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Molecules 2009, 14, 970-978; doi:10.3390/molecules14030970 

 

 



molecules 

ISSN 1420-3049 

www.mdpi.com/journal/molecules 



Article  

 

Antioxidant Activity and Phenolic Content of Paederia foetida 

and Syzygium aqueum 

 

Hasnah Osman *, Afidah A. Rahim, Norhafizah M. Isa

 

and Nornaemah M. Bakhir                           

 

Universiti Sains Malaysia, 11800 Gelugor, Pulau Pinang, Malaysia 



 

*   Author to whom correspondence should be addressed; E-mail: ohasnah@usm.my; Tel: +604-

6533262; Fax: +604-6574854 

 

Received: 8 December 2008; in revised form: 13 January 2008 / Accepted: 15 January 2009 / 

Published: 3 March 2009 

 

 



Abstract: The antioxidant activity of fresh and dried plant extracts of Paederia foetida and 

Syzygium aqueum were studied using β-carotene bleaching and the 2,2’-azinobis(3-ethyl-

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

2

 = 0.9849) with those of the 



ABTS assay.  

 

KeywordsPaederia foetidaSyzygium aqueum; Antioxidant activity; Phenolic content.  

 

 



Introduction 

 

Plants consumed by humans may contain thousands of different phenolic compounds. The effect of 



dietary phenolics are of great current interest, due to their antioxidative and possible anticarcinogenic 

activity [1]. Phenolic compounds also function as free-radical scavengers, reducing agents, and 

quenchers of singlet-oxygen formation [2].  Antioxidant compounds that scavenge free radicals help 

protect against degenerative diseases [3].  



OPEN ACCESS

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971

Paederia foetida L. (P. foetida) is locally known in Malaysia as akar sekuntut. This aromatic 

climbing plant is a leafy vegetable that can be eaten raw or steamed [4]. This popular plant is used as a 

remedy for diarrhoea and dysentery in Bangladesh [5] and to inhibit intestinal motility [6]. 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.  



Syzygium aqueum  (S. aqueum), also known as watery rose apple or water apple, has thirst-relieving 

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 [9].  

The volatile oils isolated by vacuum distillation from Syzygium species contain a high percentage of 

terpenoids and γ-terpinene [10], with tannins and related compounds are also found in the leaves of 

Syzygium species [11].  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.  



 

Results and Discussion  

 

Extraction  

 

The percentage of the crude extracts in methanol was between 8.8 and 10.1% w/v and they were 



used in its form after dilution.  Methanol was used as the extraction solvent for fresh and dried samples 

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 [14].   

 

 Antioxidant activity 



 

 Coupled oxidation of β-carotene and linoleic acid 

 

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 



rapidly bleaches the typically orange colour of β-carotene which is monitored spectrophotometrically 

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 [15].  The total antioxidant activities of the crude 

extracts of 

DL

-α-tocopherol, fresh P. foetida, fresh S. aqueum, dried P. foetida, dried S. aqueum, and 



quercetin after 160 h reaction time were 79.69 ± 3.16%, 78.13 ± 2.90%, 73.77 ± 2.95%, 66.67 ± 

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 

DL

-α-tocopherol > fresh P. foetida > 



fresh S. aqueum > dried P. foetida > dried S. aqueum > quercetin.  The antioxidant activity of fresh P. 

Molecules 2009, 14   

 

           



 

 

 



 

 

 



         

 

 



972

foetida was the highest and was comparable to 

DL

-α-tocopherol. All of the tested samples more 



efficiently slowed the bleaching of β-carotene than did quercetin. 

 

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 



  

ABTS free radical-scavenging activity 

 

ABTS assay

 

is commonly used to assess radical scavenging or antioxidant activity. The scavenging 



activity is measured by the absorbance at 414 nm, which decreased as the ABTS radical is scavenged. 

The free radical scavenging activity of fresh and dried extracts along with reference standards, such as 

quercetin and (

L

)-(+) 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 

(

L

)-(+) ascorbic acid. The difference in the antioxidant activity profiles of the various extracts is 

consistent with previous reports [16] 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].  



P. foetida has higher levels of phenolic compounds than does S. aqueum,  which may have 

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 [19].  

Scavenging activity increased with the extracts concentration (Figure 3). At 0.035 mg mL

-1

, the order 



of scavenging activity extracts was: fresh P. foetida > fresh S. aqueum > dried P. foetida > dried S. 

aqueum. A plateau was reached at 0.055 mg mL

-1

 with the scavenging activity  > 90% for all extracts. 



 

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Figure 2. Values of free radical-scavenging activity of sample extracts in ABTS assay. 

Variations were significant at level p<0.05. 

 

 

 



 

 

 



 

 

 



 

 

 



Figure 3.   Free radical-scavenging activity of extract samples at different concentration 

using ABTS ‘assay’. (



L

)-(+) Ascorbic acid and quercetin were used as references. 

 

0

20



40

60

80



100

120


0

0.02


0.04

0.06


0.08

0.1


Concentration (mg/mL)

ABT

S

 Scaven

g

in

g

 act

ivit

y (

%

)

(L)-(+) ascorbic acid

dried pf

fresh pf


dried sa

fresh sa


Quercetin

 

 Total phenolic content of the plant extracts 

 

The total phenolic content for all of the extracts decreased after drying (Table 1). The amount of 



total phenolics varied widely between 20.80 and 63.00 mg/g sample weight. Previous investigations 

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 [4], 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 

0

10

20



30

40

50



60

70

80



90

dried pf


fresh pf

dried sa


fresh sa

quercetin

(L)-(+)

ascorbic


acid

Samples

A

n

ti

o

x

id

an

t act

iv

it



(%

)

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974

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. 



Samples FA

b

 



P. foetida leaves (dried) 

35.52 ± 1.64 



P. foetida leaves (fresh) 

62.64 ± 1.32 



S. aqueum leaves (dried) 

20.77 ± 0.34 



S. aqueum leaves (fresh) 

52.96 ± 1.62 



P. foetida twig (dried) 

20.8 ± 3.25 



P. foetida twig (fresh) 

60.93 ± 3.40 

All analyses were mean of triplicate measurements ± 

standard deviation 

b

Results expressed in mg ferulic acid equivalent/g 



sample weight 

 

Correlation between two methods of antioxidant activity 

 

Extracted samples from leaves at a concentration of 0.02 ppm were chosen to test the correlation 



between the two methods. The percentage of antioxidant activity were between 65 and 80% for both 

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

2

 = 0.9878. 



 

Table 2. Comparison of oxidation (%) determination between oxidation β-

carotene and ABTS method 



Samples 

Antioxidant (%) 

(β-carotene) 

method 

Antioxidant (%) 

(ABTS) method 

S. aqueum Leaves (fresh) 

73.77 70.31 



S. aqueum Leaves (dried) 

58.73 65.08 



P. foetida Leaves (fresh) 

78.13 75.38 



P. foetida Leaves (dried) 

66.67 67.74 



 

Correlation between phenolic content and antioxidant activity 

 

Some studies report a strong correlation between phenolic content and antioxidant activity in fruits, 



vegetables and grains [14] while other reports do not [12,16].  In this study the antioxidant activity of 

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). 

 


Molecules 2009, 14   

 

           



 

 

 



 

 

 



         

 

 



975

Figure 4. Correlation between phenolic content and antioxidant activity. 

 

 



 

 

 



 

 

 



 

 

 

 

 

 

Conclusions 

 

This study clearly indicated that  P. foetida and S.  aqueum, both  have high  antioxidant activity. 



Fresh samples had higher phenolic contents and better antioxidant activity than did dried samples.  

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

2

 = 



0.9878.  The present study confirms that P. foetida and S.  aqueum could be significant sources of 

natural antioxidant compounds that may have potent beneficial health effects. 



 

Experimental  

 

Preparation of samples 

 

Plant materials were collected from trees growing in home gardens in Gelugor, Penang, Malaysia. 



Upon arrival at the laboratory, samples were washed with water to remove debris. The leaves and 

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 


14 days until a constant weight was achieved. Fresh 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 



solution was then filtered through Whatman No. 4 filter paper and the solvent was removed in vacuo.  

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

-1

). The extracts were stored at 



-20 ºC.  These crude leave extracts of P. foetida and of S. aqueum  were subjected to ABTS free 

0

10



20

30

40



50

60

70



80

90

dried sa



dried pf

fresh sa


fresh pf

A

n

ti

o

x

id

a

n

t a

c

ti

v

ity

 (

%

)

0

10



20

30

40



50

60

70



P

h

en

o

li

c

 co

n

te

n

t (

m

g

/g

)

 β-carotene

ABTS

Phenolic content



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976

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. 

 

Antioxidant activity 



 

Coupled oxidation of β-carotene and linoleic acid 

 

The β-carotene bleaching assays were conducted as previously described with slight modifications  



[21,22]. A mixture of β-carotene (60 mg, Sigma Chemical Co.), linoleic acid (1.0 g, Sigma Chemical 

Co.) and Tween

®

 40 (20 mL, Sigma Chemical Co.) were dissolved in chloroform (20 mL, Merck).  



Chloroform was removed at 40 

°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 

DL

-α-tocopherol (Sigma 



Chemical Co.) and quercetin (Sigma Chemical Co.)  as standards.  A blank solution without β-carotene 

was prepared containing the same concentration of sample.  The total antioxidant activity was 

calculated based on the following equation: 

AA = [1 – (

Α

s

0

 - 

Α



160

)/(


Α

c

160

 - 

Α

c



160

)] X 100 

where A

s

0



 is the absorbance of sample at 0 min, A

s

160



 is the absorbance of sample at 160 min, A

c

0



 is the 

absorbance of control sample at 0 min, and A

c

160


 is the absorbance of control sample at 160 min. 

 

 ABTS free radical scavenging activity 

 

Radical scavenging activity was measured as previously described [23, 24] with minor 



modifications. 2,2’-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was used as the free 

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 (

L

)-(+) 


ascorbic acid standards (0.005 g mL

-1

 – 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. 



aqueum extracts. A control sample (without antioxidants or extract), containing the same amount of 

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

0

 – A) / A



0

] x 100 


where A

0

 is the absorbance of the control reaction and A is the absorbance in the presence of samples 



at 60 min.   

 

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977

Quantitative determination of total phenolic content 

 

The total phenolic content of the crude methanol extract was determined by using a modified Folin-



Ciocalteu method with ferulic acid (Sigma Chemical Co.) as a standard [25].  Folin-Ciocalteu reagent 

(0.25 mL, Fluka) was added to methanolic extract solution of (1.0 mg mL

-1

, 10 mL), then 20% 



aqueous sodium carbonate solution (1.2 mL) was added and the tube vortexed and then incubated for 

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.  



 

Acknowledgements 

 

This research was supported by RU research grant No. 1001/PKIMIA/811016 and 



304/229/PKIMIA/637059. 

 

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© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. 



This article is an open-access article distributed under the terms and conditions of the Creative 

Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). 



 


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