Food Research International 44 (2011) 672676

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Food Research International

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Total phenolics, avonoids and antioxidant activity of tropical fruit pineapple

M. Amzad Hossain a,, S.M. Mizanur Rahman b
a
b

Biotechnology Research Institute, Universiti Malaysia Sabah, Locked Bag No. 2073, 88999 Kotakinabalu, Sabah, Malaysia
Department of Chemistry, University of Dhaka, Dhaka-1000, Bangladesh

a r t i c l e

i n f o

Article history:
Received 3 September 2010
Accepted 26 November 2010
Keywords:
Total phenolics
Flavonoids
Antioxidant activity
Pineapple

a b s t r a c t
Pineapple has several benecial properties including antioxidant activity. The fruit of pineapple was extracted
with ethyl acetate, methanol and water. The phenolic content of the extracts was determined by Folin
Ciocalteu method and antioxidant activity was assayed through some in vitro models such as antioxidant
capacity by phosphomolybdenum, -carotene-linoleate, and radical scavenging activity using ,-diphenyl-picrylhydrazyl (DPPH) method. The phenolic contents of the extracts as caffeic acid equivalents were found
to be highest in methanol (51.1%) followed by ethyl acetate (13.8%) and water extract (2.6%). Antioxidant
capacity of the extracts as equivalent to ascorbic acid (mol/g of the extract) was in the order of methanol
extract N ethyl acetate extract N water extract. In comparison with butylated hydroxyanisole (BHA), at
100 ppm of concentration, the antioxidant and free radical scavenging activities of the extracts assayed
through -carotene-linoleate and DPPH method were also found to be highest with methanol extract
followed by ethyl acetate and water extracts. The results indicated that the extent of antioxidant activity of the
extract is in accordance with the amount of phenolics present in that extract and the pineapple fruit being rich
in phenolics may provide a good source of antioxidant.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
In recent years, consumers desire to reduce the risk of or to
manage a specic health condition through improved diet. Plants
have evolved different phytochemicals and enzymes as antioxidant
defense to maintain growth and metabolism (Pandhair & Sekhon,
2006). Concern about improving health, involving agricultural
products with potential benets, has enhanced research on antioxidants (Moore, Hao, Zhou, Luther, & Costa, 2005). Many degenerative
human diseases including cancer, cardio- and cerebro-vascular
diseases have been recognized as being a possible consequence of
free radical damage to lipids, proteins and nucleic acids (Choi & Lee,
2009).
A possible way to ght these diseases is to improve our body's
antioxidant defenses. High consumption of fruits and vegetables has
been associated with a lowered incidence of such degenerative
diseases (Bajpai, Yoon, & Kang, 2009). Fruits also help to improve
health in other ways. Pineapple juice, for example, can be taken to
alleviate sore throat and seasickness. The functional bioactivity of a
plant extract, in general, depends upon the presence of compounds
such as polyphenols, carotenoids and chlorophyll (Choi & Lee, 2009).
Plants contribute in this area primarily due to the antioxidant activity

Corresponding author.
E-mail address: dramzadh@gmail.com (M.A. Hossain).
0963-9969/$ see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2010.11.036

of polyphenolic compounds (Higdon & Frei, 2003; Jacob, Hakimuddin,

Paliyath, & Fisher, 2008).
Flavonoids, mainly present as colouring pigments in plants also
function as potent antioxidants at various levels. Some studies
showed that avonoids could protect membrane lipids from oxidation
(Terao, Piskula, & Yao, 1994). Pineapple, Ananas comosus L., Merr. is an
important tropical fruit, that is consumed in many parts of the world
as fresh fruit, juice, jam, jelly and dried product. It has a high nutritive
value and is a rich source of vitamins A, B and C besides several
minerals such as calcium, phosphorus and iron. Though there are
some reports on the antioxidant activities of pineapple in relation to
other fruits (Gardner, White, McPhail, & Duthie, 2000), they deal with
only one or two parameters and not in detail or do not suggest
possible components/mechanisms.
It is known that plants protect themselves against pathogens by
various defense responses which include the production of antimicrobial peptides. Antimicrobial proteins are short cationic amphiphilic
peptides which form an important component of immune defenses
(Hancock & Sahl, 2006). The antimicrobial magainin gene MSI-99
isolated from skin secretions of the African clawed frog (Xenopus
laevis), is known to confer resistance against a broad spectrum of
fungal and bacterial pathogens. Recently, a few reports have been
made available on the expression of different analogues of magainin
in tobacco (Li et al., 2001), tomato (Allan, Blowers, & Earle, 2004), and
potato (O'Callaghan et al., 2004) for obtaining enhanced disease
resistance. Pineapple too is susceptible to diseases. One such synthetic
substitution analogue of magainin, MSI-168, was used to transform

M.A. Hossain, S.M.M. Rahman / Food Research International 44 (2011) 672676

leaf bases of pineapple in order to instill in-built resistance against

microbial infections. Transgenic plants of pineapple were thus
produced by using the Agrobacterium strain EHA 105 harboring the
plasmid pMSI-168 for transformation (Mhatre, Nagi, & Ganapathi,
2009; Mhatre, Tilak-Jain, De, & Devasagayam, 2009). The successful
expression of this synthetic peptide in pineapple was obtained
resulting in normal growth and fruiting of transgenic plants. Such
an alteration in the genetic makeup could introduce changes in the
biochemical processes of the plant, namely the antioxidant makeup.
To rule out this possibility we sought to study the possible changes in
the antioxidant status of various parts of transformed pineapple fruit
extracts in comparison with non-transformed ones.
Several studies have revealed that the majority of the antioxidant
activity may be from compounds such as avonoids, isoavones,
avones, anthocyanins, catechins and other phenolics (Kahkonen,
Hopia, & Vuorela, 1999; Alothman, Bhat, & Karim, 2009; Mhatre, Nagi
et al., 2009; Mhatre, Tilak-Jain, et al., 2009; Isabelle et al., 2010;
Danino, Gottlieb, Grossman, & Bergman, 2009). Oxidative stress has
been linked to various diseases (Halliwell, 1994) while food industry
long has been concerned with issues such as rancidity and oxidative
spoilage of foodstuffs (Shahidi & Wanasundara, 1992). The enzymatic
oxidation as well as auto oxidation of lipids during storage and
processing is the major reaction responsible for the deterioration in
food quality affecting the colour, avour, texture and nutritive value
of the foods. Antioxidants are often added to foods to prevent the
radical chain reactions of oxidation by inhibiting the initiation and
propagation step leading to the termination of the reaction and a
delay in the oxidation process.
However, the commonly used synthetic antioxidants such as
butylated hydroxyanisole (BHA) and butylated hydroxy toluene
(BHT) are restricted by legislative rules because they are suspected
to have some toxic effects and as possible carcinogens (Imaida et al.,
1983). Therefore, there has been a considerable interest by the
industry and a growing trend in consumer preferences for natural
antioxidants over synthetic compounds and elimination of synthetic
antioxidants in food applications has given more impetus to explore
natural source of antioxidants. Thus, antioxidants are of interest to
both food scientists and health professionals and there has been a
convergence of interest among researchers in these elds as the role
of antioxidants in the diet and their impact on human health has come
under attention. In the present study, the antioxidant activity of the
extracts of pineapple fruit was assayed through various in vitro
models. So far we know, this is the rst report on antioxidant activity
of tropical pineapple fruit.
2. Experimental
2.1. Materials
-Carotene, linoleic acid, ,-diphenyl--picrylhadrazyl (DPPH)
and butylated hydroxyanisole (BHA) were obtained from E. Merck
(Germany). Solvents used for extraction were methanol (GC grade)
and ethanol, obtained from Merck (Darmstadt, Germany). The
puried water was obtained from water distillation plants in our
laboratory. All other chemicals were of analytical grade or GC grade.
UV spectra UVVisible spectra measurements were done using a
HITACHI, U-2000 spectrophotometer.
2.2. Sample collection
There are different types of pineapples available in Bangladeshi
markets all year round. Calendar is one of the most popular pineapple
cultivars grown in Indian region. For our experiment we used the
calendar varieties. Calendar pineapples were collected from local
markets of different districts of Bangladesh. The skin of pineapples is
initially green, but turns yellowish when ripe. The colour of the

673

pineapple ranges from pale green to yellow. After harvesting, the

apples are stored at 2 C until analysis.
2.3. Extraction
Ripe pineapples were cut into pieces and washed with deionised
water. The small pieces were homogenised in a grinder for 3 min to
40-mesh size paste. Twenty ve grams of paste samples was extracted
with 150 mL of ethyl acetate by mixing, using a magnetic stirrer at
30 C for 2 h. The extract was ltered through Whatman No. 41 lter
paper to obtain particle free extract. The residue was reextracted
twice and ltered. The extracts were pooled and concentrated and
dried under vacuum. The same procedure was followed for the
other solvents such as methanol and water for antioxidant fractions
(Jena et al., 2002) and the extracts were used to explore their antioxidant activity.
2.4. Determination of total phenolics
The concentration of total phenols in the extracts was determined
by using FolinCiocalteu reagent and external calibration with caffeic
acid. Briey, 0.2 mL of extract solution and 0.2 mL of FolinCiocalteu
reagent were added and the contents mixed thoroughly. After 4 min,
1 mL of 15% Na2CO3 was added, and then the mixture was allowed to
stand for 2 h at room temperature. The absorbance was measured at
760 nm using a HITACHI, U-2000 spectrophotometer. The concentration of the total phenolics was determined as mg of caffeic acid
equivalent by using an equation obtained from caffeic acid calibration
curve. The estimation of phenolic compounds in the fractions was
carried out in triplicate and the results were averaged.
2.5. Determination of total avonoids
Total avonoid contents of pineapples were determined by using
the aluminium chloride colorimetric method as described by Willet
(2002), with some modications. Methanol extracts (0.5 mL), 10%
aluminium chloride (0.1 mL), 1 M potassium acetate (0.1 mL) and
distilled water (4.3 mL) were mixed after incubation at room
temperature for 30 min. The absorbance was measured at 415 nm
using a HITACHI, U-2000 spectrophotometer. Quercetin was used to
make the calibration curve. The estimation of total avonoids in the
extracts was carried out in triplicate and the results were averaged.
2.6. Evaluation of antioxidant capacity by phosphomolybdenum method
The total antioxidant capacity of ethyl acetate, methanol and
water extracts of pineapple was evaluated by the method of Prieto,
Pineda, and Aguilar (1999). An aliquot of 0.1 mL of sample solution
(100 g/mL) was combined with 1 mL of reagent solution (0.6 M
sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium
molybdate). The tubes were capped and incubated in a boiling
water bath at 95 C for 90 min. After the samples had cooled to room
temperature, the absorbance of the aqueous solution of each was
measured at 695 nm against blank. A typical blank solution contained
1 mL of reagent solution and the appropriate volume of the same
solvent used for the sample and it was incubated under same
conditions as rest of the sample. For samples of unknown composition, water soluble antioxidant capacity was expressed as equivalents
of ascorbic acid (mol/g of extract).
2.7. Antioxidant assay using -carotene-linoleate model system
The antioxidant activity of the pineapple extracts was evaluated using -carotene-linoleate model system as described by
Jayaprakasha, Singh, and Sakariah (2001) with some modication.
0.1 mg of -carotene in 0.2 mL of chloroform, 10 mg of linoleic acid

674

M.A. Hossain, S.M.M. Rahman / Food Research International 44 (2011) 672676

and 100 mg of Tween-20 (polyoxyethylene sorbitan monopalmitate)

were mixed. Chloroform was removed at 40 C under vacuum and the
resulting mixture was diluted with 10 mL of water and was mixed
well. To this emulsion, 20 mL of oxygenated water was added. Four
milliliter aliquots of the emulsion were pipetted into different test
tubes containing 0.2 mL of extracts (50 and 100 ppm) and BHA (50
and 100 g) in ethanol. BHA was used for comparative purposes. A
control containing 0.2 mL of ethanol and 4 mL of the above emulsion
was prepared. The tubes were placed at 50 C in a water bath and the
absorbance at 470 nm was taken at zero time (t = 0). Measurement of
absorbance was continued till the colour of -carotene disappeared in
the control tubes (t = 60 min) at an interval of 15 min. A mixture
prepared as mentioned above without -carotene served as blank. All
determinations were carried out in triplicate.
The antioxidant activity (AA) of the extracts was evaluated in
terms of bleaching of the -carotene using the following formula,

o
o

AA = 100 1Ao At = Ao At
where Ao and Aoo are the absorbance values measured at zero time of
the incubation for test sample and control, respectively. At and Aot are
the absorbance measured in the test sample and control, respectively,
after incubation for 60 min. The results were expressed in % basis of
preventing bleaching of -carotene.
2.8. Radical scavenging activity using DPPH method
Radical scavenging activity of the extracts was determined
essentially as described by Blois (1958) with some modication.
Different concentrations (25, 50 and 100 L equivalent to 25, 50 and
100 g, respectively) of the extracts and BHA (25, 50 and 100 g) were
taken in different test tubes. The volume was adjusted to 100 L by
adding MeOH. Five milliliters of 0.1 mM methanolic solution of
DPPH was added to these tubes and shaken vigorously. The tubes
were allowed to stand at 27 C for 20 min. The control was prepared
as above without any extract and MeOH was used for the baseline
correction. The changes in the absorbance of the samples were measured at 517 nm. Radical scavenging activity was expressed as the
inhibition percentage and was calculated using the following formula,
% radical scavenging activity = Control ODsample OD = Control OD 100:

2.9. Statistical analyses

Experimental results were mean S.D. of three parallel measurements and analyzed by SPSS 10 (SPSS Inc. Chicago, IL). Differences
between means were determined using Tukey multiple comparisons
and least signicant difference (LSD). Correlations were obtained by
Pearson correlation coefcient in bivariate correlations. P values b 0.05
were regarded signicant.
3. Results and discussion
The yields of ethyl acetate, methanol and water extracts of
pineapples were 4.9%, 21.5% and 4.3%, respectively. The total phenolic
contents of the extracts as determined by FolinCiocalteu method, are

Table 1
Phenolic contents (as caffeic acid equivalent) of pineapple extracts.
Extract

Phenolics (mg/g)

Water extract
Ethyl extract
Methanol extract

2.6 0.1
13.8 0.3
51.1 0.2

The values are means SD of three replicates.

reported as caffeic acid equivalents (Table 1). Among the three

extracts, methanol extract was containing highest (51.1%) amount of
phenolic compounds followed by ethyl acetate extract (13. 8%) and
water extract (2.6%). In recent studies, it has been reported that the
yield of extractable compounds was highest in methanol extract from
the peel and seeds of pomegranate in comparison with the solvents
such as ethyl acetate and water (Negi, Jayaprakasha, & Jena, 2002).
Furthermore, the extraction of phenolic compounds from the fruit is
commonly achieved with methanol or aqueous methanol (Antolovich,
Prenzler, Robards, & Ryan, 2000).
The levels of total phenolics determined in this way are not
absolute measurements of the amounts of phenolic compounds, but
are in fact based on their chemical reducing capacity relative to
caffeic acid. It has been observed that the phenol antioxidant index, a
combined measure of the quality and quantity of antioxidants in
vegetables (Elliot, 1999). In the present study the responses of the
extracts in this assay may arise from the variety and/or quantity of
phenolics found in three different extracts of pineapple. Fruit and
vegetables are the main sources of antioxidant vitamins (vitamin E,
vitamin C, precursor of vitamin A i.e., -carotene), which act as free
radical scavengers, making these foods essential to human health
(Elliot, 1999). However, more than 80% of the total antioxidant
capacity in fruits and vegetables comes from the ingredients other
than antioxidant vitamins, indicating the presence of other potentially
important antioxidants in these foods (Miller & Rice-Evans, 1997).
The phenolic compounds are the dominant antioxidants that exhibit
scavenging efciency on free radicals and reactive oxygen species are
numerous and widely distributed in the plant kingdom (Prior & Cao,
2000). In the present study, the relative antioxidant ability of the
pineapple extracts was investigated through some in vitro models
such as antioxidant capacity by phosphomolybdenum method, carotene-linoleate system, and radical scavenging activity using, , diphenyl--picrylhydrazyl (DPPH) method.
The result of total avonoid contents of the extracts of pineapple is
given in Table 2. The total avonoid contents varied from 39.4 to
55.2 mg quercetin/g weight. The variation may be due environmental
conditions, which can modify the constituents of the plant.
The antioxidant capacity of the extracts was measured spectrophotometrically through phosphomolybdenum method, which is
based on the reduction of Mo (IV) to Mo (V) by the sample analyte
and the subsequent formation of green phosphate/Mo (V) compounds
with a maximum absorption at 695 nm. The antioxidant capacity of
the extracts of pineapple was found to decrease in the order,
methanol extract N ethyl acetate extract N water extract (Table 3).
The antioxidant activity through -carotene-linoleate system of
pineapple extracts at 50 and 100 g/mL concentrations compared
with butylated hydroxyanisole is presented in (Table 4). The addition
of the extracts of pineapple and butylated hydroxyanisole at 50 g/mL
concentrations prevented the bleaching of -carotene to different
degrees. -Carotene in this model system undergoes rapid discolouration in the absence of an antioxidant. This is because of the coupled
oxidation of -carotene and linoleic acid, which generates free
radicals. The linoleic acid free radical formed upon the abstraction
of a hydrogen atom from one of its diallylic methylene groups attacks
the highly unsaturated -carotene molecules. As a result, -carotene
was oxidised and broken down in part, subsequently the system loses
its chromophore and characteristic orange colour, which can be
monitored spectrophotometrically. In our present study, the extracts
from pineapple fruits were found to hinder the extent of -carotene
bleaching by neutralizing the linoleate free radical and other free
radicals formed in the system. Methanol extract, ethyl acetate extract
and water extracts showed 84.3%, 55.4% and 51.8% antioxidant
activity, respectively, at 100 g/mL concentration.
The free radical scavenging activity of the fruit extracts of
pineapple was tested through DPPH method and the results are
presented in the (Fig. 1). The role of antioxidants is their interaction

M.A. Hossain, S.M.M. Rahman / Food Research International 44 (2011) 672676

Table 2
Total avonoids contents of pineapple extracts.
Extract

Total avonoids (mg quercetin/g)

Water extract
Ethyl extract
Methanol extract

39.4 1.1
37.9 0.3
55.2 0.2

The values are means SD of three replicates.

with oxidative free radicals. The essence of DPPH method is that

the antioxidants react with the stable free radical i.e., ,-diphenyl-picrylhydrazyl (deep violet colour) and convert it to ,-diphenyl-picrylhydrazine with discolouration. The degree of discolouration
indicates the scavenging potentials of the sample antioxidant. In
the present study the extracts of pineapple fruit were able to
decolourise DPPH and the free radical scavenging potentials of
the extracts were found to be in the order of methanol extract N ethyl
acetate extract N water extract. It has been found that cysteine,
glutathione, ascorbic acid, tocopherol, polyhydroxy aromatic compounds (hydroquinone, pyrogallol, etc.), and aromatic amines (pphenylene diamine, p-aminophenol etc.), reduce and decolourise
,-diphenyl--picrylhydrazyl by their hydrogen donating ability
(Blois, 1958). It appears that the extracts from the fruits of pineapple
possess hydrogen donating capabilities to act as antioxidant.
In our present study, the decreasing order of antioxidant activity
among the pineapple fruit extracts assayed through all the three
methods was found to be methanol extract N ethyl acetate extract N water extract. This order is similar to the phenolic contents of the
extracts that showed the extent of antioxidant activity of the extract is
in accordance with the amount of phenolics present in that extract. In
the present study it is found that the methanol extract of pineapple
fruit contains substantial amount of phenolics and it is the extent of
phenolics present in this extract being responsible for its marked
antioxidant activity as assayed through various in vitro models.
Several reports have conclusively shown close relationship
between total phenolic contents and antioxidative activity of the
fruits and vegetables (Deighton, Brennan, Finn, & Davies, 2000). Since
the chemical composition and structures of active extract components
are important factors governing the efcacy of natural antioxidants,
the antioxidant activity of an extract could not be explained on the
basis of their phenolic content, which also needs their characterization (Heinonen, Lehtonen, & Hopla, 1998). For instance, it has been
reported that phenolic compounds with ortho- and para-dihydroxylation or a hydroxy and a methoxy group are more effective than
simple phenolics (Shahidi & Naczk, 1995). However, synergistic or
additive actions of the phenolics present in the extracts cannot be
ruled out. So far we know this is the rst report that envisages the
antioxidant activities of tropical pineapple extracts. Hence the fruits of
pineapple could be a good source of antioxidant phenolics. Further
studies are warranted for the isolation and identication of individual
phenolic compounds and also in vivo studies are needed for a better
understanding of their mechanism of action as antioxidant.
Acknowledgements
The authors are grateful to the Chairman, Department of
Chemistry, Dhaka University, Dhaka for her continuous encourage-

Table 3
Antioxidant capacity of pineapple extracts by phosphomolybdenum method.
Extract

Antioxidant capacity (%) as equivalent to ascorbic

Water extract
Ethyl acetate extract
Methanol extract

612.1 12.4
1051.8 19.6
1933.0 9.1

The values are means SD of three replicates.

675

Table 4
Antioxidant activity of pineapple extracts and BHA by -carotene-linoleate model
system (% inhibition of bleaching of -carotene).
Extract/BHA

50 g

100 g

BHA
Water extract
Ethyl acetate extract
Methanol extract

93.0 0.2
28.7 1.1
43.9 0.4
62.4 0.2

97.5 0.1
51.8 0.5
55.4 0.3
84.3 0.2

Fig. 1. Radical scavenging activity of pineapple fruit extract by DPPH method.

ment during the work and the use of all laboratory facilities. Thanks
are also due to Mr. Shahidul Alam, M. Sc. student, Department of
Chemistry, Dhaka University, Dhaka, for his help to collect and
prepare the pineapple samples.

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