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Nutritional and biochemical evaluation of Averrhoa bilimbi L.

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Nutritional and biochemical evaluation of Averrhoa bilimbi L.

Peris C.1, Singh K.2*, D'souza M.2
1 DOS in Nutrition and Dietetics, Mount Carmel College, Palace Road, Bangalore, India- 560052.
2 Department of Chemistry, Mount Carmel College, Palace Road, Bangalore, India- 560052.

A RT I C L E H I S T O RY ABSTRACT

Received: 15-06-2013 Averrhoa bilimbi is a nutrition-packed, starchy fruit that grows mostly on the trunk of tall
trees and is a rich source of ascorbic acid. Other than the vitamins, the fruit also consists of
Accepted: 18-07-2013 fibre, ash, protein and moisture as well as minerals. Phytochemical screenings have
shown the presence of carbohydrates, flavonoids, tannins and hydrolysable tannins. The
Available online: 31-08-2013 total antioxidant activity (% inhibition of DPPH) in the Averrhoa bilimbi fruit and fruit
juice was found to be 43.89 ± 0.69 % and 34.47 ± 0.98 % respectively. Ascorbic acid
content in the fresh fruit and fruit juice was found to be 15.42 ± 1.44 mg/100 g and 10.83 ±
0.72 mg/100ml respectively. The amount of ß-carotene in the fresh fruit and fruit
concentrate samples was found to be around 0.031 ± 0.001 mg/100g and 0.028 ± 0.003
mg/100 ml respectively. The phenol content was found to be approximately 275 ± 2.24
KEYWORDS:
mg/100g and 130 ± 1.74 mg/100ml of the fresh fruit and juice respectively. The flavonoid
Antioxidants, Ascorbic acid, content was found to be higher in the fresh fruit sample containing around 155 ± 1.83 mg/
Averrhoa bilimbi, ß-carotene, 100g and 125 ± 1.36 mg / 100 ml of the fruit concentrate made into a juice. The values
calcium, flavonoids, phenols, seen in both samples give us an insight into its antioxidant potential and hence the need to
tannins. increase its consumption in order to obtain health benefits.

INTRODUCTION cancer and cardiovascular diseases are irrefutable, there is a real

Since time immemorial human beings have been dependant
on plants for food, medicine and shelter. Plants provide a wealth
of bioactive compounds. They are the main source of drugs that
have been used from ancient times as herbal remedies, prevention
and cure of various diseases and ailments. Many Indian plants are
used therapeutically for their antidiabetic effect, antihyper
lipidemic activity and antibacterial activities[1]. Trees in particular
have played a significant role in our culture, yet today we pay far
less importance to them. There exist several beneficial fruit
bearing trees in different parts of the world that still remain
unexplored nutritionally. Averrhoa bilimbi, a tropical tree
domesticated in home gardens commonly known as the
Cucumber tree or Tree sorrel is one such evergreen tree bearing a
rare and exotic fruit locally known as 'bilimbi'. In India today, the
Averrhoa bilimbi tree is found mainly in coastal areas with a
humid climate such as Karnataka, Kerala, Maharashtra and Goa
where it is popularly known by the names 'bilimbi', 'Irumban puli'
'bimbul' or 'bimli'. Averrhoa bilimbi belongs to the Order
Oxalidales and is part of the Wood Sorrel family known as
Oxalidaceae. It is commonly known by its binomial name
Averrhoa bilimbi L.
The current concept and trend today, is towards total health
management, the emphasis being on prevention rather than cure.
Since the connection between diet and some chronic diseases like
need for development of disease-fighting foods[2,3]. Diet is
believed to play an important role in the four major diseases of
advanced and transitional economies viz. cardiovascular disease,
cancer, hypertension, and obesity which can be elicited by the
overproduction of free radicals causing oxidative damage to
biomolecules like lipids, proteins and DNA.
Fruits are also diverse in antioxidant composition and those
with high antioxidant activity generally contain more
antioxidants[4]. Antioxidants are defined as a class of compounds
which include vitamins, enzymes and phytochemicals. Numerous
epidemiological studies suggest that diets rich in antioxidants
execute a protective role in health and disease[5,6]. Antioxidants
may serve the task of reducing oxidative damage in humans
induced by free radicals and reactive oxygen species (ROS) under
oxidative stress conditions. Reactive oxygen species (ROS)
causes DNA and protein damage, lipid peroxidation, cancer,
ageing and inflammatory activity [7]. Averrhoa bilimbi is a
nutrition-packed, starchy fruit that grows mostly on the trunk of
tall trees and is known to be a rich source of ascorbic acid. Other
than vitamins, the fruit also consists of fibre, ash, protein and
moisture as well as minerals[8]. It is known to possess a large
number of health and medicinal benefits which include
hypoglycemic, hypotriglyceridemic, antioxidant, antibacterial,
hepato-protective and antifertility[9] to name a few. It is due to the

*CORRESPONDING AUTHOR:
Singh K., Department of Chemistry, Mount Carmel College, Palace Road, Bangalore, India- 560052.
Email : kavi182@yahoo.co.in Phone No. +91 9980490272

58

biochemical constituents that the consumption of bilimbi may aid
in the prevention of the above mentioned maladies.
The use of bilimbi for commercial purposes especially as
antioxidants remains low, due to its lack of popularity and
research. It is an underutilized fruit, not grown on a commercial
basis in India today and is yet to become popular with a large
number of consumers. Also, grocery store sales are limited in
most markets. Hence, the objective of this study was to prepare a
fruit concentrate from the Averrhoa bilimbi using traditional
methods; and to determine the antioxidant potential (free radical
scavenging activity, flavonoids, ascorbic acid, ß-carotene, and
phenols) in both fruit and fruit juice with a goal to reintroduce the
consumption of this fruit.
MATERIALS AND METHOD
Plant Material
The fruits of Averrhoa bilimbi were collected from Fraser
Town, Bangalore, India in August 2012. After collection, fruits
were thoroughly washed with water, sliced with a knife and
utilized in the experiments.
Fruit Concentrate Preparation
500 g of Averrhoa bilimbi fruit was taken and homogenized in
a liquidizer with 250 ml water. The pulp was strained through a
sieve and the filtrate was kept aside. 500 ml of water was
separately boiled along with 750 g sugar and 15 g of citric acid
and made into syrup. The above mixture was added to the
Averrhoa bilimbi filtrate. The filtrate was left to cool and then
packed in sterilized glass bottles. A fruit concentrate was also
prepared by traditional methods, diluted in the ratio of 1:2 and
estimated for the different parameters.
Total Antioxidants
The total antioxidant activity of the samples was estimated by
the method described by Braca[7]. A stock solution of ascorbic acid
(1000 µg/ml) was diluted suitably in order to obtain different
concentrations of ascorbic acid ranging from 10µg-100µg. 0.1 ml
of each of the above prepared concentrations of ascorbic acid was
taken in clean dry test-tubes tubes. The volume in each tube was
made up to 3 ml with DPPH. The test tubes were incubated for 10
min at room temperature. The contents of the tubes were mixed
well and the absorbance read at 517 nm against a suitable blank.
3.1 ml DPPH was used as control. A standard curve was plotted to
determine the total antioxidant content in the samples. The %
inhibition of DPPH by the samples was calculated as follows:
% Inhibition of DPPH by the sample = Ac-As x 100
Ac
where, Ac is the Optical Density (O.D.) of the control, As is the
Optical Density (O.D.) of the sample.
Ascorbic acid
Ascorbic acid content was estimated using the method
described by Osborne and Boogt [10] 0.5 g of the sample was
homogenized with 10 ml of oxalic acid-acetic acid mixture and
filtered through a muslin cloth. The filtrate was then centrifuged
at 4000 rpm for 5 minutes and the resultant supernatant was
collected and used for the estimation of ascorbic acid. To 2.5 ml of
the supernatant taken in a conical flask, 5 ml of the oxalic acid-
Arch. of Pharm. and Bio Sci. | May-Aug 2013 | Vol-1 | Issue-2
acetic acid mixture was added and titrated against indophenol
dye. The volume of indophenol dye used to neutralize the acid
was noted and was used for the calculation of the amount of
ascorbic acid in the sample.
β-Carotene
β-carotene was estimated using the method described by
Mangels[11]. β-carotene was extracted from 0.5 g of fresh tissue in
10 ml of 1:1 petroleum ether and acetone. The extract was then
filtered through Whatman No. 1 filter paper and left undisturbed
overnight. The extract was centrifuged and the centrifugate
evaporated till half its original volume. A pinch of sodium
sulphate was added to all the solutions and again centrifuged. β-
carotene thus obtained was read at 470 nm in a calorimeter.
Total Phenols
The total phenol content of the samples was estimated using
the method described by Singleton and Rossi[12] 1g of the fresh
sample was weighed accurately and macerated in a mortar and
pestle with 10 ml of methanol and water (4:1). The mixture was
filtered using a Whatman filter paper and the filtrate was left
exposed to air. Once partially evaporated, 6 drops of 2 M
sulphuric acid was added to it followed by the addition of
chloroform to the mixture in a 3:1 ratio. The chloroform layer was
separated out using a separating funnel and was then exposed to
air for about 12 h. Once completely evaporated, the residue was
dissolved in minimal amount of methanol and used for the
estimation of total phenols.
Flavonoids
The flavonoid content of the samples was estimated using the
method described by Yang[13]. 0.5 to 2.0 ml aliquots of standard
Quercetin solution (1 mg/1 ml) was pipetted out into different test
tubes. The volume in each of the tubes was made up to 2 ml with
methanol. 2 ml of methanol served as the blank. 0.1 ml of 10 %
aluminium chloride reagent was added to each of the tubes
followed by 0.1 ml of 1M potassium acetate solution and 2.8 ml of
distilled water. The test tubes were incubated for 30 min at room
temperature. The contents of each tube were mixed well and the
absorbance was read at 670 nm against the blank.
Tannins
0.5 g of the freshly ground sample was macerated in distilled
water and boiled for 30 min. The mixture was then centrifuged at
2000 rpm for 20 minutes. 5 ml of the supernatant made up to 100
ml with distilled water was used for the estimation of tannins.
Determination of tannin was based on the method of Schanderi[14].
Aliquots of standard tannic acid solution (50µg/ml) were pipetted
out into different test tubes and made upto 10 ml with distilled
water. 0.5ml of Folin Denis reagent followed by 1.0 ml of sodium
carbonate was added to each test tube. The tubes were incubated
for 30 minutes at room temperature and the absorbance was read
at 760 nm against a suitable blank. A standard curve was plotted to
determine the tannic acid content in the samples.
Carbohydrate
100 mg of the fresh sample was macerated with a small
amount of 0.2 N sulphuric acid and refluxed for 30 minutes in a
flask. The mixture was then cooled and neutralized with a pinch of
sodium carbonate. The mixture was then made up to 50 ml with
0.2 N sulphuric acid and the contents were centrifuged. The
59

supernatant was collected and used for the estimation of the
amount of total carbohydrates present in the sample. The total
carbohydrate was measured according to the procedure of Roe[15].
0.2 to 1.0 ml aliquots of standard glucose solution (1mg/ml) was
pipetted out into different test tubes. 1.0 ml of the sample solution
was used for estimation. The volume in each of the tubes was
made up to 1.0 ml with distilled water followed by 4 ml of
Anthrone reagent. The tubes were incubated in a boiling water
bath for 10 minutes. 7 ml of distilled water was added to each tube
and the absorbance was read at 630 nm against a blank.
Statistical analysis
Samples were triplicated for the determination of each of the
biochemical components. The average value for each of the
triplicate samples was used for data analysis. The data were
presented as Mean ± SD for each group.
RESULTS
The physicochemical characteristics of bilimbi fruits and fruit
juice are as shown in Table 1. These values refer to the averages of
the determinations made during August/ September 2012. The
total carbohydrates in bilimbi were found to be 5.04 ± 1.17. The
antioxidant potential in terms of % inhibition of DPPH was found
to be 44 % in the fresh fruit and 34 % in the fruit juice. The juice
concentrate was diluted (1:2) but still showed good antioxidant
potential. The estimation of ascorbic acid indicates that the fresh
fruit has about 15 mg/100 g of the fruit. The amount was shown to
be comparatively lesser in the fruit juice in having 10 mg/100 ml.
The amount of ß-carotene estimated in the fresh fruit and fruit
juice and was found to be around 0.031 mg/100 g and 0.287
mg/100 ml respectively. Polyphenolic compounds like flavonoids
and tannins usually found in plants have been reported to have
multiple biological effects, including antioxidant activity. The
results of total phenol content, tannins and flavonoids are as
presented in Table 1. The phenol content was found to be
approximately 275 mg/100 g and 130 mg/ 100 ml of the fresh fruit
and juice respectively. The flavonoid content was found to be
higher in the fresh fruit sample containing around 155 mg/100g
Table 1: Nutrient content in Averrhoa bilimbi fruit and fruit juice samples
60
Arch. of Pharm. and Bio Sci. | May-Aug 2013 | Vol-1 | Issue-2
and 125 mg/100 ml of the fruit juice. While tannin levels
estimated in the fresh fruit was 5.07 ± 0.59 that in the fruit juice
was negligible.
DISCUSSION
Preliminary studies have shown that harvesting should not be
done at full maturity as it has an influence on the physicochemical
characteristics of the fruit. The carbohydrate levels were lower
than those reported for a similar fruit of the same family, Averrhoa
carambola (~ 8 mg/100g)[16]. Ripe bilimbi fruits had higher levels
of carbohydrate than half-ripe fruits, independently of the season
in which they were harvested[17]. This happens because during
maturation the starch is converted to simple sugars.
The juice concentrate was diluted (1:2) but yet showed good
antioxidant potential as determined by DPPH inhibition. The fruit
extract of Averrhoa bilimbi has potential antioxidant capacity and
its consumption may contribute substantial amount of
antioxidants to the diet [18,19].
Ripe bilimbi harvested during dry season showed maximum
level of ascorbic acid than the raw or half-ripe fruit. Similar
pattern was observed by Lima et al[17]. Therefore, the medicinal
use of this fruit against scurvy, which was recommended by
Corrêa[20] and Wong & Wong[21], can be justified.
Consumption of ß-carotene is known to be beneficial in
prevention of a number of cancers[22] and cardiovascular
diseases23. Fruits containing ß-carotene, vitamin E and lycopene
constitute natural sources of antioxidants. Fruits form a major part
of the daily consumption in both healthy and diseased people and
have a variety of pleasant and attractive flavours. Cooking and
dehydration at high temperatures of 50 °C-70 °C may lead to
greater destruction of carotenoids in Averrhoa bilimbi. The
principle cause of deterioration of the carotenoid is oxidation, this
being more severe once cellular integrity has been lost due to the
high degree of unsaturation of the carotenoid [24,25,26,27]. Hence,
consumption of the fruit in its raw form or in a beverage that has
not been subjected to excess cooking is preferred.

Bilimbi fruits are rich in polyphenolic antioxidants with
strong radical scavenging capacity [28]. Unripe fruits have a higher
amount of polyphenolics when compared to the ripe fruits. This is
because during the ripening process, a number of free radicals are
generated which have to be converted to harmless reduced
substances. This is done at the cost of antioxidants like phenols,
ascorbic acid etc [29]. The time chosen for harvesting bilimbi
ensures maximum antioxidant levels in the fruit as well as the
fruit juice.
The flavonoid content was found to be higher in fresh fruit
when compared to the fruit juice. The presence of phenols,
flavonoids and tannins is responsible for maintaining an
antioxidant pool within the body that has a role in free radical
scavenging[30]. Thereby, it has been associated with decreased risk
of some age related and chronic diseases in humans.
CONCLUSION
The current concept and trend today, is towards total health
management, the emphasis being on prevention rather than cure
and since the connection between diets and some chronic diseases
like cancer and cardiovascular diseases are irrefutable, there is a
real need for development of disease-fighting foods.
Antioxidants today have found a significant place in commercial
food products which strive to be healthy and natural at the same
time. It is well established that fruit and fruit products are a rich
source of vitamins, minerals and antioxidants. The food industry
is coming out with new innovations in terms of products to draw
the attention of the growing number of consumers today. Food
products in various forms are made easily available to consumers
with a view to retain the taste and sensory characteristics of the
food in its natural form. Keeping this in mind, a fruit concentrate
was prepared from the Averrhoa bilimbi fruit with the extra
addition of sugar and a mild preservative (citric acid). It was
shown to be close in flavour to the fruit in its fresh form and
proved to be a good thirst quencher at the same time.
It can be inferred from our studies that the consumption of the
Averrhoa bilimbi fruit plucked during the semi-ripe stage shows
the maximum nutritional properties, however the refreshing
beverage that can be made from it does not fall far behind in
providing similar benefits. It was found to have significant
medicinal and nutritional properties. Hence, it deserves more
attention and it would be highly beneficial to tap its potential in
order to obtain maximum benefits in the future. Increased
consumption of Averrhoa bilimbi among the population is
possible, but will require an integrated approach to improving
consumer awareness and bringing about behavioural change.
ACKNOWLEDGEMENT
The authors would like to thank Mount Carmel College, Sr.
Juanita and Dr Radhe Kale for providing the opportunity to carry
out this research work.
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