Universiti Sains Malaysia, 11800 Gelugor, Pulau Pinang, Malaysia
* Author to whom correspondence should be addressed; E-mail: email@example.com; Tel: +604-
6533262; Fax: +604-6574854
Received: 8 December 2008; in revised form: 13 January 2008 / Accepted: 15 January 2009 /
Published: 3 March 2009
benzothiazoline-6-sulfonic acid) (ABTS) radical cation assay. The percentage of
antioxidant activity for all extract samples using both assays was between 58 and 80%. The
fresh samples of both plants had higher antioxidant activity than the dried samples. The
results of the β-carotene bleaching assay were correlated (R
= 0.9849) with those of the
Plants consumed by humans may contain thousands of different phenolic compounds. The effect of
activity . Phenolic compounds also function as free-radical scavengers, reducing agents, and
quenchers of singlet-oxygen formation . Antioxidant compounds that scavenge free radicals help
protect against degenerative diseases .
climbing plant is a leafy vegetable that can be eaten raw or steamed . This popular plant is used as a
remedy for diarrhoea and dysentery in Bangladesh  and to inhibit intestinal motility . Iridiod
glycosides, paederolone, paederone, paederine and paederenine were the phytochemicals identified in
this plant [5,7]. Previous studies [7,8] also identified a number of steroids and terpenoids and 77
constituents in the volatile oils of the leaves, stems and flowers of P. foetida, some at high levels.
properties and is usually consumed raw. In Malaysia the powdered dried leaves are used to treat a
cracked tongue and a preparation from the root is used to relieve itching and to reduce swelling .
The volatile oils isolated by vacuum distillation from Syzygium species contain a high percentage of
terpenoids and γ-terpinene , with tannins and related compounds are also found in the leaves of
Syzygium species . Even though there are reports on the antioxidant activity of P. foetida and S.
aqueum, different analytical methods were used [12,13]. Therefore, this study was undertaken to
evaluate the antioxidant capability of these plants using two methods: coupled oxidation of β-carotene-
linoliec acid and an ABTS assay. The comparison on the percentage of antioxidant activity of fresh
and dried samples would also be studied.
The percentage of the crude extracts in methanol was between 8.8 and 10.1% w/v and they were
of P. foetida and S. aqueum due to its polarity and its known ability to extract compounds such as
phenolics, flavonoids and other polar materials .
In the β-carotene-linoleic acid coupled oxidation model system, the linoleic acid free radical (LOO
formed attacks the highly unsaturated β-carotene molecules and in the absence of an antioxidant
at 450 nm. The extracts reduced the extent of β-carotene bleaching by neutralising the linoleate-free
radical and other free radicals formed in the system . The total antioxidant activities of the crude
-α-tocopherol, fresh P. foetida, fresh S. aqueum, dried P. foetida, dried S. aqueum, and
3.30%, 55.73 ± 2.82% and 42.37 ± 3.25%, respectively (Figure 1). Variations were significant
(p<0.05). The fresh samples had higher antioxidant activity than did the dried samples. In this study,
the order of antioxidant activity towards β-carotene oxidation was
-α-tocopherol > fresh P. foetida >
-α-tocopherol. All of the tested samples more
Figure 1. Antioxidant activity of the different extracts and standard samples at 0.02 ppm
in the β-carotene-linoleate system. Variations were significant at level p<0.05.
pf = Paederia foetida sa = Syzygium aqueum
is commonly used to assess radical scavenging or antioxidant activity. The scavenging
The free radical scavenging activity of fresh and dried extracts along with reference standards, such as
quercetin and (
)-(+) ascorbic acid were determined by ABTS assay and the results are shown in
Figure 2. All of the extracts had strong antioxidant abilities that exceeded the control, quercetin except
)-(+) ascorbic acid. The difference in the antioxidant activity profiles of the various extracts is
consistent with previous reports  of different constituents in the extracts. Phenolic compounds
scavenge free radicals by forming a stable ABTS-H. The scavenging activity of the extracts also could
be due to the presence of
steroids and terpenoids which are known to occur in P. foetida plant [7, 8].
contributed to its high antioxidant activity [6, 9, 17, 18]. The fresh P. foetida and S. aqueum extracts
had 70-76% antioxidant activity and the dried samples had 65-68% antioxidant activity (Figure 2)
which was higher than the activity of the standard commercial antioxidant, quercetin. The decrease in
antioxidant activity in the dried samples could be due to degradation of the antioxidants during drying.
The storage, processing and preparation conditions are known to alter the content of antioxidants but
little information is known about the impact of drying on the antioxidant activity of vegetables .
Scavenging activity increased with the extracts concentration (Figure 3). At 0.035 mg mL
, the order
with the scavenging activity > 90% for all extracts.
Variations were significant at level p<0.05.
using ABTS ‘assay’. (
)-(+) Ascorbic acid and quercetin were used as references.
(L)-(+) ascorbic acid
Total phenolic content of the plant extracts
The total phenolic content for all of the extracts decreased after drying (Table 1). The amount of
showed that P. foetida and S. aqueum are low in total phenolic content compared to other fruit and leaf
samples of several plants[12, 13]. Total phenolic content in ferulic acid equivalent gave the highest
levels of 62.64 ± 1.32 and 60.93 ± 3.4 mg/g sample weight for the fresh leaves and twigs of P. foetida,
respectively. P. foetida leaves and twigs often are consumed raw , and the total phenolic content of
the twigs was slightly lower than that of the leaves, (Table 1). It is expected that the total antioxidant
activity of the twigs should be as good as the leaves sample since antioxidant activity increases
proportionally with the phenolic content [1,20].
Table 1. Total phenolic contents of P. foetida and S. aqueum.
35.52 ± 1.64
62.64 ± 1.32
20.77 ± 0.34
52.96 ± 1.62
20.8 ± 3.25
60.93 ± 3.40
All analyses were mean of triplicate measurements ±
Results expressed in mg ferulic acid equivalent/g
Correlation between two methods of antioxidant activity
Extracted samples from leaves at a concentration of 0.02 ppm were chosen to test the correlation
methods (Table 2). Fresh P. foetida and S. aqueum both had better antioxidant activity than the dried
samples. The correlation between the β-carotene oxidation and ABTS methods had R
carotene and ABTS method
Some studies report a strong correlation between phenolic content and antioxidant activity in fruits,
the plant extracts correlated well with the total phenolic content. A positive correlation was observed,
whereby the antioxidant activity increased when the total phenolic content increased (Figure 4).
This study clearly indicated that P. foetida and S. aqueum, both have high antioxidant activity.
There is not much difference in the total phenolic content between the leaves and twig of P. foetida. A
good correlation between the β-carotene oxidation and ABTS methods was observed, with an R
natural antioxidant compounds that may have potent beneficial health effects.
Plant materials were collected from trees growing in home gardens in Gelugor, Penang, Malaysia.
twigs of P. foetida and leaves of S. aqueum were stripped from the plants. Plant materials (500 g) were
divided into fresh and dried samples. The dried samples were air dried at room temperature (30
°C) for 24 hr and were immediately immersed in the solvents used for extraction.
Preparation of extracts
All fresh and dried samples were extracted with methanol: water (1:10). The aqueous methanol
The crude extract was transferred into a 100 mL volumetric flask and ethanol was added up to the
mark to prepare solutions at different concentrations (0.005 - 0.1 mg mL
). The extracts were stored at
radical scavenging activity and antioxidant activity towards β-carotene oxidation, while all crude
leaves and twig extracts were used for the analysis of total phenolic content.
The β-carotene bleaching assays were conducted as previously described with slight modifications
Co.) and Tween
40 (20 mL, Sigma Chemical Co.) were dissolved in chloroform (20 mL, Merck).
°C with a rotary evaporator. After evaporation, the mixture was
immediately added to oxygenated distilled water (25 mL) to form an emulsion. The emulsion (25 mL)
was transferred to test tubes containing extracts (1.0 mL) and the mixture was then gently mixed. One
mL of the mixture was pipetted and mixed with 95% ethanol (5 mL) at 0
°C. Absorbance of the
samples at 450 nm were measured in triplicates every 20 min. for a duration of 160 min. with a Hitachi
U-2000 Spectrophotometer. The above procedure was repeated using
was prepared containing the same concentration of sample. The total antioxidant activity was
calculated based on the following equation:
AA = [1 – (
)] X 100
absorbance of control sample at 0 min, and A
ABTS free radical scavenging activity
Radical scavenging activity was measured as previously described [23, 24] with minor
radical source and prepared by reacting 3.75 mM ABTS diammonium salt (Fluka) and 1.225 mM
potassium persulphate (BDH chemicals) overnight at 30
°C. The mixture was diluted 10-fold with
99.5% ethanol (Merck) before use. The diluted ABTS radical solution (3.0 mL) was added to (
– 0.1 mg mL
1, 1.0 mL, Merck), and the mixtures were
incubated for 60 minutes. The absorbance at 414 nm was then measured at 30 ºC. The procedure was
repeated with quercetin (Sigma Chemical Co.) standard, followed by fresh and dried P. foetida and S.
ethanol and ABTS radical was prepared and measured daily. The scavenging ability of antioxidants
was calculated according to the following equation :
ABTS scavenging activity (%) = [(A
– A) / A
] x 100
is the absorbance of the control reaction and A is the absorbance in the presence of samples
The total phenolic content of the crude methanol extract was determined by using a modified Folin-
(0.25 mL, Fluka) was added to methanolic extract solution of (1.0 mg mL
, 10 mL), then 20%
40 minutes. A blue color appeared and the absorbance was measured at 725 nm with a Hitachi U-2000
Spectrophotometer. All measurements were made in triplicates and the results expressed as mg of
ferulic acid per gram of sample.
This research was supported by RU research grant No. 1001/PKIMIA/811016 and
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Sample Availability: Not available.
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Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).