International Journal of Food Properties

ISSN: 1094-2912 (Print) 1532-2386 (Online) Journal homepage: http://www.tandfonline.com/loi/ljfp20

Antioxidant Activity of Different Extracts of Red

Pitaya (Hylocereus polyrhizus) Seed

Liana Adnan , Azizah Osman & Azizah Abdul Hamid

To cite this article: Liana Adnan , Azizah Osman & Azizah Abdul Hamid (2011) Antioxidant Activity
of Different Extracts of Red Pitaya (Hylocereus polyrhizus) Seed, International Journal of Food
Properties, 14:6, 1171-1181, DOI: 10.1080/10942911003592787

To link to this article: https://doi.org/10.1080/10942911003592787

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International Journal of Food Properties, 14:1171–1181, 2011
Copyright © Taylor & Francis Group, LLC
ISSN: 1094-2912 print / 1532-2386 online
DOI: 10.1080/10942911003592787

ANTIOXIDANT ACTIVITY OF DIFFERENT EXTRACTS

OF RED PITAYA (HYLOCEREUS POLYRHIZUS) SEED

Liana Adnan, Azizah Osman, and Azizah Abdul Hamid

Department of Food Science, Faculty of Food Science and Technology, Universiti
Putra Malaysia, Serdang, Selangor, Malaysia

Antioxidant activity of three different extracts (ethanolic, chloroformic, and hexanic) of

red flesh pitaya (Hylocereus polyrhizus) seed using free radical scavenging assay, linoleic
acid model system, and ferric thiocyanate (FTC) method was determined. Ethanolic extract
inhibit 74.76% of free radicals at 1000 µg/mL, while chloroformic extract gave the high-
est inhibition using linoleic acid model system (98.90% at 100 µg/mL) and FTC (96.34%)
method. Total phenolic and ascorbic acid contents of the seed were 13.56 ± 2.04 and 0.36 ±
0.01 mg/g, respectively, while catechin was the major flavonoid detected. In conclusion,
the study showed that both polar and non-polar compounds contribute to the antioxidative
activity measured.

Keywords: Red pitaya seed, Antioxidant activity, Free radical scavenging assay, Linoleic
acid model system, Ferric thiocyanate method.

INTRODUCTION
Cardiovascular diseases, diabetes, cancer, arthritis, and many other diseases suf-
fered by the patients may be caused by genetic or the age factors.[1] However, personal
lifestyle that includes dietary intakes and physical activity, along with environmental
surroundings, may also contribute to the risk of the diseases. In addition, free radicals
produced by our body inducing oxidative stress is related to several other harmful effects,
including coronary heart diseases, neodegenerative disorders, and aging.[2] Consumption
of fresh fruits and vegetables are inevitably important to help prevent the action of free
radicals.[2]
For many years, fruits have become the main subject for researchers to investigate for
their bioactive compounds that are beneficial for human health and red pitaya fruit is a new
promising one. The red pitaya (Hylocereus polyrhizus) fruit, that is known also as pitahaya,
and dragon fruit fall under the Cactaceae family under the genus Hylocereus.[3] This exotic
fruit is gaining much interest nowadays due to its unique taste and shape, along with the
attractive colour. Currently, researchers are more interested in the red pigment of pitaya
fruits, known as betacyanin that falls under the group of betalain, which can be used to

Received 17 September 2009; accepted 1 January 2010.

Address correspondence to Azizah Osman, Department of Food Science, Faculty of Food Science
and Technology, Universiti Putra Malaysia, Serdang, Selangor, 43400 Malaysia. E-mail: azosman@putra.upm.
edu.my; azizahmasahuling@yahoo.com

1171
1172 ADNAN, OSMAN, AND ABDUL HAMID

replace synthetic dyes. Extensive research had been carried out on the pigment[4,5] includ-
ing the beneficial effect of betalains as antiradical and antioxidative agent.[6–8] However,
the studies conducted on the antioxidative activity of red pitaya fruit[9,10] were mainly on
its pulp and peel. Recently, there was a report on the essential fatty acids found in the
red and white pitaya seeds, in particular, linoleic acid.[11] Nevertheless, there is negligi-
ble report on the antioxidative activity of pitaya seeds, as well as their potential bioactive
compounds.
Seeds from other fruits, such as the grape seeds,[12,13] berries,[14] and from a
Cactaceae family, Opuntia sp.,[15] are potent antioxidative agent. They are abundant with
various compounds ranging from hydrophilic to lipophilic, such as flavonoids, phenolic
acids, carotenoids, tocopherols, and essential fatty acids that may prevent the oxidative
damage done by the free radicals. The grape seed procyanidin is renowned as a good
antioxidative agent,[16] and the grape seed oil were available commercially due to its good-
ness comparable to vitamin E in reducing lipid oxidation and scavenging free radicals.
Hence, this study was conducted to determine the antioxidant activity of the red pitaya
seed through extraction using solvents of different polarities and to examine the potential
bioactive compounds responsible for the activity.

MATERIALS
Chemicals
Tween 20, 3-t-butyl-4-hydroxyanisole (BHA), ferrous chloride, 1,1-diphenyl-2-
picrylhydrazyl (DPPH), disodium hydrogen phosphate, sodium dihydrogen phosphate,
and α-tocopherol were obtained from Sigma (St. Louis, MO, USA). Ammonium thio-
cyanate, Folin-Ciocalteu reagent, sodium carbonate, citric acid, and trifluoroacetic acid
(HPLC grade) were purchased from Fisher Scientific (Leicestershire, UK). Both analytical
and HPLC grade methanol and 95% ethanol were from BDH (VWR International Ltd.,
England), while chloroform and n-hexane were from Merck (Darmstadt, Germany). Gallic
acid and linoleic acid were from Acros Organic (NJ, USA).

METHODS
Sample Preparation and Extraction
The red pitaya fruits were obtained from a pitaya farm in Sepang, Selangor. Upon
arrival at the laboratory, the fruits were peeled and the seeds were separated from the pulp
manually and washed with tap water several times to remove mucilages and pulp left-
over. The seeds were air-dried, ground, and sieved (<0.85 mm particle sized). Some of the
ground seed were kept in a capped-bottle for determination of ascorbic acid and flavonoids
content, while the others were extracted with solvents of different polarity, namely 95%
ethanol, chloroform, and n-hexane. Extraction was carried out, according to Siddhuraju
and Manian,[17] with some modifications. Briefly, 20 g of the ground seed were extracted
with 200 mL solvent and extraction were carried out in an Innova 4000 incubator shaker
(New Brunswick Scientific, NJ, USA) at 30◦ C, 150 rpm for 24 h. The extracts were vacuum
filtered through Whatman No. 4 filter paper and rotary evaporated (Büchi, Switzerland)
at 40◦ C to remove the solvents. The extracts were stored at −20◦ C until further
analysis.
 ANTIOXIDANT ACTIVITY OF RED PITAYA SEED 1173

DPPH Free Radical Scavenging Activity

The antioxidative activities of the different extracts were measured using the
DPPH free radical scavenging assay.[18] 100 µL of extracts with different concentrations
(200–1000 µg/mL) were mixed with 3.9 mL DPPH• (25 mg/L) in methanolic solution
and the reaction mixture were allowed to stand for 30 min in the dark before measuring the
optical density at 515 nm. Methanol served as a blank, whereas the control is prepared with
100 µL methanol with DPPH solution. The antioxidant activity is expressed as percentage
of inhibition of the free radical calculated using this equation:

% inhibition = (Acontrol − Asample ) × 100/Acontrol

A higher percentage of inhibition indicated a better antioxidant. BHA served as a positive

control.

Antioxidant Activity in Linoleic Acid Model System

Formation of conjugated diene was determined to measure the antioxidative activity
of the extracts.[19] The range of concentration used in this assay was 20–100 µg/mL. BHA
served as a positive control. The linoleic acid emulsion was prepared by mixing 10 mM
linoleic acid and Tween 20 (with the same amount as linoleic acid) with 0.2 M sodium
phosphate buffer, pH 6.5. The antioxidant activity (AOA%) was calculated as follow:

AOA% = [(A234 control − 234 sample)/234 control] × 100%

where A234 control and 234 sample is the differences in absorbance of the control and
test sample respectively, measured before and after incubation.

Ferric Thiocyanate Assay

The linoleic acid peroxidation was determined using the ferric thiocyanate (FTC)
method.[20] Preparation of linoleic acid emulsion was made by mixing 500 µL of 1 mg/mL
of different extracts, α-tocopherol, and BHA with 2.5 mL linoleic acid emulsion and
2.5 mL phosphate buffer pH 7.0 in an amber capped-vial. Reaction mixture without the
test sample served as a control. All the test samples were incubated at 40◦ C and measured
every day until the absorbance of the control reached a maximum. The measurement of the
antioxidant activity was carried out by withdrawing 100 µL aliquots from the test samples
and mixed with 5 mL of 75% ethanol, 100 µL of ammonium thiocyanate, and 100 µL of
20 mM ferrous chloride in 3.5% HCl. The changes in colour of the mixtures were mea-
sured at 500 nm exactly after 3 min. Lipid peroxidation inhibition (LPI) was calculated up
until day three of measurement as follows:

LPI(%) = [1 − (Absorbance of sample/Absorbance of control)] × 100

Determination of Total Phenolic Content

Determination of total phenolic content in the seed was carried out following the
Folin-Ciocalteu method by Singleton and Rossi[21] as described by Peschel et al.[22]
1174 ADNAN, OSMAN, AND ABDUL HAMID

100 µL of the ethanolic extract (1 mg/mL) was mixed up with 7.9 mL distilled water and
0.5 mL Folin-Ciocalteu’s reagent. After 2 min, 1.5 mL of 7.5% sodium carbonate solution
was added in and mixed thoroughly. The sample was measured spectrophotometrically
(Shimadzu, Japan) at 765 nm after 2 h of incubation. The amount of total phenolics was
determined as gallic acid equivalent (GAE) and expressed as mg GAE/g seed dry weight.

Ascorbic Acid Content

Determination of ascorbic acid content in the seed was carried out using Waters
2487 dual wavelength absorbance detector HPLC system (Waters Associates, Milford,
MA). Quantification of the ascorbic acid was performed using the Waters reversed-phase
Symmetry C18 column (150 × 3.9 mm i.d., 5 µm) at 25◦ C. Mobile phase used was acetoni-
trile:methanol (70:30 v/v) that was run isocratically at flow rate of 1 mL/min by Waters
600 pump. The wavelength was set at 254 nm with 20 µL injection volume. Extraction
of ascorbic acid was carried out by homogenizing 1 g dried seed with 20 mL 3% citric
acid for 3 min.[23] The extract and ascorbic acid standard were filtered through Waters C18
Sep-Pak cartridge before refiltered with 0.45 µm Whatman nylon membrane filter prior to
HPLC analysis. Solvents used were of HPLC grade. Identification was done based on the
retention time and absorbance spectra of ascorbic acid standard used while quantification
was done using a standard curve (0–100 µg/mL).

Flavonoids Content
Several flavonoids in the seed (catechin, epicatechin, quercetin, myricetin,
kaempferol, and rutin) were detected and quantified according to the modified method[24]
of Hertog et al.[25] using the HPLC system and column mentioned before. The flavonoids
content were compared with a known standard and expressed as mg flavonoid/g seed
dry weight. Two mobile phases were used. Solvent A consisted of triflouroacetic acid in
deionized water, pH 2.5 whereas solvent B is made up of 100% methanol and runs using
a linear gradient: at 0 min, 100% solvent A; at 20 min, 50% solvent A and 50% solvent
B; at 30 min, 100% solvent B; and at 35–40 min, 100% solvent A. The wavelength was
set at 280 nm with 20 µL injection volume. Extraction of the compounds was done by
hydrolyzing 1 g of seed with 12 mL methanol, 8 mL deionized water, and 5 mL 6 M HCl at
95◦ C for 2 h. The test sample was allowed to cool down to room temperature before being
filtered with 0.45 µm Whatman nylon membrane filter. Solvents used were of HPLC grade.

Statistical Analysis
Statistical analysis of the data were subjected to a one way analysis of variance and
the significant difference was determined by Tukey’s multiple range test (P < 0.05) using
the Minitab Release 14 (Minitab Inc., PA, USA). All analyses were done in triplicate and
data were expressed as means ± SD.

RESULTS AND DISCUSSION

Extraction Yield
Three solvents with different polarities were chosen for extraction of the red pitaya
seed. Extraction using ethanol attained both solid and oil fraction since the seed was not
defatted before analysis, whereas for non-polar solvents, chloroform and hexane, only the
 ANTIOXIDANT ACTIVITY OF RED PITAYA SEED 1175

Table 1 Extraction and percent yield of the red pitaya seed extracts using different
solvents.

Extract Extraction yielda (g) Yieldb (%)

Ethanol 3.08 ± 0.51 15.4c

Chloroform 6.99 ± 0.51 34.9d
Hexane 5.38 ± 0.34 26.9e
a Each value is expressed as means ± SD of three independent extractions (n = 3).
within a column are significantly different (P < 0.05)
b Means with different letters(c,d,e)

as determined by Tukey’s multiple range test.

oil fractions were extracted out. Defatting of sample may reduce the bioactive compounds
in the seed since some of the compounds were probably eliminated out and lessen the total
phenolic content as reported by Yilmaz and Toledo[16] against the de-oiling of Muscadine
seed. Result obtained in Table 1 show the extraction yield; in decreasing order with chlo-
roform > hexane > ethanol. From the result, around 27–35% of oil in the seed was
extracted through extraction using chloroform and hexane. Chloroformic extract gained
the highest (p < 0.05) yield with 34.9% and 2.27-fold to that of ethanolic extract (15.4%).
Hexanic oil extract was 26.9% and even higher than the caneberry seed oil (11–19% oil)
extracted using the same solvent.[14] Nevertheless, high yield does not necessarily imply
that it will also give high antioxidative activity as proven by several researchers,[12 ,13 ,26]
since the antioxidative activity depends on the potent antioxidant present in the
extract.

DPPH Free Radical Scavenging Activity of the Extracts

DPPH assay is one of the widely used methods to test the antioxidative activity of the
sample due to its stability, simplicity, and the short time required for analysis. Basically,
the antioxidant capacity of the samples are measured through their ability to reduce the
DPPH• by donating the hydrogen atom and can be determined through the discoloration of
the mixture using the spectrophotometer.[18] It can be seen in Fig. 1 that only the ethanolic

120

100

80
% inhibition

60

40

20

0
0 200 400 600 800 1000
Concentration (µg/ml)

Ethanolic extract Chloroformic extract Hexanic extract BHA

Figure 1 The antioxidant activity of the red pitaya seed extracts as measured by DPPH• assay.
1176 ADNAN, OSMAN, AND ABDUL HAMID

extract showed the inhibitory effect towards the free radicals with high antioxidant activ-
ity in a dose-dependent behaviour as the chloroformic and hexanic extracts showed a very
limited activity. At 1000 µg/mL, the ethanolic extract scavenged 74.76% of DPPH free
radicals and is significantly (p < 0.05) different as compared to the chloroformic and hex-
anic extract with only 17.53% and 18.28%, respectively. However, the reducing ability of
the ethanolic extract at that concentration is still not equivalent to BHA.
High phenolic content were usually correlated with high radical scavenging
activity.[9] Moreover, phenolic compounds were more extractable using polar solvents as
stated elsewhere.[17 ,26] Hence, antioxidant activity of the pitaya seed ethanolic extract was
most probably due to the presence of polyphenols, such as flavonoids and phenolic acids in
the extract, which have the hydrogen-donor ability to scavenge the free radicals. It has been
suggested that flavonoids, such as procyanidin B2 , epicatechin, and epigallocatechin gal-
late, which were found mainly in the ethanolic extract of loquat seed, contributed towards
its high antioxidative activity against the free radicals.[27] Reports by Brand-Williams
et al.[18] stated that the free radical scavenging activity of phenolics is dependable on their
structure and the antioxidant activity was proportional with the number of hydroxyl group
present.
Even though this method might not affected by substrate polarity,[28] the less polar
compounds extracted in the chloroformic and hexanic extracts may not have the com-
pounds that can easily donate the hydrogen atom/electron to reduce the DPPH• . Their
complex mechanism of action may also be the contributor of low antioxidant activity, as
proven by Xu et al.[29] They observed that there was a slow reaction in the kinetic behaviour
of sesame seed n-hexane extract in contrast to other polar solvents’ extracts. Hence, it is
necessary to conduct more than one antioxidative test to evaluate the antioxidant activity
of the samples due to the differences in reaction behaviour.

Antioxidant Activity of the Extracts in Linoleic Acid Model System

Formation of conjugated diene is an indication of lipid oxidation during the initiation
phase and can be measured spectrophotometrically at 234 nm. A good antioxidant should
have the ability to act as a chain breaker during that phase and inhibiting the oxidation
process.[30] All the extracts possessed good inhibitory effect towards the oxidation process,
as shown in Fig. 2. At 20 µg/mL, all extracts showed inhibition of 60–80% and were not
significantly different among each other except with BHA (97.25%). At 100 µg/mL, all
the samples tested showed relatively the same level of inhibitory effect with 95.60, 98.90,
and 88.46% for ethanolic, chloroformic, and hexanic extract, respectively, and were not
significantly different with that of BHA.
Using this method, the trend in the antioxidant activity of the extracts were not sim-
ilar with that of the DPPH• assay. All the extracts possessed nearly the same inhibitory
effect towards the formation of conjugated diene, while in the previous assay only ethano-
lic extract exhibited strong inhibitory activity. Furthermore, the concentrations used were
much smaller as compared to DPPH• method indicating the effectiveness of the extracts
in inhibiting the oxidation process in lipid model system. Emulsified media was able to
reveal the extracts antioxidative ability due to their potential interactions with the emulsion
system. According to Koleva et al.,[31] lipophilic antioxidants were concentrated in the oil
phase that gives powerful protection of the emulsions against oxidation that showed the
occurrences of polar paradox.
 ANTIOXIDANT ACTIVITY OF RED PITAYA SEED 1177

120

100

80
% inhibition
60

40

20

0
0 20 40 60 80 100
Concentration (µg/ml)

Ethanolic extract Chloroformic extract Hexanic extract BHA

Figure 2 The antioxidant activity of the red pitaya seed extracts as measured by linoleic acid model system.

Ferric Thiocyanate Method

Peroxides formed during the initial stage of linoleic acid oxidation can also be mea-
sured using the FTC assay. Peroxides will oxidize Fe2+ to Fe3+ and reaction with SCN-
will give the red colour due to formation of thiocyanate.[22] In Fig. 3, the control gave max-
imum absorbance up to day 3 and started to decrease since the linoleic acid was already
used up and no more hydroperoxides were available to oxidize Fe2+ until this stage.[12]
Chloroformic extract (96.34%) was slightly higher than ethanolic extract (95.52%) in terms
of their lipid peroxidation inhibition using this assay. The ability of ethanolic and chloro-
formic extracts to slow down the oxidation of linoleic acid is comparable to BHA and
α-tocopherol and even better than the latter. Even though hexanic extract displayed the
lowest antioxidative activity with 66.02% inhibition, the differences are not significant

2,5

2,0

1,5
A500

1,0

0,5

0,0
0 1 2 3 4
Incubation time (day)

Control Ethanolic extract Chloroformic extract

Hexanic extract BHA a-tocopherol

Figure 3 The antioxidant activity of the red pitaya seed extracts as measured by FTC method.
1178 ADNAN, OSMAN, AND ABDUL HAMID

between all the samples tested. Xu et al.[29] also reported a similar trend referring to the
n-hexane extract of sesame seed using the same assay. Inhibition of lipid peroxidation may
be due to the seed coat, as proposed by Siddhuraju and Manian,[17] that protects the seed
internally by inducing endogenous antioxidants.
These results revealed that the extracts exhibited their efficacy as an antioxidant more
in the linoleic acid or lipid emulsion. This may be due to the emulsion properties itself as
linoleic acid is likely to control the level of dissociation of the extracts, especially the
non-polar compounds that exhibited an interaction between the extract and emulsion.[31]
Apart from the hydrophilic compounds present in the ethanolic extract, the lipophilic com-
pounds, such as essential fatty acids, phospholipids, carotenoids, and tocopherols, that may
be present in all the extract also possessed a good antioxidant activity.

Total Phenolic, Ascorbic Acid, and Flavonoid Content

Phenolic compounds are widely known for their beneficial effects, such as preventing
hormone-related cancers, potent antioxidant, and antibacterial agents.[32] Results obtained
by Chang et al.[15] also suggested that phenolic acids and flavonoids were the main contrib-
utors in antioxidant activity and anti-LDL peroxidation. The total phenolic content of the
pitaya seed, as shown in Table 2, is 13.56 ± 2.04 mg GAE/g dry weight. This amount
of total phenolic was much lower if compared to the grape seed, which were around
27.35–46.69 mg GAE/g dry weight depending on the varieties.[16] Drying or heating of the
seed affect the total phenolic content. Heating at certain degrees of temperature increase
the extractable phenolic compounds, but at higher temperature it will start to reduce the
compounds, as reported by Soong and Barlow.[33]
Table 2 shows that the ascorbic acid content of the seed is 0.36 ± 0.01 mg/g dry
weight. This is interesting as the ascorbic acid content of the pitaya pulp itself is 7–11
mg/100 g fresh weight [34] and considered low in comparison to other cactus family, such
as Opuntia sp. Nonetheless, there is no ascorbic acid detected in the seed of the Opuntia
dillenii Haw fruit.[15] Selected flavonoids in the seed were measured and compared with
external standards using HPLC system (Fig. 4). Results obtained are shown in Table 3 and
it was expressed as mg/g seed dry weight. Catechin was the major flavonoid detected in
the red pitaya seed with 3.60 ± 2.33 mg/g dry weight. This was followed by quercetin,
myricetin, and epicatechin with 1.31 ± 0.45, 0.63 ± 0.30, and 0.60 ± 0.11 mg/g dry
weight, respectively. On the other hand, the amount of rutin found in the red pitaya seed
was the lowest with 0.53 ± 0.02 mg/g dry weight. The amount of kaempferol in the
seed was below detection limit. Catechin, epicatechin, and quercetin were several major
flavonoids found in the Opuntia dillenii Haw seed.[15]

Table 2 Total phenolic and ascorbic acid contents of the red pitaya seed.

mg/g DWa seed

Total phenolicb Ascorbic acid

13.56 ± 2.04 0.36 ± 0.01

a DW, dry weight.
b Expressed as gallic acid equivalent (GAE).
Each value is expressed as means ± SD of three independent samples
with three measurements per sample (n = 9).
 ANTIOXIDANT ACTIVITY OF RED PITAYA SEED 1179

1
3
6
4

(a)

4
3
5
2
6

(b)

Figure 4 HPLC chromatograms of (a) a mixture of flavonoid standard; (b) red pitaya seed [absorbance at 280
nm vs. time (min)]: catechin (1), epicatechin (2), rutin (3), quercetin (4), myricetin (5), and kaempferol (6).

CONCLUSION
Result of the study showed that total phenolic compound and ascorbic acid content
of red pitaya seed were found to be 13.56 ± 2.04 and 0.36 ± 0.01 mg/g, respectively.
Catechin (3.60 ± 2.33 mg/g) was found to be the major flavonoid detected. The extracts
showed different trends of antioxidant activity when determined using different methods,
reflecting the different mechanisms of action involved. Chloroformic and hexanic extracts
only showed potent antioxidant activity using the linoleic acid model system and FTC
method. Ethanolic extract exhibited high radical scavenging activity at 1000 µg/mL with
1180 ADNAN, OSMAN, AND ABDUL HAMID

Table 3 Flavonoids content of the red pitaya seed.

mg/g DWa
Catechin Epicatechin Rutin Quercetin Myricetin Kaempferol

3.60 ± 2.33 0.60 ± 0.11 0.53 ± 0.02 1.31 ± 0.45 0.63 ± 0.30 NDb
a Dryweight.
b Notdetected (below detection limit).
Each value is expressed as means ± SD of three independent samples with three measurements per
sample (n = 9).

74.76% that was significantly (p < 0.05) different than that of chloroformic (17.53%) and
hexanic extract (18.28%). Overall, the study revealed the health benefits and potential use
of red pitaya seed as a source of natural antioxidant.

ACKNOWLEDGMENTS
The authors would like to thank the Universiti Putra Malaysia for the financial support under
Research University Grant Scheme (RUGS) (Project No. 02/01/07/0015RU).

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