全 文 ：天然产物研究与开发 Nat Prod Res Dev 2012，24:219-223
文章编号:1001-6880(2012)02-0219-05
Received September 16，2010;Accepted December 31，2010
Foundation Item:This work was supported by the National Science ＆
Technology Pillar Program of China (2007BAI27B04)and National
Nonprofit Institute Research Grant of CATAS-ITBB (No．
ITBBZD1041)．
* Corresponding author Tel:86-898-66961869;E-mail:hfdai2001@ ya-
hoo． com． cn
香露兜叶的抗氧化活性
王 辉1，3，罗 应1，3，梅文莉1，3，谭乐和2，徐雪荣1，3，戴好富1，3*
1海南省药资源天然产物研究与利用重点实验室 中国热带农业科学院热带生物技术研究所，海口 571101;
2中国热带农业科学院香料饮料研究所，万宁 571533;3 海口市热带天然产物研究与利用重点实验室，海口 571101
摘 要:本文评价了香露兜叶的乙醇提取物以及石油醚、乙酸乙酯、正丁醇和水相组分的抗氧化活性。在体外
测定了提取物和组分的总抗氧化活性，二苯代苦味肼基(DPPH)自由基清除活性，超氧阴离子自由基清除活性
和铁离子还原能力，并利用分光光度法测定了其总酚和总黄酮的含量。结果表明，所有的提取物和组分均表现
出抗氧化和自由基清除活性。抗氧化活性的大小如下:正丁醇部分 ＞乙酸乙酯部分 ＞乙醇提取物 ＞水部分 ＞
石油醚部分。总酚含量的顺序和其相似，说明提取物和组分中的酚类化合物使其具有抗氧化活性。香露兜叶
提取物可能会成为有价值的天然抗氧化资源，并将在保健品和食品中得到应用。
关键词:香露兜;抗氧化活性;自由基清除活性;酚类化合物;黄酮类化合物
中图分类号:R284. 2 文献标识码:A
Antioxidant Properties of Pandanus amaryllifolius Leaves
WANG Hui1，3，LUO Ying1，3，MEI Wen-li1，3，TAN Le-he2，XU Xue-rong1，3，DAI Hao-fu1，3*
1Hainan key laboratory for research and development of natural products from Li folk medicine，
Institute of Tropical Bioscience and Biotechnology，Chinese Academy of Tropical Agricultural Sciences，Haikou
571101，China;2 Institue of spice and Beverage Resear，Chinese Acadamy of Agricultural Sciences，Wanning 571533，China;
3Haikou key laboratory for research and development of tropical natural products，Haikou 571101，China
Abstract:The antioxidant activities of the ethanol extract and petroleum ether-，ethyl acetate-，n-butanol-，and aqueous-
soluble fractions from Pandanus amaryllifolius leaves were investigated in this study． Total antioxidant capacity，1，1-di-
phenyl-2-picryhydrazyl (DPPH)scavenging activities，superoxide anion radical scavenging activities，and ferric reduc-
ing /antioxidant power (FRAP)were tested in vitro． The amounts of total phenolics and total flavonoids in the extracts
and fractions were also determined by spectrophotometric methods． The results showed that all the extracts and fractions
exhibited antioxidant and radical-scavenging activities． The decreasing order of antioxidant and radical-scavenging activi-
ties were found to be n-butanol fraction ＞ ethyl acetate fraction ＞ ethanol extract ＞ aqueous extract ＞ petroleum ether frac-
tion． This similar order of the amounts of total phenolics indicated that phenolics contributed to the antioxidant activities
of extract and fractions． It suggests that the extract of Pandanus amaryllifolius leaves may be valuable antioxidant natural
sources and applicable in both healthcare products and food industry．
Key words:Pandanus amaryllifolius;antioxidant activities;radical-scavenging activity;phenolics;flavonoids
Introduction
Reactive oxygen species (ROS) ，such as superoxide
anion radical，hydroxyl radical，and H2O2，are produced
in oxidative metabolism of cell． Excessive ROS can
cause cell death and tissue damage，resulting in many
diseases such as cardiovascular diseases，neurodegener-
ative disorders and cancer ［1，2］． Antioxidants，which
can reduce oxidative damage of cells，are gaining more
and more attention in recent years． As synthetic antioxi-
dants are thought to have possible toxicity，natural an-
tioxidants are attracting public interest，and an increas-
ing number of antioxidants from plants have been used
in drug，food as well as cosmetic industry．
Pandanus amaryllifolius，originated from Indonesia，is
widely distributed in Africa，South Asia，South East A-
DOI:10.16333/j.1001-6880.2012.02.021
sia and Australia． Its leaves，which emit a pleasant aro-
ma，are used as traditional flavoring agents in food
wrapping，or in cooking to enhance the fragrance of
food in Southeast Asian． Abas et al． evaluated the an-
tioxidant activities of the methanol extracts of P． ama-
ryllifolius leaves using ferric thiocyanate and thiobarbi-
turic acid methods，but no antioxidant activity was
found ［3］． In contrast，Yan and Asmah reported the high
total phenolic contents，antioxidant and scavenging ac-
tivities of its methanol extracts［4］，and Fatihanim et al．
reported the excellent heat-stable antioxidant capacity
of its ethanol extracts［5］． However，only the crude ex-
tracts of P． amaryllifolius leaves were evaluated，the an-
tioxidant properties of its different fractions have not
been systematically studied．
The aim of the present study was to evaluate the antiox-
idant and radical-scavenging activities of the petroleum
ether-，ethyl acetate-，n-butanol-，and aqueous-soluble
fractions of P． amaryllifolius leaves． The amounts of to-
tal phenolics and total flavonoids in the extract / frac-
tions were also determined due to the important roles of
phenolics and flavonoids as antioxidants．
Materials and Methods
Chemicals
Folin-Ciocalteu’s reagent，1，1-diphenyl-2-picrylhydra-
zyl (DPPH) ，2，2-azinobis-(3-ethylbenzothiazoline-6-
sulfonate) (ABTS· + ) ，rutin，gallic acid，ascorbic
acid，6-hydroxy-2，5，7，8-tetramethychroman-2-carbox-
ylic acid (Trolox ) ，2，4，6-tripyridyl-s-triazine
(TPTZ) ，α-tocopherol，ascorbic acid，xanthine，and
xanthine oxidase (XOD)were purchased from Sigma
Chemical Co．(St． Louis，MO，USA)． All other reagents
were of analytical grade．
Plant materials
Leaves of P． amaryllifolius were collected in Xinglong
botanical garden，Wanning，Hainan，China，in Septem-
ber 2009，and authenticated by Prof． Zhengfu Dai，In-
stitute of Tropical Bioscience and Biotechnology，Chi-
nese Academy of Tropical Agriculture Sciences，where
a voucher specimen of the collection (No． ME-
090912)has been deposited．
Extraction
The fresh leaves of P． amaryllifolius (200 g)were ex-
haustively extracted with 95% ethanol at room temper-
ature，and concentrated under reduced pressure to re-
move ethanol． The concentrate was suspended in H2O
and partitioned with petroleum ether，ethyl acetate and
n-butanol successively． All the extracts and the aqueous
layer were separately combined and evaporated to dry-
ness under reduced pressure． These four fractions were
designated as petroleum ether fraction (PEF，6． 0 g) ，
ethyl acetate fraction (EAF，4． 2 g) ，n-butanol fraction
(BUF，3． 1 g)and aqueous fraction (AF，6． 3 g) ，re-
spectively．
Total antioxidant capacity
The total antioxidant capacity was determined following
the method described by Siddhuraju［6］ with some minor
modifications． Potassium persulfate was added into 7
mM of ABTS· + and kept for 12 – 16 h at room tem-
perature in the dark． The ABTS· + solution was diluted
with PBS (potassium phosphate-buffered saline，pH 7．
4)to an absorbance of 0． 70 ± 0． 02 at 734 nm before
analysis． ABTS· + solution (3． 0 mL)was added to 0．
1 mL of the sample dissolved in DMSO at different con-
centrations and mixed thoroughly． The reaction mixture
was kept at room temperature for 6 min，and the absor-
bance was recorded at 734 nm on the Shimadzu UV-2550
spectrophotometer． Trolox was used as the positive con-
trol． The percentage scavenging effect was calculated as
Scavenging rate = ［1 – (A1– A2)/ A0］ × 100%
where A0 was the absorbance of the control (without
extract) ，A1 was the absorbance in the presence of the
extract，A2 was the absorbance without ABTS
· + ．
DPPH˙ scavenging capacity
The DPPH˙ scavenging capacity was measured using
the method of Blois［7］ with slight modifications． A 0． 1
mM solution of DPPH˙ in ethanol was prepared and 2
mL of this solution was added to 0． 1 mL of the sample
dissolved in DMSO at different concentrations． After
gentle mixing and 30 min of incubation at room temper-
ature，the absorbance of the resulting solutions was
measured at 517 nm． Ascorbic acid was used as the
positive control． The percentage scavenging effect was
calculated as Scavenging rate = ［1 – (A1– A2)/
A0］ × 100%
022 Nat Prod Res Dev Vol. 24
Where A0 was the absorbance of the control (without
extract) ，A1 was the absorbance at the presence of the
extract，A2 was the absorbance without DPPH˙．
Antioxidant activity by the ferric reducing /antioxi-
dant power (FRAP)assay
The procedure described by Benzie and Strain was fol-
lowed［8］． Brieμy，the ferric reducing /antioxidant power
(FRAP)reagent containing 2． 5 mL of 10 mM 2，4，6-
tripyridyl-S-triazine (TPTZ)solution in 40 mM HCl，
2． 5 mL of 20 mM FeCl3 and 2． 5 mL of 300 mM ace-
tate buffer at pH 3． 6 was prepared freshly and warmed
at 37 ℃ before analysis． Aliquots (40 μL)of 0． 1mg /
mL sample were mixed with 0． 2 mL of distilled water
and 1． 8 mL of FRAP reagent，and the absorbance of
the reaction mixture was measured at 593 nm after in-
cubation at 37 ℃ for 30 min． FeSO4 was used as the
positive control，and its linear gradient concentrations
were tested to establish the standard curve． The final
results were expressed as the concentration of antioxi-
dants having a ferric reducing ability equivalent to that
of 1． 0 mM FeSO4，particularly expressed as mmol Fe
(Ⅱ)equivalent /g sample in dry weight．
Superoxide anion radical scavenging activity
The superoxide anion radical scavenging activities of
the samples were carried out by the method of Sakana-
ka et al with slight modifications［9］． The mixture con-
taining 0． 5 mM xanthine，0． 6 mM hydroxylammonium
chloride solution in 65 mM phosphate buffer solution
(pH7． 8)was prepared． Then 0． 1 mL sample solutions
were added into 1． 5 mL of the mixture，and 0． 3 mL of
200 μg /mL xanthine oxidase solution were added to
start the reaction at 25 ℃ for 20 min． The reactive liq-
uid was sampled 0． 5 mL，and added 0． 5 mL of 19 mM
p-aminobenzene sulfonic acid and 0． 5mL of 1． 0% α-
naphthylamine solution and mixed fully，then placed at
room temperature to react for 20 min，and at last the
absorbency (A1)was tested at 530 nm． The sample
was substituted by redistilled water and repeated the
procedures mentioned above to test the absorbency (A0)
of the blank． α-Tocopherol was used as the positive con-
trol，and its linear gradient concentrations were tested to
establish the standard curve． The superoxide radical-scav-
enging activity was shown with α-tocopherol equivalent
antioxidant capacity (μmol α-tocopherol /g DW)．
Determination of the amounts of total phenolics
The amount of total phenolics was measured by the
method described by Hai-yun Li et al．［10］． Briefly，0． 4
mL of extract or standard solution of gallic acid was
added to a 10 mL volumetric flask containing 3 mL of
distilled water，and then 1 mL of Folin and Ciocalteu’s
phenol reagent was added to the mixture and shaken．
After 5 min reaction，4 mL of 7% Na2CO3 solution was
added with mixing． The solution was then immediately
diluted to 10 mL with distilled water and mixed thor-
oughly． After incubation for 90 min at room tempera-
ture，the absorbance versus a blank was determined at
750 nm． Total phenolic contents of extracts or fractions
were expressed as mg gallic acid equivalents (GAE)/ g
dry weight．
Determination of the amounts of total flavonoids
The amount of total flavonoids was measured by a col-
orimetric assay developed by Jia et al．［11］． An aliquot
(1 mL)of extract or standard solution of rutin was
added to a 10 mL volumetric flask containing 4 mL of
distilled water． Initially，0． 3 mL of 5% NaNO2 was
added to the flask． After 5 min，0． 3 mL of 10% AlCl3
was added． At 6 min，2 mL of 1 M NaOH was added to
the mixture，and the reaction flask was diluted to vol-
ume with distilled water immediately and mixed thor-
oughly． Absorbance of the mixture，pink in color，was
determined at 510 nm versus a blank． Total flavonoids
of the extracts or fractions were expressed as mg rutin
equivalents (RE)/ g dry weight．
Results and Discussion
Total antioxidant capacity
It is well known that free radical scavenging activity of
antioxidants against ABTS· + is attributed to their hy-
drogen donating ability． Derived fractions of the extract
of P． amaryllifolius leaves were examined for their
ABTS· + scavenging activity，and the results were com-
pared to that of Vc and Trolox． The ability to scavenge
ABTS· + of various fractions was in the order of Trolox
(99． 5) ＞ Vc (98． 6%) ＞ n-butanol fraction
(61. 5%) ＞ ethyl acetate fraction (55． 3%) ＞ a-
queous fraction (14． 0%) ＞ petroleum ether fraction
122Vol. 24 WANG Hui，et al:Antioxidant Properties of Pandanus amaryllifolius Leaves
(3． 47%)at the concentration of 350 mg /L，as shown
in Fig． 1． The correlation between ABTS· + scavenging
activity and concentration of the fractions is almost lin-
ear，indicating that the radical-scavenging activity is re-
lated to the amount of antioxidant from P． amaryllifolius
leaves．
Fig. 1 ABTS· + scavenging activities of extracts or
fractions of P． amaryllifolius leaves．
AF:aqueous fraction;EAF:ethyl acetate fraction;BUF:n-butanol
fraction;PEF:petroleum ether fraction
DPPH scavenging activities
DPPH radical，which can be scavenged by antioxidants
by hydrogen donation，is usually used as a substrate to
evaluate antioxidant activity． The scavenging effects of
the fractions of P． amaryllifolius leaves on DPPH radi-
cal were shown in Fig． 2． It can be found that the radi-
cal-scavenging activities increased with the increasing
concentration． The ability to scavenge DPPH radicals of
various fractions was in the order of Vc (99． 1%) ＞
n-butanol fraction (75． 0%) ＞ ethyl acetate fraction
(51． 2%)＞ aqueous fraction (37． 0%)＞ petroleum
ether fraction (10． 3%)at the concentration of 250
mg /L． The DPPH radical scavenging ability of the frac-
tions is similar to that of ABTS· +，but a little higher
then that of ABTS· +，suggesting that the method is
more sensitive．
Fig. 2 DPPH ˙ scavenging activities of fractions of
the extract of P． amaryllifolius leaves．
AF:aqueous fraction;EAF:ethyl acetate fraction;BUF:n-butanol
fraction;PEF:petroleum ether fraction
Superoxide anion radical scavenging activity
Superoxide anion radical is a precursor of more reactive
oxygen species，such as hydroxyl radical，hydrogen per-
oxide，or singlet oxygen in living systems［12］，leading to
tissue damage and various diseases． The scavenging ac-
tivities of the extract or fractions on superoxide radicals
were demonstrated in Table 1． The n-butanol fraction
exhibited the highest activity，while the petroleum ether
fraction and the aqueous fraction showed no activity．
The ability to scavenge superoxide anion radical was in
the order of n-butanol fraction (1711 ± 23) ＞ ethanol
extract (1256 ± 34) ＞ ethyl acetate fraction (971 ±
32) ＞ aqueous fraction /petroleum ether fraction．
Antioxidant activity by the ferric reducing /antioxi-
dant power (FRAP)assay
Fe3 + reduction is often used as an indicator of electron-
donating activity，and can be closely associated with
antioxidant properties of compounds［13］． In this assay，
ferric-tripyridyltriazine was reduced to the ferrous com-
plex and formed an intense blue color which can be
measured at 593 nm． The intensity of the color is pro-
portional to the antioxidant activities of the samples．
The ferric ion-reducing activities of the extract / frac-
tions are similar to the results obtained from the three
methods above，as shown in Table 1． The highest activi-
ty was for the n-butanol fraction，followed by the etha-
nol extract，ethyl acetate fraction and aqueous fraction．
No activity was detected in the petroleum ether frac-
tion．
Table 1 Ferric reducing /antioxidant power (FRAP)and
O-·2 radical scavenging activity of extract / frac-
tions of P． amaryllifolius leaves
Extract / fractions
Antioxidant activity
(mmol FeSO4 /gDW)
O-·2 radical
scavenging activity
(μmol α-
Tocopherol /gDW)
Ethanol extract 468 ± 32 971 ± 32
Petroleum ether Not detected Not detected
Ethyl acetate 267 ± 20 1256 ± 34
n-butanol 861 ± 19 1711 ± 23
Water 224 ± 19 Not detected
The amounts of total phenolics and flavonoids
Phenolic compounds contribute to the overall antioxi-
222 Nat Prod Res Dev Vol. 24
dant activity because of their hydrogen donating abili-
ty． The amounts of total phenolics and flavonoids in ex-
tract and fractions of P． amaryllifolius were demonstra-
ted in Table． 2． The amount of total phenolics in the
ethanol extract was 38． 4 GAE mg /g DW，while in its
n-butanol fraction，the content was 63． 02 GAE mg /g
DW，the highest among the fractions． The content of to-
tal flavonoids was the highest in the ethyl acetate frac-
tion，453． 99 RE mg /g DW，and the lowest in the aque-
ous fraction，only 15． 06 RE mg /g DW． It is worth
pointing out that the highest content of total phenolics
was detected in the n-butanol fraction，while the con-
tent of total flavonoids was the highest in the ethyl ace-
tate fraction． Generally，flavonoids tend to concentrate
in the ethyl acetate fraction，while polyphenols are en-
riched in the aqueous fraction． Therefore，it can be de-
duced that polyphenols contribute to the antioxidant ac-
tivity of P． amaryllifolius leaves．
Table 1 Amounts of total phenolics and flavonoids in ex-
tracts / fractions of P． amaryllifolius leaves
Extract / fractions Total phenolics
(GAE mg /g DW)
Total flavonoids
(RE mg /g DW)
Ethanol extract 38． 40 ± 0． 21 239． 82 ± 2． 40
Petroleum ether Not detected Not detected
Ethyl acetate 30． 90 ± 1． 87 453． 99 ± 4． 79
n-butanol 63． 02 ± 2． 26 52． 20 ± 0． 52
Water 10． 08 ± 0． 05 15． 06 ± 0． 19
Conclusion
As a conclusion，the results obtained in this study
clearly demonstrate that all the tested extracts / fractions
of P． amaryllifolius leaves showed antioxidant and radi-
cal-scavenging activities． The decreasing order of an-
tioxidant and radical-scavenging activities among the
extracts assayed through all the four methods was found
to be n-butanol fraction ＞ ethyl acetate fraction ＞ a-
queous fraction ＞ petroleum ether fraction． This order
is similar to the amount of total phenolics of the ex-
tracts / fractions． Extracts of Pandanus amaryllifolius
leaves might be valuable antioxidant natural sources
and applicable in both healthy medicine and food in-
dustry． However，the components responsible for the an
tioxidant activities of its extracts are currently unclear，
further work should be performed on the isolation andi-
dentification of the components in the extracts，espe-
cially in the n-butanol and ethyl acetate fractions of
ethanol extract．
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