South African Journal of Botany 93 (2014) 98–104

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Antioxidant and tyrosinase inhibition activities of the ethanol-insoluble

fraction of water extract of Sapium sebiferum (L.) Roxb. leaves
Rao. Fu, Yuting. Zhang, Yiran. Guo ⁎, Fang. Chen ⁎⁎
College of Life Sciences, Sichuan University, Sichuan Key Laboratory of Resource Biology and Biopharmaceutical Engineering, Key Laboratory of Bio-resource and Eco-environment, Ministry of
Education, Chengdu 610064, PR China

a r t i c l e i n f o a b s t r a c t

Article history: The ethanol-insoluble fraction of water extracts of Sapium sebiferum leaves (new and fallen) were screened for
Received 8 December 2013 their in vitro antioxidant and tyrosinase (TYR) inhibition activities. Fourier Transmission-Infra red was adopted
Received in revised form 26 March 2014 for analysis of the major functional groups; common methods (scavenging of DPPH, ABTS, nitrite and hydroxyl
Accepted 1 April 2014
radicals; ORAC; reducing power; β-carotene bleaching; and Fe2+ chelating assays) were employed to evaluate
Available online xxxx
the antioxidant properties; and L-DOPA method was used to determine the TYR inhibition effects, and the copper
Edited by AR Ndhlala chelating activities were also determined. Our results showed that fraction of water extracts of new and fallen
S. sebiferum leaves have great antioxidant and TYR inhibition activities, even better than those of the positive con-
Keywords: trol (BHT and arbutin). The TYR inhibition effect was significantly (P b 0.05) and positive (r = 0.8605) correlated
Antioxidant activity with its copper chelating activity, which can be proposed as the mechanism of TYR inhibition. These results dem-
Copper chelating onstrate that S. sebiferum leaves have great potential for being further studied and used, and no strict collection
Ethanol-insoluble time limited. Altogether, potential antioxidant activity and TYR inhibitory property of S. sebiferum leaves could be
Sapium sebiferum applied to the industry as natural antioxidants and TYR inhibitors.
Tyrosinase inhibition
© 2014 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction lack of antioxidants facilitates the development of cardiovascular dis-

Reactive oxygen species (ROS) as well as reactive nitrogen species
(RNS) are inherent parts of the anabolism and catabolism processes in
tissues, including skin (Pinnell, 2003). These species play a dual role as
both toxic and beneficial. At a low level, ROS and RNS exert favorable
effects by assisting the immune system and mediating cell signaling;
however, at high concentrations, they generate oxidative stress that
can damage cell structures (Flora, 2006). The excessive ROS/RNS should
be eliminated by the antioxidant system. Mechanisms of antioxidant
action include serving as (1) chemical traps/sinks that “absorb” energy
and electrons, quenching ROS/RNS; (2) binding/inactivation of metal
ions to prevent generation of ROS/RNS; and (3) chain-breaking antiox-
idants, which scavenge and destroy ROS/RNS (Karadag et al., 2009). A
Abbreviations: TYR, tyrosinase; FT-IR, Fourier Transmission-Infrared; DPPH, 2,2-
diphenyl-1-picrylhydrazyl; ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid);
ORAC, oxygen radical absorbance capacity; L-DOPA, L-3,4-dihydroxyphenylalanine; Vc,
ascorbic acid; BHT, 2,6-di-tert-butyl-4-methylphenol; TCM, Traditional Chinese
Medicine; ROS, reactive oxygen species; RNS, reactive nitrogen species; AAPH, 2,2′-
azobis(2-amidinopropane) dihydrochloride; N, ethanol-insoluble fraction of water ex-
tracts of new leaves; F, ethanol-insoluble fraction of water extracts of fallen leaves; DW,
distilled water; AUC, area under curve; TE, Trolox Equivalents; EDTA, ethylene diamine
tetraacetic acid; PV, pyrocatechol violet.
⁎ Corresponding author. Tel./fax: +86 28 85412182.
⁎⁎ Corresponding author. Tel./fax: +86 28 85417281.
E-mail addresses: ran@scu.edu.cn (Y. Guo), fangchen1112@gmail.com (F. Chen).
eases, cancers, neurodegenerative diseases and inflammatory diseases
(Krishnaiah et al., 2011).
A compound with an antioxidant activity usually has a whitening
effect. Tyrosinase (TYR) is a multifunctional copper-containing enzyme
distributed ubiquitously in organisms. TYR catalyzes two distinct reac-
tions of melanin synthesis, the hydroxylation of L-tyrosine to L-DOPA
and the oxidation of L-DOPA to dopaquinone (Lontie, 1984; Solano
et al., 2006). Copper is the active site of TYR. Therefore, a compound
with copper chelating activity may be serviceable for the inhibition of
TYR. TYR inhibitors may be clinically applicable for the treatment of
some dermatological disorders associated with melanin hyperpigmenta-
tion and important in cosmetics for depigmentation (Shiino et al., 2001).
Sapium sebiferum (L.) Roxb. (Euphorbiaceae), which originated in
China, now grows primarily in the subtropical regions of the world
and is cultivated as an ornamental plant and has been regarded as an in-
vasive species in the U.S. The seeds contain 45–60% oil (Atabani et al.,
2013; Liu et al., 2009). Currently, it has been considered useful in the
production of biodiesel because it is the third most oil enriched crop,
just after algae and oil palm (Sanford et al., 2009). The leaves of
S. sebiferum have been long used in Traditional Chinese Medicine
(TCM) as an anti-inflammatory medication for the treatment of skin
disease, including eczema, shingles, edema, swelling, and ascites, among
other maladies. It has been reported that the leaves of S. sebiferum contain
gallic acid, kaempferol, quercetin, β-sitosterol glycoside, astragalin, 6-O-
galloyl-D-glucose, methyl gallate, and methyl-3,4,5-trihydroxybenzoate

http://dx.doi.org/10.1016/j.sajb.2014.04.003
0254-6299/© 2014 SAAB. Published by Elsevier B.V. All rights reserved.
 R. Fu et al. / South African Journal of Botany 93 (2014) 98–104
(Hsu et al., 1994; Kane et al., 1988; Liu et al., 1988). Meanwhile, Gao
Yinyu et al. had reported that the soluble glycoprotein from
S. sebiferum leaves consisted of saccharide and protein with a ratio
94:6 (Gao et al., 2003). Modern pharmacology studies showed that
extracts of S. sebiferum leaves, including methanol, ethanol, water,
ethyl acetate fraction of ethanol, and ethanol soluble fraction of water
extracts exhibited antioxidant, anti-microbial, anti-inflammatory, anti-
hypertensive, and analgesic activities (Bin-xue et al., 2004; Chaudhary
et al., 2011; Fu et al., 2013; Xiao-lie et al., 2011). Our preliminary
work has revealed that extracts of S. sebiferum leaves at different devel-
opment stages exhibited distinct bioactivity and chemical composition
(data not shown here).
The aim of this study was to evaluate the antioxidant and TYR inhi-
bition activities of the ethanol-insoluble fraction of water extract of
S. sebiferum leaves at two different development stages (new and
fallen). Both extracts exhibited a potential antioxidant activity that
could be applied to the industry as natural antioxidants. And its TYR
inhibitory property made it suitable for the cosmetic industry.
2. Materials and methods
2.1. Reagents
Linoleic acid, ascorbic acid (Vc), 2,2′-azino-bis(3-ethylbenzthiazoline-
6-sulfonic acid) (ABTS), β-carotene, 2,6-di-tert-butyl-4-methylphenol
(BHT), tyrosinase from mushroom, L-DOPA, arbutin and 2,2-diphenyl-1-
picrylhydrazyl (DPPH), fluorescein disodium salt and 2,2′-azobis(2-
amidinopropane) dihydrochloride (AAPH) were purchased from
Sigma-Aldrich Chemical Co. (St Louis, MO, USA). All other chemicals
and reagents used in this study were of analytical grade.
2.2. Preparation of the ethanol-insoluble fraction of water extracts
Fresh new and fallen (collected in April and December respectively)
leaves of S. sebiferum were dried under the condition of shade and
ventilation at room temperature (~25 °C), and ground into powder. A
3 g portion of powder was weighed and extracted with 90 ml water at
90 °C (heating in a water bath) for 2 h in a round-bottom flask. After
vacuum filtration, the solutions were concentrated using a vacuum
rotary to about 10 ml and mixed with absolute ethanol (1:5, v/v) for
24 h at 4 °C. The precipitates were collected after centrifugation and
washed with ethanol (five times) and acetone (twice). 0.3325 g and
0.2195 g of ethanol-insoluble fraction of water extracts were obtained
from new (for short, N) and fallen leaves (for short, F), after drying
under an infrared lamp (~50 °C) . They were then ground into powders
and stored in −20 °C, freshly dissolved in distilled water (DW) to per-
form different antioxidant assays; and in the buffer for TYR inhibition
and copper chelating assays.
2.3. Fourier Transmission-Infrared (FT-IR) spectral analysis
IR spectra were scanned using an FT-IR spectrophotometer (Thermo
Nicolet NEXUS 670 FTIR, USA) with KBr pellets in the range of 4000–
400 cm−1.
2.4. Evaluation of in vitro antioxidant activity
2.4.1. DPPH radical scavenging assay
The DPPH radical scavenging assay was conducted using the method
of Eldeen et al. (2011). Briefly, 100 μl of the sample solution (1–16 μg/ml)
was mixed with 100 μl of DPPH solution (0.1 mM, in methanol). The
reaction mixture was shaken and incubated for 30 min at 25 °C, and
the absorbance was measured at 517 nm. The radical scavenging activity
was calculated using formula (1) as follows:
DPPH radical scavenging activityð% Þ ¼ ½1−ðAi−AsÞ=Ac  100
99
where Ac represents the absorption of the negative control, Ai represents
the absorption of the experimental group and As represents the absorp-
tion of the sample background. Vc served as a reference.
2.4.2. ABTS radical scavenging assay
The ABTS radical scavenging activity was evaluated using the method
of Adewusi et al. (2011) with minor modifications. The ABTS•+ solution
was produced by reacting 7 mM ABTS with 2.45 mM potassium persul-
fate (described as final concentrations and dissolved in phosphate buffer
(0.2 M, pH 7.4)) at 25 °C in the dark for 12–16 h. This solution was then
diluted with phosphate buffer to obtain an absorbance of 0.7 ± 0.02 at
734 nm. Aliquots of 50 μl of samples (0.5–32 μg/ml) were mixed with
150 μl of the diluted ABTS•+ solution. After the mixture had been incu-
bated at room temperature for 3 min, the absorbance was measured at
734 nm. The ABTS radical scavenging activity was calculated by formula
(1). Vc was used as a reference.
2.4.3. Oxygen radical absorbance capacity (ORAC) assay
The method of the ORAC assay was adapted from Alañón et al.
(2011) and Bernaert et al. (2012) with minor modifications. The assay
was performed on a microplate reader (Spectra Max M2, Molecular
Devices, USA) and 96-well plates. The perimeter wells were not used
for experiments, but were all filled with 200 μl phosphate buffer
(75 mM, pH 7.4) to ensure that all sample wells were surrounded
by full wells. To each well, 40 μl of Trolox dilutions, 40 μl of samples
(dilution by phosphate buffer to 50 μg/ml) or 40 μl of phosphate buff-
er was pipetted in triplicate for the calibration curve, samples or the
blank, respectively. 40 μl of fluorescein solution (44.782 nM) was then
added to each well. The microplate was incubated at 37 °C for 20 min.
170 μl of 2,2′-azobis-2-methyl-propanimidamide dihydrochloride
(AAPH, 122.499 mM) was added, and the fluorescence was recorded
immediately at an excitation wavelength of 485 nm and an emission
wavelength of 538 nm every 2 min thereafter for 120 min. Calculations
were based on the area under the fluorescence decay curve (AUC).
ORAC values were calculated using a regression equation for a linear
regression on the range of 59.193–236.526 μM Trolox standards. The net
AUC was obtained by subtracting the area under the curve for the blank
values from the curves of samples and standards. ORAC values were
expressed in μmol Trolox Equivalents per mg (μmol TE/mg).
2.4.4. Reducing power assay
The reducing powers of N and F were measured according to the
method of Chen et al. (2011) with slight modifications. Aliquots of
samples (25 μl) with various concentrations (2–32 μg/ml) were mixed
with 50 μl of phosphate buffer (0.2 M, pH 6.6) and 25 μl of 1% potassium
ferricyanide [K3Fe(CN)6]. After incubation at 45 °C for 30 min, 40 μl of
10% trichloroacetic acid (TCA) and 60 μl of 0.1% ferric chloride (FeCl3)
were added. The absorbance was measured at 700 nm. Vc was used as
a reference.
2.4.5. Nitrite scavenging assay
The nitrite scavenging activities of the samples were evaluated using
the method of Debnath et al. (2011) with minor modifications. Briefly, 1
ml of samples at various concentrations (10–320 μg/ml) were mixed
with 0.3 ml of NaNO2 (5 μg/ml), and then 0.1 N HCl was added dropwise
until the pH was 2.0. Next, the mixture was incubated at 37 °C for 30
min. After incubation, the reaction mixture was immediately mixed
with 0.3 ml of sulfanilic acid (0.4%), and then incubated at room temper-
ature for 5 min. Then, 0.3 ml of N-ethylenediamine (0.2%) and 2.0 ml of
distilled water were added to the above mixture, and incubated again
for 15 min at room temperature. The absorbance was measured at 538
nm. The nitrite scavenging activity was calculated using formula (1).
ð1Þ Vc was used as a reference.
100 R. Fu et al. / South African Journal of Botany 93 (2014) 98–104

2.4.6. OH radical scavenging assay
The abilities of N and F to scavenge the OH radical were determined
using the method of Liu et al. (2012). Briefly, 150 μl of samples
(10–640 μg/ml) was mixed with 25 μl FeSO4 (1.5 mM) and 10 μl
salicylic acid (20 mM, in ethanol), then 15 μl H2O2 (6 mM) was added
to start the reaction. The mixture was incubated at 37 °C for 1 h. The ab-
sorbance was measured at 520 nm. The OH radical scavenging activity
was calculated using formula (1). Vc was used as a reference.
sodium acetate buffer (pH = 6.0) and 200 μl of 2 mM CuSO4. The
mixture was incubated at room temperature for 10 min. Then
200 μl of 2 mM PV was added. After 20 min, the absorbance was mea-
sured at 632 nm. The copper chelating activity was calculated using
the formula:
Copper chelating activityð% Þ ¼ ½1−ðA−BÞ=ðC−DÞ  100

2.4.7. β-Carotene bleaching assay

where A is the A632 nm of sample + buffer + CuSO4 + PV, B is the
β-Carotene–linoleic acid system according to the modified method
A632 nm of sample + buffer + buffer + PV, C is the A632 nm of
used by Zovko Končić et al. (2010). Briefly, 3 ml of β-carotene solution
buffer + buffer + CuSO4 + PV, and D is the A632 nm of buffer + buffer
in chloroform (0.2 mg/ml) was pipetted into a round-bottom flask,
+ buffer + PV.
and then 0.05 ml of linoleic acid and 0.5 ml of Tween 40 were added.
After removing the chloroform in a rotary evaporator at 30 °C, 200 ml
of distilled water was added to wash out the residue. The reaction 2.6. Statistical analysis
mixtures contained 5 ml of the above emulsion and 200 μl of samples
(0.03 mg/ml) and incubated at 50 °C for 90 min. BHT was used as a All the previous experiments were performed at least three times.
positive control. The antioxidant activity of the extracts or standard All data are reported as the mean ± SD. Differences were found by the
was calculated as: Student's t-test. Significant difference was assumed at ⁎P b 0.05. IC50
(defined as the dose of extract that is required for 50% scavenging or
Linoleic peroxidation inhibition ðLPIÞð% Þ
chelating activity in vitro) for antioxidant assays and Pearson correla-
¼ ½1−ðAi0 −Ai90 Þ=ðAc0 −Ac90 Þ  100 tion coefficients for the association between the TYR inhibition effect
and the copper chelating activity was established using SPSS.
where Ai0 and Ai90 were the absorbance of test samples at t = 0 min and
t = 90 min, respectively, Ac0 and Ac90 were the absorbance of control at
t = 0 min and t = 90 min, respectively. 3. Results and discussion

2.4.8. Fe2+-chelating activity 3.1. FT-IR analysis

The chelating of ferrous ions by N and F was estimated by the method
of Ganesan et al. (2011) with slight modifications. Briefly, 1 ml of differ-
ent concentrations (20–1280 μg/ml) of samples were mixed with 2.75
ml distilled water and 0.05 ml FeCl2 (2 mM). The reaction was initiated
by the addition of 0.2 ml ferrozine (5 mM) and the mixture was shaken
vigorously and left standing at room temperature for 10 min. The absor-
bance at 562 nm was measured. The Fe2+-chelating activity was calcu-
lated using the formula:
2þ
Fe ‐chelating activityð% Þ ¼ ð1−Ai=AcÞ  100
where Ai is the absorbance of the test group, and Ac is the absorbance of
the negative control. EDTA was used as a reference.
2.5. Determination of the TYR inhibition activities
The TYR inhibition effect was determined according to the method
previously described by Maisuthisakul and Gordon (2009) and Wang
et al. (2010) with minor modifications. Arbutin was used as a positive
control. Samples were dissolved in phosphate buffer (0.1 M,
pH 6.8), and then 160 μl of different concentration samples was
mixed with 10 μl aqueous solution of mushroom TYR (500 units/ml)
and incubated at 30 °C for 5 min, and then 30 μl of aqueous solution
of L-DOPA (5 mM) was added and the mixture was incubated at 30 °C
for 30 min. The absorbance was measured at 470 nm with reference
at 700 nm. TYR inhibitory activity was determined by the following
equation:
FT-IR spectra of N and F are shown in Fig. 1. They are polysaccharide
like spectra in a strong stretching peak around 3410 cm− 1 for O\H
stretching vibrations and a weak absorption peak of about 2925 cm−1
for C-H stretching vibrations, respectively. The O\H group also sug-
gested that there may be some phenolic compounds in the extract.
1715 cm− 1, 1615 cm− 1 were attributed to carbonyl group bending
vibration and 1211 cm− 1, 765 cm− 1 were C\O\C vibration (Zhao
et al., 2012). Around 1072 cm− 1 region is dominated by sugar ring
vibrations overlapping with stretching vibrations of (C\OH) side
groups and the glycosidic (C\O\C) bond vibration (Peng et al.,
2010). N and F have almost the same FT-IR spectra. Their intensity dif-
ferences may be due to the origin and technique of the measurement
procedure. The results of FT-IR demonstrated that the extracts were
composed of polysaccharide, phenolic and others. As already known,
polysaccharide and phenolic compounds exhibit various bioactivities,
and according to traditional uses of S. sebiferum leaves for the treatment
of skin diseases, antioxidant activities of extracts were detected.

TYR inhibitionð% Þ ¼ ½1−ðC−DÞ=ðA−BÞ  100

where A is the optical density (OD470) without samples or TYR, B is the

OD470 without samples but with TYR, C is the OD470 with samples and
TYR, and D is the OD470 with samples but without TYR.
The copper chelating activity was also determined using the pyro-
catechol violet (PV) method (Sánchez-Vioque et al., 2013) with minor Fig. 1. FT-IR spectra of the ethanol-insoluble fraction of water extracts. N, the ethanol-
modifications to understand the possible mechanism of the TYR inhibi- insoluble fraction of water extract of new S. sebiferum leaves; F, the ethanol-insoluble fraction
tion effect further. N or F (0.5 ml) was mixed with 2 ml of 50 mM of water extract of fallen S. sebiferum leaves.
 R. Fu et al. / South African Journal of Botany 93 (2014) 98–104 101

The DPPH radical scavenging activities of N and F are presented in

Fig. 2. Antioxidant properties of Sapium sebiferum leaves ethanol-insoluble fraction of
water extracts determined using (A) a DPPH radical scavenging assay; and (B) an ABTS
radical scavenging assay. Each bar represents the mean ± SD (n = 3). N, the ethanol-
insoluble fraction of water extract of new S. sebiferum leaves; F, the ethanol-insoluble
fraction of water extract of fallen S. sebiferum leaves; Vc, ascorbic acid.
3.2. In vitro antioxidant activities
Antioxidants reduce the oxidative stress in cells and are therefore
useful in the treatment of human diseases. Antioxidant derived from
plant has been widely researched due to its relative safe than synthetic
antioxidants (Krishnaiah et al., 2011). Many methods have been
established to determine antioxidant activity, while each method has
its own merits and drawbacks. Therefore, it is pertinent to use several
assays instead of only one to determine the antioxidant (Petlevski
et al., 2013). In the present study, several common methods, as men-
tioned earlier, were used for the determination of in vitro antioxidant
of N and F.
Fig. 2(A). Both samples and the positive control exhibited a dose-
dependent activity. In the present experiment, the IC50 for N and F
were 4.84 ± 0.04 μg/ml and 5.38 ± 0.25 μg/ml, respectively (Table 1).
N and F exhibited strong ABTS radical scavenging activities as described
in Fig. 2(B). The IC50 values of N, F and Vc were represented in Table 1.
The IC50 values of N and F were in the same order of magnitudes com-
pared with reference Vc for DPPH and ABTS radical scavenging assays,
indicating that they are powerful antioxidants.
The ORAC method is believed to be a preferable method as its biolog-
ical relevance to the in vivo antioxidant efficacy. Fig. 3 shows the kinetic
curves of Trolox standard and samples. The highest concentration of
Trolox (247.012 μM) provided virtually full protection for approximate-
ly 40 min, before the fluorescence intensity began to diminish. The AUC
was calculated from these kinetic curves by SoftMax_Pro 5 software.
The ORAC values of N and F were determined from the regression equa-
tion of a calibration cure from net AUCs of Trolox against concentrations.
In our duplicate tests, the correlation coefficients (R2) of regression
equations were 0.9933, 0.9902 and 0.9892, respectively. The ORAC
values of N and F were 3.00 ± 0.12 μmol TE/mg and 3.33 ± 0.07 μmol
TE/mg, respectively (Table 1).
The reducing capacity of a compound may serve as a significant indi-
cator of its potential antioxidant activity. Fig. 4(A) shows the reducing
power of N and F determined using the Fe3+–Fe2+ system with higher
absorbance at 700 nm which indicates a stronger reducing power. The
reducing capacities of samples and the positive control increased with
the increase of concentration. The results indicated that the order of
reductive potential was Vc N N N F.
The nitrite scavenging activities of the samples and standard are
presented in Fig. 4(B). They all exhibited a dose-dependent activity at
a concentration ranging from 10 to 320 μg/ml. The IC50 values of N
and F were 43.91 ± 3.69 μg/ml and 46.52 ± 1.44 μg/ml (Table 1),
respectively. The results of OH radical scavenging activities of N
and F are given in Fig. 4(C). All the samples exhibited a concentration-
dependent activity. The IC50 values were represented in Table 1.
The basis of the β-carotene bleaching assay is the discoloration of
β-carotene in a reaction with a linoleic acid free radical, while the
antioxidants present in the reaction solution can prevent the degrada-
tion of β-carotene. The abilities of N and F to slow down the rate of
discoloration of β-carotene were calculated. All samples showed an in-
hibition effect on bleaching of β-carotene in comparison with the nega-
tive control. The inhibition effects of N and F were 67.71 ± 2.56% and
48.78 ± 4.63% at a concentration of 30 μg/ml, respectively. As a positive
control, BHT exhibited an inhibition rate of 7.22 ± 1.21% (Table 1).
Metal chelating activity is claimed as one of the antioxidant mecha-
nisms, since it reduces the concentration of the catalyzing transition
metal in lipid peroxidation. In this assay, ferrozine quantitatively
formed complexes with Fe2+. With the existence of chelating agents,
the complex is disrupted resulting in fading of the red color. Fig. 4(D)
shows the ferrous ion chelating activities of N and F, indicating that

Table 1
Antioxidant activities of the ethanol-insoluble fraction of water extracts of Sapium sebiferum leaves determined using various methods.

Samples LPI (%) IC50 (μg/ml) ORAC (μmol TE/mg)

a a 2+ a b b b
β-Carotene bleaching ABTS Fe -chelating DPPH Nitrite OH ORAC assayc
a a
N 67.71 ± 2.56% 8.32 ± 0.35 584.34 ± 7.00 4.84 ± 0.04 43.91 ± 3.69 229.15 ± 18.04 3.00 ± 0.12
F 48.78 ± 4.63% 9.52 ± 0.63 964.36 ± 56.31 5.38 ± 0.25 46.52 ± 1.44 189.48 ± 9.62 3.33 ± 0.07
Vc nd 6.89 ± 0.02 nd 3.71 ± 0.26 6.82 ± 0.39 119.50 ± 20.18 nd
BHT 7.22 ± 1.21% nd nd nd nd nd nd
EDTA nd nd 49.48 ± 0.93 nd nd nd nd

LPI, IC50 and ORAC values represent the mean ± SD (n = 3).

nd, not determined.
a
N exhibited a significantly stronger antioxidant activity than F (P b 0.05).
b
There is no significant difference between N and F with respect to the antioxidant activity (P N 0.05).
c
F exhibited a significant higher ORAC value than N (P b 0.05).
102 R. Fu et al. / South African Journal of Botany 93 (2014) 98–104

Fig. 3. ORAC assay kinetic curves. Representative kinetic curves for Trolox standards from 46.315 to 247.012 μM and samples are shown. Data are expressed as the mean only (n = 3). N,
the ethanol-insoluble fraction of water extract of new S. sebiferum leaves; F, the ethanol-insoluble fraction of water extract of fallen S. sebiferum leaves.

Fe2 + could be chelated by samples. The IC50 values all samples were
represented in Table 1.
The IC50 values obtained from scavenging of DPPH, ABTS, nitrite and
hydroxyl radicals, and Fe2 +-chelating assays; ORAC and LPI values
acquired from ORAC and β-carotene bleaching assays were taken as
parameters for statistical analyses between the different samples
(Table 1). The antioxidant activities of N and F in different assays were
significantly weaker than the corresponding reference (P b 0.05) with
the exception of β-carotene bleaching assay, which N and F showed sig-
nificantly stronger inhibition effects than the reference BHT (P b 0.05).
In scavenging of DPPH, nitrite and OH radicals assays, there is no signif-
icant difference between N and F (P N 0.05). F had a significantly higher
ORAC value than N (P b 0.05). However, N exhibited significantly
stronger antioxidant activity than F in the β-carotene bleaching,
ABTS radical scavenging, and Fe2+-chelating assays (P b 0.05). N also
had a higher reducing power than F.
From the above results, we can see that whether ethanol-insoluble
fraction of water extract of new or fallen S. sebiferum leaves exhibited
a strong and potential antioxidant activity. The antioxidant activities
of N and F were distinct in some assays, suggesting that the chemical
compositions and contents of N and F were different. In addition,
based on two facts that S. sebiferum leaves have been applied for exter-
nal use for the treatment of skin diseases, and a high level of ROS is
proposed to contribute to the development of various skin disorders
(Bickers and Athar, 2006), our results indicated that the strong antioxi-
dant activity of the ethanol-insoluble fraction of water extract of
S. sebiferum leaves is responsible, at least in part, for its treatment effect
on skin diseases. Excessive ultraviolet radiation will induce various skin

Fig. 4. Determination of the antioxidant activities of N and F using (A) a reducing power assay; (B) a nitrite-scavenging assay; (C) an OH radical scavenging assay; and (D) a ferrous-ion
chelation assay. Data are expressed as the mean ± SD (n = 3). N, the ethanol-insoluble fraction of water extract of new S. sebiferum leaves; F, the ethanol-insoluble fraction of
water extract of fallen S. sebiferum leaves; Vc, ascorbic acid; BHT, 2,6-di-tert-butyl-4-methylphenol.
 R. Fu et al. / South African Journal of Botany 93 (2014) 98–104
diseases, meanwhile too much melanin will also be produced, in this
situation, usually a whitening agent is required. A compound with
antioxidant activity usually has a whitening effect, therefore we then
preliminary determined the whitening effects of ethanol-insoluble frac-
tion of water extract using TYR inhibition assay and related mechanism.
3.3. TYR inhibition effects of N and F and possible mechanism
TYR inhibitors are chemical agents capable of reducing enzymatic
reactions, such as food browning and melanization of human skin.
Therefore, these agents have a good commercial potential in cosmetic
industries. Fig. 5(A) shows the mushroom TYR inhibition effects of N
Fig. 5. (A) Tyrosinase inhibition effects of N and F. Arbutin was used as a positive control,
data are expressed as the mean ± SD (n = 3); (B) copper chelating activities of N and F,
data are expressed as the mean ± SD (n = 3); and (C) correlation established between
the percent inhibition of TYR and copper chelating activity. N, the ethanol-insoluble fraction
of water extract of new S. sebiferum leaves; F, the ethanol-insoluble fraction of water extract
of fallen S. sebiferum leaves.
103
and F; they reduced mushroom TYR activity in a dose-dependent
manner. At a concentration of 2 mg/ml, the inhibition effects of N and
F were 39.10 ± 3.88% and 34.37 ± 5.68%, respectively. While at the
same condition, the standard arbutin showed an inhibition effect as
17.20 ± 0.64%. N and F have significant potential inhibition effects
than arbutin (P b 0.05). As already known, copper is the active center
of TYR and N and F have shown some metal chelating activities, so we
examined the copper chelating activities of N and F, trying to under-
stand the inner mechanism of the TYR inhibition effects.
The copper chelating activities of N and F were presented in
Fig. 5(B), the doses (converted to mg) were the same as used in the
TYR inhibition assay. They exhibited copper chelating activities in a
dose-dependent manner. In order to understand the interrelationship
between TYR inhibition effect and copper chelating activity, both N
and F were used in an analysis of the Pearson correlation between
TYR inhibition and copper chelating activity. Fig. 5(C) illustrates the sig-
nificant positive linear correlations that were observed (P b 0.05). TYR
inhibition effects of ethanol-insoluble fraction of water extract of
S. sebiferum leaves are positively (r = 0.8605) correlated to its copper
chelating activity. As already known, copper is the active site of TYR,
thus this result indicated that the ethanol-insoluble fraction of water
extract of S. sebiferum leaves might exert their TYR inhibition effects
by chelating copper.
In this part, TYR inhibition effects of N and F were examined and the
possible mechanism was proposed. Our results demonstrated that
S. sebiferum leaves may be able to exhibit a whitening effect when
used for the treatment of skin diseases as already known.
4. Conclusion
In conclusion, the present study has clearly demonstrated that both
N and F, the ethanol-insoluble fraction of water extracts of new and
fallen leaves of S. sebiferum, have strong antioxidant and TYR inhibition
activities, even better than the positive control (BHT and arbutin). The
possible mechanism of TYR inhibition effect is its copper chelating
property. New leaves exhibit a greater potential activity than fallen
leaves which generally indicates that new leaves may be more suitable
for being employed, while fallen leaves can be also selected with respect
to the integrated utilization of natural resources. In a word, the ethanol-
insoluble fraction of water extract of S. sebiferum leaves has a great po-
tential for further study and in industrial applications due to its antioxi-
dant and TYR inhibition activities. We are currently attempting to
isolate and identify the antioxidant and TYR inhibition components in
these active extracts.
Acknowledgment
This work was supported by Special Key Projects of the Basic National
Science and Technology of China (No. 2008FY110400-2) and the
National “12th Five-Year” Science and Technology Support project
(No. 2011BAD22B08).
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