overall nutrient retention

Vitamin C content was estimated by using of food products because it is highly sensitive to
various methods: Iodometry, DNPH method, DCPIP oxidation and leaching into water during cooking. It
dye and N- Bromosuccinimide titration method by begins to degrade immediately after harvest and
using lemon juice as sample and ascorbic acid tablet as degrades steadily during storage and other processes.
standard (Table 1, Figure 1, 2 & 5). The results obtained The study done clearly highlights that Vitamin C
showed N- Bromosuccinimide method estimation was activity drops on treatment with heat. The loss of
most efficient and sensitive in estimating the amount of Vitamin C in green leafy vegetables is due to the
Vitamin C as it quantified both ascorbic acid and processing method employed in its preparation. The
dehydroascorbic acid present in the sample. The other losses observed in this study are high most especially
techniques showed lower quantification of Vitamin C as when the vegetables were subjected to boiling and
the methods were specific only for estimation of microwave heating as compared to blanching (Table 2).
ascorbic acid. Hence, N-Bromosuccinimide method was Loss as a result of boiling, microwave heating and
chosen for estimation of Vitamin C in vegetables as it blanching can be justified since Vitamin C is water-
was most sensitive method for estimating the total soluble and heat labile. Thus, Vitamin C is easily
ascorbic acid present in the sample and also due to easy leached into the boiling medium, the rate of destruction
and simplicity of method. of vitamin was not uniform. The percentage of Vitamin
C activity loss was 40-50% on boiling, 20-40% on
We report here the Vitamin C content of raw, blanching and 50-75% on microwave heating (Table 3
boiled, blanched, microwave radiation exposed samples & Figure 3). The observations put forth that microwave
of vegetables namely Spinach, Methi, Lal maath, Chauli cooking of the said vegetables does not seems to be a
and Bhathua (Figure 4). The retention of Vitamin C is good cooking practice.

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Singh RR et al.; Sch J Agric Vet Sci., Oct-Nov 2016; 3(6):416-423

CONCLUSION vegetables in Davanagere city,(Karanataka)-India.

It has been observed from the analytical results International Journal of Pharmacy & Life Sciences.
that Vitamin C content is conserved maximum on 2013 Mar 1;4(3).
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ACKNOWLEDGEMENT American journal of clinical nutrition. 1999 Jun
The author would like to express her gratitude 1;69(6):1086-107.
to the management of K.C. College for providing us the 14. Yuan GF, Sun B, Yuan J, Wang QM. Effects of
necessary facilities to carry out this research work. I different cooking methods on health-promoting
would like to mention a special thanks to my mentor Dr. compounds of broccoli. Journal of Zhejiang
Anupma Harshal and Science Honours program. Lastly University Science B. 2009 Aug 1;10(8):580-8.
would like to thank my family, friends and almighty for 15. Agbemafle R, Edward AO, Genevive EA, Daniel
their support. I would also like to extend my regards to NA. Effects of boiling time on the concentrations
Ms. Shivani Tiwari for her support and guidance. of vitamin c and beta-carotene in five selected
green vegetables consumed in Ghana. Applied
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