Antioxidant analysis of different parts of Carica papaya

Abstract

This study was conducted to compare the total antioxidant activity (TAA), total phenolic content (TPC) and total flavonoid content (TFC) from the different parts of papaya tree including their ripe and unripe fruit, seeds and the young leaves. Two methods namely DPPH radical scavenging activity and β-carotene bleaching assay were used to determine the TAA, whereas TPC was determined by Folin-Ciocalteu's method while TFC by aluminium trichloride (AlCl3). For these purposes, methanolic extracts (80%) were prepared. The results showed that the highest antioxidant activity through β-carotene bleaching assay was observed in unripe fruit (90.67 ± 0.29%) followed by young leave, ripe fruit and the seed. In other hand, young leaves exhibited a significant higher scavenging effect compared to others and the dose required in reducing the absorbance of DPPH control solution by 50% (EC50) was calculated at 1.0 ± 0.08mg/ml. The EC50 values were 4.3 ± 0.01mg/ml, 6.5 ± 0.01mg/ml and 7.8 ± 0.06mg/ml for unripe fruit, ripe fruit and seeds respectively. Interestingly, both TPC and TFC also showed that young leaves had the highest antioxidant content (424.89 ± 0.22mg GAE/ 100 g dry weight and 333.14 ± 1.03mg rutin equivalent/ 100 g dry weight, respectively). Statistically, Pearson correlation showed there were positive correlations between TPC and TFC with antioxidant activity assayed by DPPH radical scavenging assay (r=0.846 and r=0.873, respectively). However there was no correlation between TPC and TFC with β-carotene bleaching activity. In brief, taken into account all the parameters measured, antioxidants were highly remarkable in the sequence of young leaves > unripe fruit > ripe fruit > seed. Nevertheless, further investigation for isolation and identification of the phytoconstituents responsible for antioxidant activity is desirable.

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*Corresponding author.
Email: asmah@medic.upm.edu.my
International Food Research Journal 20(3): 1043-1048 (2013)
Journal homepage: http://www.ifrj.upm.edu.my
1Maisarah, A.M., 1Nurul Amira, B., 1*Asmah, R. and 2Fauziah, O.
1Department of Nutrition and Dietetics, 2Department of Human Anatomy,
Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400, Serdang,
Selangor, Malaysia
Antioxidant analysis of different parts of Carica papaya
Abstract
This study was conducted to compare the total antioxidant activity (TAA), total phenolic content
(TPC) and total avonoid content (TFC) from the different parts of papaya tree including their
ripe and unripe fruit, seeds and the young leaves. Two methods namely DPPH radical scavenging
activity and β-carotene bleaching assay were used to determine the TAA, whereas TPC was
determined by Folin-Ciocalteu’s method while TFC by aluminium trichloride (AlCl3). For
these purposes, methanolic extracts (80%) were prepared. The results showed that the highest
antioxidant activity through β-carotene bleaching assay was observed in unripe fruit (90.67 ±
0.29%) followed by young leave, ripe fruit and the seed. In other hand, young leaves exhibited
a signicant higher scavenging effect compared to others and the dose required in reducing the
absorbance of DPPH control solution by 50% (EC50) was calculated at 1.0 ± 0.08mg/ml. The
EC50 values were 4.3 ± 0.01mg/ml, 6.5 ± 0.01mg/ml and 7.8 ± 0.06mg/ml for unripe fruit, ripe
fruit and seeds respectively. Interestingly, both TPC and TFC also showed that young leaves
had the highest antioxidant content (424.89 ± 0.22mg GAE/ 100 g dry weight and 333.14
± 1.03mg rutin equivalent/ 100 g dry weight, respectively). Statistically, Pearson correlation
showed there were positive correlations between TPC and TFC with antioxidant activity assayed
by DPPH radical scavenging assay (r=0.846 and r=0.873, respectively). However there was
no correlation between TPC and TFC with β-carotene bleaching activity. In brief, taken into
account all the parameters measured, antioxidants were highly remarkable in the sequence of
young leaves > unripe fruit > ripe fruit > seed. Nevertheless, further investigation for isolation
and identication of the phytoconstituents responsible for antioxidant activity is desirable.
Introduction
Carica papaya (C. papaya) belongs to the family
of Caricaceae, and several species of Caricaceae
have been used as medication against a variety of
diseases (Mello et al., 2008). It was originally derived
from the southern part of Mexico, C. papaya is a
constant plant and it is presently distributed over the
whole tropical area. All parts of the papaya plant can
be used as medicine; the fruit esh, owers, seeds
and the owers. Many scientic investigations have
been conducted to evaluate the biological activities
of various part of C. papaya including their fruits,
shoots, leaves, rinds, seeds, roots or latex.
The major groups of phytochemicals that have
been suggested as a natural source of antioxidants
may contribute to the total antioxidant activity of
plant materials including polyphenols, carotenoid
and traditional antioxidant vitamins such as vitamin
C and E. Antioxidant is any substance that when
present at low concentration compared to those of an
oxidisable substrate signicantly delays or prevents
oxidation of that substrate (Halliwell et al., 1995).
Antioxidant functions are associated with decreased
DNA damage, diminished lipid peroxidation,
maintained immune function and inhibited malignant
transformation of cells (Gropper et al., 2009). Several
studies showed that phenolic compounds are the
major bioactive phytochemicals with human health
benets (Cao et al., 1996). In fact, many authors have
reported a direct relationship between total phenolic
content and antioxidant activity in numerous seeds,
fruits and vegetables (Yang et al., 2009).
These present ndings can contribute to the
increasing database for the medicinal plant or
could be used as antioxidant in food and medicinal
preparations. Thus, the aim of the study was to
determine the total antioxidant activity (TAA), total
phenolic content (TPC) and total avonoid content
(TFC) from the different parts of papaya tree including
their ripe and unripe fruit, seeds and the young leaves.
Two methods namely DPPH radical scavenging
Keywords
Antioxidant activity
total phenolic content
total avonoid content
Carica papaya
Article history
Received: 7 August 2012
Received in revised form:
4 January 2013
Accepted: 10 January 2013

1044 Maisarah et al./IFRJ 20(3): 1043-1048
activity and β-carotene bleaching assay were used
to determine the antioxidant activity to evaluate
the relationship with the TPC and TFC. For these
purposes, methanolic extracts (80%) were prepared
and TPC were determined by Folin-Ciocalteu’s
method while TFC by aluminium trichloride (AlCl3)
method.
Materials and Method
Plant materials
Samples of the ripe, unripe, seeds, and the
young leaves were obtained from Taman Pertanian
Universiti Putra Malaysia (UPM), Serdang, Selangor,
Malaysia.
Chemicals
β-carotene, linoleic acid, Tween 20, α-tocopherol,
gallic acid, 2,2-diphenyl-2-picrylhydrazyl (DPPH)
and rutin were purchased from Sigma chemical Co.
(St. Louis, MO, USA). Folin-Ciocalteu reagent,
sodium bicarbonate, aluminum chloride and methanol
were purchased from Merck (Darmstadt, Germany),
ascorbic acid (Fluka, Switzerland) and chloroform
was from Fisher Scientic (Loughborough, UK).
Preparation of sample extracts
Before analysis, each of the samples was
immediately washed several times with tap water,
followed by rinsed it with deionized water to ensure
that all contaminants were removed. The samples
were then individually prepared where the edible
portion (ripe and unripe) were all diced or cut into
small pieces prior to packing and stored at -80°C
for three consecutive days. Subsequently they were
lyophilised in a freeze-dryer (Virtis route, Gardiner,
New York). The lyophilized samples were ground to a
ne powder and packed in air tight containers before
stored in -20°C until required for further analyses.
The ground samples were extracted with 80%
aqueous methanol (w/v, 1:25) at 200 rpm for 2 hour
at ambient temperature with continuous stirring
in a dark bottle using an orbital shaker (Heidolph
Unimax 1010, Shcwabach, Germany). The mixture
was ltered through a lter paper (Whatman No. 4).
The obtained solutions were then used for TAA and
TFC content.
Determination of antioxidant activity
β-carotene bleaching assay
The antioxidant capacity of each of the sample
extracts was estimated by the β-carotene bleaching
method following the procedure described by Velioglu
et al. (1998) with modications. One milliliter of
β-carotene (0.2mg/ml chloroform), linoleic acid
(0.02 ml) and Tween 20 (0.2 ml) were added to
0.2ml of sample extracts, standard (α-tocopherol)
and control (80% methanol). Thereafter, chloroform
was evaporated to dryness under vacuum using
rotary evaporator. After evaporation, 100 ml of
deionized water was added into the mixture and
shaken vigorously until emulsion was obtained. Two
milliliters of aliquots of the emulsions were pipetted
into the test tubes and immediately placed in water
bath at 45°C for 2 hours. The absorbance was read
at 20 min interval at 470 nm, using a UV-visible
spectrophotometer (Secomam, Anthelie Advanced
5) at initial time (t=0). Degradation rate (dr) of the
sample was calculated according to the rst order
kinetics as described by Al-Saikhan et al. (1995):
dr of sample = (ln [A0/At]) / t
where: ln = natural log; Ao = initial absorbance at time
0; At = absorbance at 20 min of incubation; t = 120
min and dr = degradation rate. Antioxidant activity
(AA) was expressed as percent of inhibition relative
to the control by using the equation:
AA% = ([dr control – dr sample] / dr control) x 100
Free radical scavenging assay
Effect of the sample extracts on DPPH radical
was measured by using a slightly modied method
previously described by Tang et al. (2002). Amount
of 200 µl of the sample extract (0.62 – 4.96 mg/ml)
or ascorbic acid (0.04 – 1.28 mg/ml) were added to 1
ml of 0.1 mM DPPH in 80% methanol. The mixture
was shaken vigorously and left to stand in dark room
for 30 min at room temperature. Absorbance of the
solution was measured spectrophotometrically at 517
nm with deionized water as blank. The capability of
sample to scavenge the DPPH radical was calculated
according to the equation as follows:
Scavenging effect (%) =
1 – Absorbance of sample at 517nm x 100
Absorbance of control at 517nm
Total phenolic content
Total phenolic content was estimated according
to the Folin Ciocalteu method following the modied
procedure described by Singleton and Rossi (1965).
Each freeze-dried sample (200 mg) was extracted
with 2 ml of 80% methanol at room temperature for
2hours by using an orbital shaker at 200 rpm. The
mixture was then centrifuged at 1000 rpm for 15 min.
An aliquot (200 µl) of the supernatant was mixed with
1.5 ml of Folin-Ciocalteu reagent (previously diluted
10 fold with distilled water) and allow standing at
room temperature. Following 5 min, 1.5 ml of 6%
(w/v) sodium bicarbonate solution was added to

Maisarah et al./IFRJ 20(3): 1043-1048 1045
the mixture. Following 90 min, the absorbance was
read spectrophotometrically at 725 nm. The standard
calibration (0.01 – 0.05 mg/ml) curve of Gallic acid
in 80% methanol curve was plotted. Results were
expressed as small cap Gallic acid equivalents (GAE)
in mg per 100 g sample extracts.
Total avonoid content
Total avonoid content was determined by the
aluminum chloride colometric assay according
to method described by Meda et al. (2005). Five
milliliters of 2% aluminum trichloride (AlCl3) in
methanol was mixed with the same volume of the
extract solution at the concentration of 0.4 mg/ml.
Following 10 min, the absorbance was taken against
a blank that consist of the same solution but without
the AlCl3 at 415 nm using UV-spectrophotometer.
Total avonoid content is expressed as mg of rutin
equivalents/ 100 g of sample extract.
Statistical analysis
Results were expressed as mean ± standard
deviation of three determinations. Independent T-test
and one way analysis of variance (ANOVA) combined
with Bonforroni’s post-hoc comparison were used
to determine the differences of means among the
samples. Pearson correlation test was used to assess
the correlation between TAA and TPC. A signicant
difference was considered at the level of p<0.05.
Results
Beta-carotene bleaching assay
The comparable β-carotene bleaching rates of
the control, α-tocopherol (standard) and methanolic
extracts of different part of papaya fruit are shown in
Figure 1. The β-carotene bleaching method is one of
the most frequently applied methods for determining
the total antioxidant property of the extracts. In the
β-carotene bleaching assay, linoleic acid produces
hydroperoxides as free radicals during incubation at
50°C and attacks the β-carotene molecules that cause
reduction in the absorbance at 470 nm. Beta-carotene
in the systems undergoes rapid discoloration in the
absence of antioxidant and vice versa in its presence.
The presence of different antioxidants can delay the
extent of β-carotene bleaching by neutralizing the
linoleate free radical and other free radicals formed
in the system (Jayaprakash et al., 2003). Thus, the
degradation rate of β-carotene–linoleate depends on
the antioxidant activity of the extracts.
The result showed the control had a substantial
and rapid oxidation of β-carotene. Accordingly, the
absorbance decreased rapidly in samples without
antioxidant, while the sample extracts with the
presence of antioxidant retained their color and also
absorbance for a longer time.
Table 1 shows the mean antioxidant activity
based on the β-carotene bleaching rate of the extracts
of different parts of the papaya plant (ripe, unripe,
young leaves and seed). The extract with the lowest
β-carotene degradation rate exhibit the highest
antioxidant activity. All extracts had lower antioxidant
activities than had standard (α-tocopherol). The
highest antioxidant activity among the samples was
observed in unripe fruit whereas seed had the lowest
antioxidant activity. Result showed that there was
considerably variation in the antioxidant activities
where it ranges from the lowest of 58% to the highest
of 91% where the orders of the antioxidant activity
are as follow: α-tocopherol > unripe fruit > young
leaves > ripe fruit > seed.
Reactive scavenging activity
The idea of a single measurement of total
antioxidant capacity is insufcient. There is various
antioxidant activity methods have been used to
evaluate and compare the antioxidant activity of
foods. Therefore, in this study, radical scavenging
activity was determined for the selected parts of
papaya plant. Being a stable free radical, the DPPH
assay is a simple and rapid method frequently used to
evaluate the ability of antioxidants to scavenge free
radicals. It gives reliable information concerning the
antioxidant ability of the tested compounds to act as
free radical scavengers or hydrogen donors (Huang
et al., 2005).
The odd electron in DPPH free radicals gives
a strong absorption maximum at 517 nm (Azizah
Figure 1. Degradation rate of different parts of papaya extracts assayed
by β-carotene bleaching assay. Values are expressed as mean ± standard
deviation (n=3). α-tocopherol was used as the standard
Table 1. Means of antioxidant activity of selected samples and standard
assayed by β-carotene linoleate bleaching
Pa pay a pla nt Antioxidant a c tiv it y (%)
Ri pe
Unripe
Seed
Leaves
Standard
88.12 ±0.41
b
90.67 ±0.29
b
58.97 ±1.08
c
90.01 ±0.44
b
96.73 ±0.08
a
Values are expressed as mean ± standard deviation (n=3). Different letters indicate there are
signicant differences (p>0.05)

1046 Maisarah et al./IFRJ 20(3): 1043-1048
et al., 1999). When DPPH free radicals becomes
paired with hydrogen from a free radical scavenging
antioxidant, its purple color fades rapidly to yellow
to form reduced DPPH-H (Yamagushi et al., 1998).
The resulting decolorization is stoichiometric with
respect to number of electrons captured.
There were reductions in the concentration of
DPPH due to the scavenging activity of the antioxidant
found in the sample extracts. At the concentration of
8mg/ml, the scavenging effects of methanol extract
of selected parts of the papaya plant and standard
decreased in the order: ascorbic acid > young leaves
> unripe fruit > ripe fruit > seed (Figure 2). The
young leaves exhibited a signicant higher (p<0.05)
scavenging effect compared to others and this was
in agreement with Runnie et al. (2004), where the
nding suggested that the methanolic leaves extract
demonstrated vasodilatory and antioxidant effects,
both implicated in the reduction of cardiovascular
disease.
As shown in Figure 2, all the sample extracts
exhibited signicant dose dependent inhibition of
DPPH activity that rapidly increase from 1 to 4mg/
ml. Scavenging effect increases as the concentration
of the sample increased until reached a plateau at 4
mg/ml. Table 2 shows the dose of young leaves extract
that required in reducing the absorbance of DPPH
control solution by 50% (EC50) was calculated at 1.0
± 0.08 mg/ml. The EC50 values were 6.5 ± 0.01 mg/
ml, 4.3 ± 0.01 mg/ml and 7.8 ± 0.06 mg/ml for ripe
fruit, unripe fruit and seeds respectively. This showed
that the young leaves exhibit a strong scavenging
activity and it has been reported that phytochemicals
especially plant phenolics constitute a major group of
compounds that act as primary antioxidant (Hatano et
al., 1989). Their protection mechanisms are through
the reaction with the oxygen radicals, superoxide
anion radicals and lipid peroxyl radicals.
Total phenolic content and total avonoid content
Phenolic compounds are widely distributed in
plants (Li et al., 2006), which have gained greatly
attention, due to their antioxidant activities and
free radical-scavenging abilities, which potentially
have benecial implications for human health
(Govindarajan et al., 2007). The TPC was determined
in comparison with standard gallic acid and the results
were expressed in terms of mg gallic aid equivalent
(GAE)/ 100 g dry sample.
This study showed that the selected parts of the
papaya plant varied signicantly. It ranged from
30.32 ± 6.90 to 424.89 ± 0.22 mg GAE/ 100 g dry
weight (Table 3). The TPC was observed in the
selected papaya plant as: young leaves > unripe > ripe
> seed. The result also indicates that the young leaves
contained high phenolic content that may provide
good sources of dietary antioxidant. For this reason,
it is obvious that TPC present in the samples have
strong effects against the scavenging activity rather
than discoloration of β-carotene. However, Khamsah
et al. (2006) found that the radicals scavenging
activity is not only due to the phenolic content itself,
but with other various antioxidant compounds. They
respond differently depending on the number of
phenolic groups that they have (Singleton and Rossi,
1965). More to the point, TPC does not incorporate
necessarily to all the antioxidants that may present in
the extracts. Therefore, sometimes there is a vague
correlation between TPC and antioxidant activity of
several plant species (Tawaha et al., 2007).
Other than that, TFC of the extracts in terms of
rutin equivalent/ 100 g dry weight (standard curve
Table 2. Means of EC50 of DPPH radical scavenging activities of selected
samples
Figure 2. Scavenging effect of different parts of C. papaya extracts on
DPPH radicals. Values are expressed as mean ± standard deviation (n=3).
Ascorbic acid was used as the standard
Pa pay a pla nt EC
50
(mg/ml)
Ri p e
Unripe
Seed
Leaves
6.5 ±0.01
c
4.3 ±0.01
b
1.0 ±0.08
a
7.8 ±0.06
d
Values are expressed as mean ± standard deviation (n=3). Different letters in columns indicate
there are signicant differences (p>0.05)
Table 3. Means of total phenolic content (TPC) of selected samples
Pa pa ya p lan t (mg GAE/100g dry weight)
R ip e
Unripe
Seed
Leaves
272.66 ±1.53 c
339.91 ±9.40 b
30.32 ±6.90 d
424.89 ±0.22 a
Values are expressed as mean ± standard deviation (n=3). Different letters in columns indicate
there are signicant differences (p>0.05)
Table 4. Means of total avonoid content (TFC) of selected samples
Values are expressed as mean ± standard deviation (n=3). Different letters in columns indicate
there are signicant differences (p>0.05)
Pa pa ya p lan t (mg GAE/100g dry weight)
Ri p e
Unripe
Seed
Leaves
92.95 ±7.1 2
a
53.44 ±6.6 3
b
59.54 ±12.23
b
333.14 ±11.02
c

Maisarah et al./IFRJ 20(3): 1043-1048 1047
equation: y = 3.021x + 0.0831, R2 = 0.9975) were
between 53.44 ± 6.64 and 333.14 ± 1.03mg rutin
equivalent/ 100 g dry weight as shown in Table 4.
In recent years, studies have shown that papaya fruit
contains not only vitamins and other nutrients but
also contains biologically avonoids (Wang et al.,
2008).
Correlations
Previous study reported that antioxidant activity
of plant material is very well correlated with the
content of phenolic compounds (Velioglu et al.,
1998). Contribution of phenolic compounds is one of
the mechanisms of the overall antioxidant activities.
This mainly due to their redox properties involve
in the plant material. Generally, the mechanisms
of phenolic compounds for antioxidant activity
are neutralizing lipid free radicals and preventing
decomposition of hydroperoxides into free radicals
(Li et al., 2006).
Pearson correlation showed there was a
positive correlation relationship between phenolic
content and antioxidant activity assayed by DPPH
radical scavenging assay (r=0.846). Conversely,
no correlation was found for antioxidant activity
assayed by β-carotene bleaching assay with phenolic
content. This was in agreement with Motalleb et al.
(2005), where they also did not nd any relationship
between antioxidant activity and phenolic content in
B. vulgaris fruit extract.
The same pattern was observed in the relationship
of TFC with the total antioxidant activity. A direct
correlation between radical scavenging activity and
TFC of the samples was successful to demonstrate by
linear regression analysis (r=0.873). It is known that
avonoid with a certain structure and particularly
hydroxyl position in the molecule can act as proton
donating and show radical scavenging activity (Hou
et al., 2003). However there was no correlation
between total avonoid content and β-carotene
bleaching activity.
Conclusion
The study clearly indicates that it is vital to measure
the antioxidant activity using various radicals and
oxidation systems and to take both phenolic content
and antioxidant activity into account while evaluating
the antioxidant potential of plant extracts. The results
obtained in this work have considerable value with
respect to the antioxidant activities of the selected
parts of the C. papaya plant. In brief, by taken into
account all the parameters measured, antioxidants
were highly remarkable in the sequence of young
leaves > unripe fruit > ripe fruit > seed. Moreover,
there is a strong positive correlation between TPC
and TFC with free radical scavenging activity, thus
showing its promising potential to be exploited as
primary antioxidant. The correlations also support
that the mechanism of action of the extracts for
the antioxidant activity may be identical, being
related with the content in phenols and avonoid
compounds, and their free-radical scavenging activity.
Nevertheless, further investigation for isolation and
identication of the phytoconstituents responsible for
antioxidant activity is desirable.
Acknowledgement
The authors thank the Universiti Putra Malaysia
under RUGS initiative 6 grant scheme (Vote number:
9199607) for the nancial support.
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