International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 3, No. 4, July 2013
Antioxidant Activities of Syzygium Cumini and Ardisia
Elliptica in Relation to Their Estimated Phenolic
Compositions and Chromatic Properties
A. M. Siti-Azima, A. Noriham, and M. Nurhuda
neurodegenerative, rheumatic arthritis and autoimmune
disorders [1]-[3]. This nutraceutical and medicinal value lies
in some phytochemical substances that produce a definite
physiological action in the human body. The antioxidant
properties of the phytochemical are always associated with
their chromophores structure that are responsible for the
bright colour of plants known as plant pigment. Anthocyanin
is water soluble pigment which is responsible for red to blue
chroma of plants.
Phenolic antioxidant which generically denominated as
ArOH contains at least one hydroxyl group (OH) attached to
the benzene ring and plays as chain breaking antioxidant [4].
Generally, there are two suggested mechanisms by which
antioxidants can play their defensive role [5], [6]. In the first
mechanism known as hydrogen atom donator (HAT), the free
radical removes a hydrogen atom from the antioxidant
(ArOH) than becoming itself a radical and terminated the
oxidation process by converting free radicals to more stable
products (1). In the second mechanism SET (single-electron
transfer), the antioxidant (ArOH) donates an electron to the
free radical becoming itself a radical cation and terminate the
oxidation chain reaction by reducing the oxidised
intermediates into the stable form (2).
Abstract—Plant extract serves as a good source of bioactive
compounds and also natural pigment which is potential to be
further applied as colourants in food and pharmaceutical
products. The aim of this study is to determine antioxidant
activities in Syzygium cumini and Ardisia elliptica based on
FRAP, ORAC, DPPH and ABTS radical scavenging assays in
relation to their total phenolic content (TPC), total flavonoids
content (TFC) and anthocyanin content (tAcy). The chromatic
properties based on colour density (CD), Lightness (L*), hue
angle (ho) and chroma value (c) of the selected plants were also
determined to evaluate their potential as natural colourants in
food. Syzygium cumini showed higher in FRAP value (25.66
mM TEAC), DPPH radical scavenging capacity (EC50=0.22
mg/ml) and TPC (43.64 mg GAE /g) as compared to Ardisia
elliptica with 19.60 mM TEAC, EC50=0.24 mg/ml and 41.75 mg
GAE /g respectively. Ardisia elliptica exhibited higher in ABTS
scavenging activity (4.63 mM TEAC), ORAC value (10.95 µM
TEAC), TFC (36.91 mg QE /g) and tAcy (9.97 mg cyanidin / g)
as compared to Syzygium cumini with 4.30 mM TEAC, 10.12
µM TEAC, 17.02 mg QE /g and 8.71 mg cyanidin /g repectively.
Ardisia elliptica showed higher in CD (1.32 AU), c (32.29) and
ho values (24.68o) but lower in L* value (50.69) as compared to
the Syzygium cumini with 1.01 AU, 12.10, 10.02o and 75.49
respectively. FRAP, ABTS and EC50 values exhibited very
strong correlation with TFC (r2= 0.974, 0.984, 0.921
respectively) and tAcy (r2= 0.953, 0.998, 0.947 respectively).
They also exhibited strong correlation with TPC (r2= 0.854,
0.779, 0.749 respectively). ORAC assay exhibited weak
correlation with TFC (r2= 0.357) and tAcy (r2= 0.229) but strong
correlation with TPC (r2=0.713). Hence, it can be concluded
that, TFC and tAcy exhibited very strong relationship with the
FRAP, ABTS and DPPH scavenging activities and work as
single electron transfer in their mechanism while Ardisia
elliptica was better in their chromatic properties as compared
to Syzygium cumini.
(1)
R ArOH R - ArO
(2)
Several methods have been established based on these two
mechanisms. Oxygen radical antioxidant capacity (ORAC)
assay and total reactive antioxidant potential (TRAP) assay
are categorised under the HAT while ferric reducing
antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical scavenging assay, ferric thiocyanate (FTC)
assay and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid) (ABTS•+) scavenging assay are categorised under SET
[7]-[9].
Syzygium cumini Skeels (syn. Eugenia jambolana Lam.,
Eugenia cumini Druce) belongs to the family of Myrtaceae is
widely found in subcontinent of Asia and other tropical
country. It is commonly known as Keriang dot or Keriang
Aceh by the Malaysian, Java plum by the Americans and
Jamun or Jambul by the Indian. The berry is oblong in shape
with dark purple skin and whitish aril. The major
anthocyanin being found in the berries are
cyanidin-diglucoside,
ptunidin-diglucoside,
delphinidin-diglucoside and malvidin-diglucoside [10]. It has
been reported to possessed several bioactivities against
Index Terms—Anthocyanin, antioxidant, ardisia elliptica,
chromatic properties, syzygium cumini.
I. INTRODUCTION
Plants are world natural ornamental that gained a wide
attention as a good source of natural antioxidant which is
associated with health benefit and well-being. Owing to its
phenolic structure plants are capable to overcome the
oxidative stress in the human cell which finally led to the
development of chronic disease such as tumor, cancer,
Manuscript received January 14, 2013; revised March 22, 2013.
A. M. Siti-Azima, A. Noriham, and M. Nurhuda are with the Universiti
Teknologi MARA, Shah Alam, 40450 Selangor, Malaysia
(e-mail:ctargh@yahoo.com,
noriham985@salam.uitm.edu.my,
nurhudamanshoor@puncakalam.uitm.edu.my).
DOI: 10.7763/IJBBB.2013.V3.221
R ArOH RH ArO
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International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 3, No. 4, July 2013
diabetic [11] and cancer [12]. Ardisia elliptica’s fruit are
roundish berries that turn shiny black when ripen, and
resemble the eye ball of chicken; hence it is known as Mata
Ayam (Chicken Eyes) among the Malaysian [13]. In Borneo
it is known as Kayu lundu, Limjong, Merjima, Pis-pis,
Sarusup, Semporna, Serusop, Serusup, Surusup, Tapiok,
Tursop laut. It is also commonly known as Buah Letus,
Cempenai, Mata Pelanduk, Mempenai, Daun Bisa Hati,
Penah, Periah, Shoebutton Ardisia and Seashore Ardisia.
Ardisia elliptica leaves have been medicinally used as
antiobiotic and antiviral [14]. Hence in this work the study on
the antioxidant activities in Syzygium cumini and Ardisia
elliptica based on FRAP, ORAC, DPPH and ABTS radical
scavenging assays in relation to their estimated phenolic
compositions which are total phenolic content (TPC), total
flavonoids content (TFC) and anthocyanin content (tAcy)
were determined. The chromatic properties based on colour
density (CD), Lightness (L*), hue angle (ho) and chroma
value (c) of the selected plants were also determined to
evaluate their potential as natural colourants in food.
was obtained. The final results were expressed in mM TEAC
/ g of fresh extract weight.
Oxygen Radical Absorbance Capacity (ORAC) assays was
conducted using the procedures outlined by the reagent
provider (Zen- Bio, Inc.; Research Triangle Park, NC). The
samples were measured at 530 nm by microplate reader
Bio-Tek Instruments, Inc provided with Gen 5 software.
D. Total Phenolic Content
Total phenolic content was evaluated based on the
Folin-Ciocalteu colorimetric method developed by [16]. The
absorbance was measured by Perkin Elmer UV-Vis
spectrophotometer
at
λmax=765nm.
Quantitative
measurements were conducted based on a Gallic acid
standard curve ranging from 0-0.5 mg/ml with r2=0.999. The
total phenolic content was expressed as Gallic acid
equivalents (GAE) in mg/g of fresh weight extract.
E. Total Flavonoids Content
Aluminium chloride colorimetric method described by [17]
was used to estimate total flavonoid content in the plant
extracts. The absorbance of orange colour of the aluminium
complex was measured by using Perkin Elmer UV-Vis
Spectrophotometer at the visible range with λmax=415 nm. A
series of quercetin standard were prepared and a linear
standard calibration curve with r2=0.998 was established.
The results were expressed as quercetin equivalent (QE) in
mg/g of fresh weight extract.
II. MATERIALS AND METHODS
A. Preparation of Raw Material
Freshly ripened Ardisia elliptica and Syzygium cumini was
collected and obtained from Santan, Perlis, Malaysia. The
fruits were washed, sealed pack and stored at -20oC for
further used.
F. Total Anthocyanin Content and Colour Properties
Total anthocyanins in the samples were measured
spectrophotometrically according to the method described by
[18]. The colour density (CD) was conducted based on the
method described by [19].
B. Extraction Procedure
The raw materials were stirred in the 100 mM citrate buffer
(pH 3.0) for 10 minutes at 100oC. The ratio of plant parts to
citrate buffer is 1:4 (v/v). The extraction procedure was
conducted twice to maximize the yield. The extracts were
filtered by using Buchner funnel and concentrated by using
rotary evaporator at 60oC and 100 mbar. The concentrated
filtrate was lyophilised and kept in amber bottle at -20oC
prior to further analysis.
G. Statistical Analysis
Analyses of data were obtained from triplicate samples.
Statistical analyses were conducted using Statistical Analysis
System 9.1.3 software package [20]. Two sample t-tests were
used to compare the mean values. Values were expressed as
means ± standard deviations. A correlation matrix was
calculated between all independent variables to examine
co-linearity.
C. Antioxidant Activities
Radical scavenging activity of the plant extracts were
conducted based on the method developed by [8]. The result
was expressed as EC50 (Efficient Concentration at 50%
scavenging activity). Ascorbic acid was used as a standard in
comparison with the extracts.
The antioxidant capability of the extract based on ferric
reducing antioxidant power (FRAP) was determined
according to the method described in [7]. The readings of the
blue (ferrous tripyridyltriazine) complex were measured by
Perkin Elmer UV-Vis spectrophotometer at λmax=593 nm.
The linear standard calibration curve ranging from 0-100 mM
Trolox with r2= 0.999 was established. The final results were
expressed in mM TEAC / g of fresh extract weight in which
TEAC is denoted for Trolox Equivalent Antioxidant
Capacity.
The ABTS·+ scavenging capacity test was carried out
according to the method described by [15]. The samples were
measured spectrophotometrically by Perkin Elmer UV-Vis
spectrophotometer at λmax=734 nm and regression r2=0.998
III. RESULTS AND DISCUSSIONS
DPPH scavenging activity is a kinetic antioxidant method
which based on the reduction of DPPH• free radical into
DPPH2 by the action of antioxidant [21]. The DPPH2 is a
stable organic radical which exhibit violet colour in alcohol
solution with an absorption peak in the visible range at
λmax=518 nm, which disappear in the presence of antioxidant.
The capacity of the compounds to scavenge the DPPH radical
has been used as the magnitude of the antioxidant capacity
[22]. The EC50 values required to scavenge 50% radical were
calculated from the regression equation prepared from series
concentration of extracts. The lower EC50 indicate the better
DPPH radical scavenging activity. Ardisia elliptica extracts
exhibited higher in EC50 value (0.24 mg/ml) of DPPH radical
scavenging activities as compared to the EC50 value (0.22
mg/ml) of Syzygium cumini extracts (Table I). This indicated
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International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 3, No. 4, July 2013
that Syzygium cumini extracts is 1.09 times greater in
scavenging effect than Ardisia elliptica extracts. Syzygium
cumini presented 1.83 times lower scavenging effect of
ascorbic acid while Ardisia elliptica exhibited 2.00 times
lower scavenging effect as compared to the ascorbic acid.
Ardisia elliptica exhibited higher ORAC value (10.95 µM
TEAC) as compared to Syzygium cumini (10.12 µM TEAC).
FRAP assay is based on the ability of the antioxidant to
reduce Fe3+ to Fe2+ in the presence of TPTZ, forming an
intense blue Fe2+–TPTZ complex with an absorption
maximum at 593 nm [23]. Based on the FRAP assay as
shown in the TABLE I, Syzygium cumini extracts (25.66 mM
TEAC) showed a significantly higher (p<0.05) in FRAP
value as compared to the Ardisia elliptica extracts (19.60 mM
TEAC). Conversely, in ABTS scavenging activities Ardisia
elliptica extract showed better ABTS scavenging activity as
compared to Syzygium cumin extract with ABTS value 4.63
µM TEAC and 4.30 µM TEAC respectively. Syzygium
cumini is insignificantly higher (p<0.05) in TPC (43.64 mg
GAE / g) but significantly lower (p<0.05) in TFC (17.02 QE /
g) and tAcy (8.71 mg cyanidin equivalent / g) as compared to
Ardisia elliptica extracts (TPC=41.75, TFC=36.91, tAcy=
9.97) (Table I).
EC50 and ABTS. The correlations strength demonstrated that
flavonoids (flavone and flavonols) and anthocyanins play a
major role in FRAP, DPPH and ABTS antioxidant activities
of Syzygium cumini and Ardisia elliptica as compared to the
phenols. Besides, a very strong correlation between FRAP
with TFC (r2=0.960) and tAcy (r2=0.953), ABTS with TFC
(r2=0.992) and tAcy (r2=0.997) and EC50 with TFC (r2=0.984)
and tAcy (r2=0.998) but low correlation between ORAC with
tAcy and TFC manifest that the flavonoids and anthoyanin
works as single electron transfer (SET) rather than hydrogen
atom donator (HAT). Previous literature reported by [24]
indicated that flavonoids owning to the subclass flavonol that
has combination of 3-hydroxy group, 2, 3-double bond and
4-oxo function in the C ring and the catechol group in the B
ring as their functional group in their chemical structure
namely quercetin, myricetin, kaempherol, rutin, galangin and
fisetin explained the reason behind reactivity towards
reducing capacity of Fe3+ to Fe2+ and the strong correlation
between FRAP assay and flavonoids. In addition, [25]
reported that only myricetin and quercetin exhibit strong
reaction with Fe3+ and the possible complexation between
flavonoids and Fe3+ occurs between the 5-hydroxyl and the
4-oxo groups. References [17] and [26] reported very strong
correlation between FRAP assay and anthocyanin and
supported the finding in this research.
TABLE I: ANTIOXIDANT ACTIVITIES AND ESTIMATION OF PHENOLIC
COMPOSITIONS IN SYZYGIUM CUMINI AND ARDISIA ELLIPTICA FRUITS
Plant samples
Antioxidant activities and phenolic
Syzygium cumini
Ardisia elliptica
compositions
fruits
fruits
b
a
FRAP mM (TEAC)
EC50 (mg/ml)
ABTS (mM TEAC)
b
ORAC (µM TEAC)
Total phenolic content (mg GAE /
g extract)
Total flavonoids content (mg QE /
g extract)
Total anthocyanin content (mg
cyanidin / g extract )
b
25.66 ± 1.40A
A
TABLE II: CHROMATIC PROPERTIES OF AQUEOUS EXTRACTION OF
SYZYGIUM CUMINI AND ARDISIA ELLIPTICA FRUITS
Hue angle
Lightness
Chroma
Plant
Colour
(ho)
(L*)
-city
samples
density
(c)
(CD)
Syzygium
1.01±
75.49±
12.10±
10.02±
cumini
0.03B
1.01A
0.66B
0.92B
fruits
Ardisia
1.32±
32.29±
50.69±
24.68±
elliptica
0.05A
1.76B
0.44A
1.02A
fruits
Note: Analysis of data were obtained from triplicate samples
Values were means ± SD
AB
Means with different capital letter within the column were significantly
different at (p<0.05)
19.60 ± 0.53B
0.22 ± 0.00
4.30 ± 0.05B
10.12 ± 1.29A
43.64±4.40A
0.24 ± 0.00A
4.63 ± 0.09A
10.95 ±1.63A
41.75±3.50A
17.02 ± 1.10B
36.91 ±2.37A
8.71 ± 0.14B
9.97 ± 0.05A
Note: Analysis of data were obtained from triplicate sample
Values were means ± SD
AB
Means with different capital letter within the row were significantly
different at (p<0.05)
a
EC50 for Ascorbic acid were 0.12 ± 0.00
b
TEAC is Trolox equivalent antioxidant capacity
TABLE III: CORRELATION BETWEEN ANTIOXIDANT ACTIVITIES OF
SYZYGIUM CUMINI AND ARDISIA ELLIPTICA FRUITS WITH THEIR PHENOLIC
COMPOSITIONS
r2
FRAP
EC50
ABTS
ORAC
TPC
0.854
0.779
0.749
0.713
TFC
0.974
0.984
0.921
0.357
tAcy
0.953
0.998
0.947
0.229
The colour properties of Ardisia elliptica extract and
Syzygium cumini extract at pH 3.0 were evaluated to
determine the potential natural colourant in food. Ardisia
elliptica extract exhibit a higher CD, c, and ho but lower in L*
as compared to the Syzygium cumini extract (Table II). Based
on the CD and L* Ardisia elliptica demonstrated a better
intensity than Syzygium cumini. Higher in c and h indicating
that Ardisia elliptica is more saturated/vivid with redder in
colour as compared to Syzygium cumini. Thus, Ardisia
elliptica has better potential as red colourant as compared to
Syzygium cumini.
The correlation analyses were conducted in this research to
determine the relationship between the phenolic composition
and four antioxidant activities studied which are FRAP,
DPPH, ABTS and ORAC as well as their antioxidative
mechanism. From the results obtained in TABLE III TFC and
tAcy showed a very strong correlation with FRAP, EC50 and
ABTS while TPC exhibited strong correlation with FRAP,
IV. CONCLUSIONS
Based on the antioxidant activities being tested, Syzygium
cumini extract exhibited higher in FRAP value and DPPH
radical scavenging activity as compared to the Ardisia
elliptica extract while Ardisia elliptica exhibited higher in
ABTS radical scavenging activity and ORAC value as
compared to the Syzygium cumini. Pearson correlation
cooficient (r2) showed that flavonoids (flavones and
flavonols) and anthocyanins exhibited very strong
relationship with SET antioxidant activities which are FRAP
and ABTS scavenging activity and weak relationship with
HAT antioxidant activity which is ORAC indicating that
flavonoids and anthocyanin favour the SET mechanism
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International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 3, No. 4, July 2013
rather than HAT. On the other hand, phenolic (phenols)
showed a strong correlation with four antioxidant activities
assays being tested. Ardisia elliptica exhibited significantly
higher in chromatic properties as compared to Syzigium
cumini and demonstrated that Ardisia elliptica has better
potential in food applications as natural colourant as
compared to Syzygium cumini.
ACKNOWLEDGMENT
The authors would like to acknowledge Faculty of Applied
Sciences, Universiti Teknologi MARA for the fund and
support
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A. M. Siti-Azima was born in June 1987. She was
graduated from Bsc. (Hons) of Food Science &
Technology (minor in Shariah Food Law), Faculty of
Applied Sciences, University of Teknologi MARA,
Shah Alam, Selangor, Malaysia in 2010. She is now
doing her postgraduate studies in Faculty of Applied
Sciences, Universiti Teknologi MARA.
A. Noriham was born in May 1959. She was
graduated from PhD of Food Science from University
of Kebangsaan Malaysia, MSc. Food Science and
Microbiology from University of Strathclyde and Bsc.
of Food Science and Technology from University of
Putra Malaysia.
Currently, she is an Assoc. Professor and Lecturer
at University of Teknologi MARA, Shah Alam
Selangor, Malaysia. Previously she was a Head of
School of Industrial Technology, Faculty of Applied Sciences, University of
Teknologi MARA Shah Alam. From 2002 until present, Noriham A. had
published 2 books, 18 Journal articles and 38 Proceeding papers. Her areas of
expertise are in food based antioxidants, phytochemicals and meat science
and technology.
M. Nurhuda was graduated from Bsc. University of
Malaya and Phd. from University of Teknologi
MARA, Malaysia. Currently she is a Lecturer in
Faculty of Pharmacy, University of Teknologi MARA,
Puncak Alam, Selangor, Malaysia. Her areas of
interest are in natural product, drug discovery,
separation science and spectroscopic analysis.
’
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