Food and Chemical Toxicology 49 (2011) 2689–2696

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Food and Chemical Toxicology

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

Evaluation to the antioxidant activity of total flavonoids extract from

persimmon (Diospyros kaki L.) leaves
Lijun Sun, Jianbao Zhang, Xiaoyun Lu, Liyu Zhang, Yali Zhang ⇑
Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, PR China

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

Article history: Persimmon leaves are commonly consumed as beverages, but are also used as a popular folk medicine in
Received 18 March 2011 China. The purpose of this work is to assess the antioxidant activity of an extract of total flavonoids from
Accepted 14 July 2011 persimmon leaves (TFPL). The effect of TFPL on total antioxidant activity, reducing power, 1,1-diphenyl-
Available online 23 July 2011
2-picrylhydrazyl (DPPH) radical scavenging, superoxide anion ( O 2 ) radical scavenging, hydroxyl (OH )


radical scavenging and metal chelating activities was examined. We found that TFPL possesses consider-
Keywords: able amounts of flavonoids (192 lg catechin equivalent/g of extract). The effect of this extract in total
Persimmon leaves
antioxidant activity, scavenging activity of superoxide anion and hydroxyl radical, reducing power and
Flavonoids
Antioxidant activity
iron chelating activity was significantly better than that of rutin. However, the effect of TFPL in free rad-
Radical-scavenging activity ical scavenging of DPPH was significantly not as good as than rutin. In addition, TFPL significantly
decreased the level of reactive oxygen species (ROS) and malondialdehyde (MDA), while increasing the
activity of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in MC3T3-
E1 cells in a dose-dependent manner. In conclusion, TFPL possess potent antioxidant and free radical
scavenging activities. These antioxidant activities could contribute, at least in part, to the traditionally
claimed therapeutic benefits of persimmon leaves.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Persimmon (Diospyros kaki L.) is a kind of plant native to China,

Oxidative stress is a result of imbalance between the antioxi-
dant defence system and the formation of reactive oxygen species
(ROS). It is believed to damage cell membranes and DNA, as well as
membrane lipid peroxidation with subsequent decreases in mem-
brane fluidity (Finkel and Holbrook, 2000; Melov et al., 2000). Oxi-
dative damage may cause cell injury, death and exacerbate the
development of several age-related chronic diseases including can-
cer, Alzheimer’s disease, Parkinson’s disease and heart disease
(Raouf et al., 2000). Therefore, antioxidants are considered to be
important nutraceuticals with many health benefits. Antioxidants
are widely used in the food industry as potential inhibitors of lipid
peroxidation (Scherer and Godoy, 2009). However, many synthetic
antioxidants used in foods, such as butylated hydroxyanisole and
butylated hydroxytoluene, may accumulate in the body, resulting
in liver damage and carcinogenesis (Valentao et al., 2002; Luo
and Fang, 2008). For this reason, more attention has been paid to
natural non-toxic antioxidants in an effort to protect the human
body from free radicals and retard the progress of many chronic
diseases.
⇑ Corresponding author. Tel.: +86 029 82668463.
E-mail address: yar.lee@mail.xjtu.edu.cn (Y. Zhang).
used traditionally for many medicinal purposes, including the
treatment of paralysis, frostbite, and burns, and to stop bleeding
(Matsuo and Ito, 1978). Flavonoid oligomers, tannins, phenols, or-
ganic acids, chlorophyll, vitamin C, and caffeine are found in per-
simmon leaves (Matsuo and Ito, 1978; Jo et al., 2003). The leaves
are commonly used for tea in Asia. Previous studies have shown
that persimmon leaves have beneficial effects on haemostasis, con-
stipation, hypertension, apoplexy and atherosclerosis (Kotani et al.,
2000; Matsumoto et al., 2002; Tanaka et al., 2003; Sakanaka et al.,
2005). In particular, flavonoid aglycones in persimmon leaves, such
as catechin, kaempferol, and quercetin, reportedly possess strong
antioxidant activities by acting as oxygen radical scavengers and
metal chelators (Morel et al., 1993; Birt et al., 2001). Chemically,
flavonoids and isoflavonoids are one-electron donors. They are
derivatives of conjugated ring structures and hydroxyl groups that
have the potential function as antioxidants in cell culture in vitro,
or in cell free systems. The previous studies suggest that the flavo-
noids present in persimmon leaves could contribute to the health
benefits attributed to persimmon (Bei et al., 2005, 2009; Lee
et al., 2006).
The balance between osteoclastic bone resorption and osteo-
blastic bone formation maintains bone mass at a homeostatic stea-
dy state. The imbalance that occurs when bone resorption is
greater than bone formation can lead to skeletal diseases, including

0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2011.07.042
2690 L. Sun et al. / Food and Chemical Toxicology 49 (2011) 2689–2696
osteoporosis. Oestrogen deficiency causes osteoporosis via in-
creased generation of ROS. Several reports have demonstrated that
osteoblast differentiation can be inhibited by oxidative stress or
induced by exogenous stimuli such as hydrogen peroxide or xan-
thine/xanthine oxidase (Mody et al., 2001; Bai et al., 2004). These
findings suggest that ROS may represent a critical target for the
treatment and/or prevention of osteoporosis. For this reason,
antioxidants may prove to be effective therapeutic candidates for
osteoporosis (Riggs et al., 2002). If a total flavonoid extract of per-
simmon leaves (TFPL) is able to prevent free radical damage in
osteoblast-like cells in vitro, it should be able to prevent cell death.
The prevention of bone loss by the inhibition of oxidative stress
and the prevention of osteoblast death in vitro could be considered
as groundwork for the therapeutic benefit of TFPL. The clinical
experiment is still needed to investigate the therapeutic effects
of TFPL administration to humans, especially those at highest risk
of osteoporosis, i.e., elderly women.
To our knowledge, few investigations have been made of the
antioxidant properties of persimmon leaves, although this medici-
nal plant is widely used by Chinese traditional healers. The aims of
this study were to determine the total flavonoid content of persim-
mon leaves and to evaluate the properties of TFPL using widely ac-
cepted antioxidative and free radical-scavenging model systems.
Rutin, a lipid-soluble analogue of flavonoids, was used as a refer-
ence antioxidant compound (Yang et al., 2008). In addition, the lev-
els of ROS and antioxidant enzymes such as superoxide-dismutase
(SOD) and glutathione (GSH-Px) were also determined in MC3T3-
E1 cells after treatment with TFPL. The results suggest that TFPL
could be useful as a potential natural antioxidant in the functional
food and pharmaceutical industries.
2. Materials and methods
2.1. Materials
Persimmon (D. kaki L. folium) leaves were harvested in Shaanxi Province, China
in October 2010, and identified according to the identification standard of the Phar-
macopeia of the People’s Republic of China. Persimmon (D. kaki L. folium) leaves
were dried in the shade for a week and powdered and passed through 60 mesh
sieves. The raw material was stored at 20 °C before extraction. The reagents 1,
1-diphenyl-2-picrylhydrazyl (DPPH), ferrozine, Dimethylsulphoxide (DMSO), 3-
(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) and 2’,7’-dichlo-
rofluorescin diacetate (DCFH-DA) were purchased from Sigma Chemicals (St Louis,
MO, USA). Assay kits for superoxide anion radical ( O  method. Hydroxyl radicals were generated in a solution of 2 mM EDTA–Fe
2 ), hydroxyl radical (OH ),
superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), pro-
tein, and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengi-
neering Institute (Nanjing, China). Dulbecco’s modified Eagle’s medium (DMEM),
foetal bovine serum (FBS) was purchased from Gibco BRL (Grand Island, NY, USA).
All other reagents were of analytical grade and made in China.
2.2. Preparation of extracts of persimmon leaves
Persimmon leaves (50 g) were cut into pieces approximately 2 cm in width and
dried. Leaves were soaked in a 70% ethanol solvent (1:10, w/v) for 2.5 h and then
placed in an ultrasonic bath and sonicated at 200 kHz at 55 °C for 45 min. Samples
were then filtered through a 0.45 lm microporous membrane (Shanghai Wanzi Shi-
ye Co. Ltd., Shanghai, China). The filtrate was collected, and the solid was extracted
two more times with the same volume of fresh solvent. Solutions were combined
and concentrated to dryness under reduced pressure in a rotary evaporator to yield
dried crude total extracts. The extract was added to distilled water and defatted
with petroleum ether and ethyl acetate.
2.3. Determination of total flavonoid content
The total flavonoid content of the extract was determined by the method de-
scribed in the Chinese Pharmacopoeia (Chinese Pharmacopoeia Committee, 2005).
The 500 ll extract was diluted appropriately and mixed with 1 mL NaNO2 (5%).
After standing for 6 min, 1 mL of 10% AlCl3 and 10 mL of NaOH (1 M) were added
to the mixture. The mixture was adjusted to 25 mL with 70% ethanol and allowed
to rest for 15 min. The absorbance (A) was measured at 510 nm, with 70% ethanol
as a blank control. Rutin was used as a reference standard and the total flavonoid
content was expressed as rutin equivalents (RE, lg/mg extract). All determinations
were performed in triplicate.
2.4. Antioxidant activity assays
2.4.1. Total antioxidant capacity (TAOC)
Suitable working standards (0.24, 1.0, 5.0, and 10 mg/mL) were prepared by dis-
solving the TFPL in distilled water. Aliquots (0.30 mL) were mixed with 3 mL of the
reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammo-
nium molybdate). The tubes were capped with aluminium foil and incubated at
95 °C for 90 min (Umamaheswari and Chatterjee, 2008). The tubes were cooled to
room temperature and absorbance was measured at 695 nm against a blank. Ascor-
bic acid was used as a standard. Total antioxidant capacity was expressed as equiv-
alents of ascorbic acid (Raghavan et al., 2003).
2.4.2. Radical scavenging activity
2.4.2.1. DPPH radical scavenging activity. The ability of the TFPL to scavenge the
DPPH free radical was assayed according to the method of Shimada et al. (1992).
Briefly, a 0.1 mM solution of DPPH in 100% MeOH was prepared. To 1 ml of this
solution was added 4 ml of sample solution in 40% MeOH at different concentra-
tions (1–160 lg/mL). The mixture was shaken vigorously and incubated for
15 min in the dark at room temperature until stable absorption values were ob-
tained. The reduction of the DPPH radical was measured by continuously monitor-
ing the decrease in absorption at 517 nm. In the control, 40% MeOH was substituted
for samples. Lower absorbances of the reaction mixture indicated higher free radical
scavenging activity. The DPPH radical scavenging activity was calculated by the fol-
lowing equation:
Scavenging effect ð%Þ ¼ ð1  Asample517 =Acontrol517 Þ  100
The EC50 value is the concentration of the sample required to scavenge 50% of
the DPPH free radical.
2.4.2.2. Superoxide anion radical scavenging activity. The measurement of superoxide
anion scavenging activity was based on the method described by Nishikimi et al.
(1972) with slight modifications. Superoxide radicals were generated in the PMS–
NADH system containing 3 mL Tris–HCl buffer (16 mM, pH 8.0), 338 lM NADH
(adenine dinucleotide), 72 lM NBT (nitroblue tetrazolium), and 30 lM PMS (phen-
azine methosulphate). Varying concentrations of samples ranging from 25 to
400 lg/mL were added to the PMS–NADH system for free radical scavenging. These
reaction mixtures were incubated at room temperature for 5 min before the absor-
bance was read at 560 nm against the blank. In the control, the sample was substi-
tuted with Tris–HCl buffer. Decreased absorbance of the reaction mixture indicated
increased superoxide anion scavenging activity. The ability of TFPL to scavenge
superoxide radical was calculated using the following equation:
Scavenging effect ð%Þ ¼ ð1  Asample560 =Acontrol560 Þ  100
EC50 value (mg/ml) is the concentration at which the scavenging activity is 50%.
2.4.2.3. Hydroxyl radical scavenging activity. The hydroxyl radical scavenging activity
of samples of TFPL was measured using a modified Smirnoff and Cumbes’ (1989)
(0.5 mL), 3% H2O2 (1 mL), and 360 lg/mL crocus in 4.5 mL sodium phosphate buffer
(150 mM, pH 7.4). Samples at concentrations ranging from 25 to 400 lg/mL were
incubated for 30 min at 37 °C and hydroxyl radicals were detected by monitoring
absorbance at 520 nm. In the control, the sample was substituted with distilled
water and sodium phosphate buffer replaced H2O2. The capability of hydroxyl rad-
ical scavenging was calculated using the following equation:
Scavenging effect ð%Þ ¼ ð1  Asample520 =Acontrol520 Þ  100
EC50 value (mg/ml) is the concentration at which the scavenging activity is 50%.
2.4.3. Reducing power
The reducing power of TFPL was determined using the method of Yen and Chen
(1995). Briefly, 0.13 mL of different concentrations of samples (25–400 lg/mL) sus-
pended in phosphate buffer (0.2 M, pH 6.6) were mixed with 0.125 mL of potassium
ferricyanide (1%, w/v) and incubated at 50 °C. At 20 min, 0.125 mL of trichloroacetic
acid (10%, w/v) was added to the mixture to terminate the reaction. The solution
was mixed with 1.5 ml of ferric chloride (0.1%, w/v), and the absorbance was mea-
sured at 700 nm. An increased absorbance of the reaction mixture indicated in-
creased reducing power.
2.4.4. Chelating activity on ferrous ions
The chelating activities of test compounds on Fe2+ were estimated based on the
decrease in the maximal absorbance of the iron (Fe2+)–ferrozine complex assayed
following previously reported methods (Dinis et al., 1994), with some modifica-
tions. Briefly, 1.0 ml of test compounds dissolved in ethanol were incubated with
0.5 ml of FeCl24H2O (1.0 mmol/L). The reaction was initiated by the addition of
1 ml of ferrozine (5.0 mmol/L), and the total reaction volume was adjusted to
4 ml with ethanol. After the mixture had reached equilibrium (10 min), the
 L. Sun et al. / Food and Chemical Toxicology 49 (2011) 2689–2696
absorbance at 562 nm was read. The negative control was prepared without the test
compound, and EDTA was used as the positive control. The chelating activity of the
test compound on Fe2+ was calculated as follows:
Chelating activity ð%Þ ¼ ðAControl562 =ASample562  1Þ  100
2.5. Antioxidant activities of TFPL in MC3T3-E1 cells
2.5.1. Cell culture and treatment
Murine osteoblast MC3T3-E1 cells were maintained in DMEM supplemented
with 10% heat-inactivated fatal bovine serum (FBS), 100 U/ml penicillin and
100 lg/ml streptomycin in a humidified 5% CO2 incubator at 37 °C. The medium
was changed every 2 days. For experimental treatments, TFPL was added to the cul-
tured cells. After 24 h of co-culturing, the cells were harvested, counted, and used
for further assays.
2.5.2. Cell viability
The cell survival rate was quantified using a colorimetric MTT assay to measure
mitochondrial activity in viable cells. This method is based on the conversion of
MTT to formazan crystals by mitochondrial enzymes. Briefly, cells seeded at a den-
sity of 5  104 cells/well in a 96-well plate were left to adhere overnight. MTT was
prepared at 2.5 mg/mL in PBS. Aliquots (20 ll) of the MTT stock solution were
pipetted into each well and the plate was incubated at 37 °C in a humidified 5%
CO2 incubator. After 4 h, the medium was removed and DMSO (200 ll) was added
to each well to dissolve the formazan. After 10 min, the optical density of each well
was measured at 570 nm by spectrophotometry.
2.5.3. Characterisation of oxidative status
Cellular ROS levels were measured by the dichlorofluorescein assay, as de-
scribed by Zuo and Clanton (2002). Accumulations of intracellular ROS can be
detected using DCFH-DA, which crosses cell membranes and is hydrolysed by intra-
cellular esterases to non-fluorescent DCFH. In the presence of ROS, DCFH is oxidised
to the highly fluorescent dichlorofluorescein (DCF), which is readily detected in a
fluorescent microplate reader. The cells (105 cells/well) were incubated with 100–
500 lg/mL of a test sample for 24 h. Subsequently, 200 mM ethanol was added to
the cells, which were then incubated with 25 lM DCFH-DA for an additional
30 min. The fluorescence intensity of the cells was measured on a fluorescence
microplate reader (BioTek Instruments, Winooski, VT) with an excitation wave-
length of 488 nm and an emission wavelength of 525 nm.
Activities of SOD, CAT, MDA and GSH-Px were determined spectrophotometri-
cally using commercially available assay kits following the manufacturer’s
protocols.
The level of lipid peroxidation was indicated by the amount of MDA in the cells
as assayed using the thiobarbituric acid reaction (TBARS). Values of MDA were ex-
pressed as nmol/ml of cells. Protein concentration was measured using the BCA pro-
tein assay kit (Pierce) with BSA as standard. The activity of CAT was determined by
measuring the decrease in absorbance at 405 nm due to the degradation of H2O2.
The results of this enzymatic assay were reported in units of CAT activity per mil-
ligram of protein (U/mg protein), where 1 U of CAT is defined as the amount of en-
zyme that decomposes 1 mmol of H2O2 per second. The activity of SOD was
determined spectrophotometrically at 550 nm, indicating the inhibition of the oxi-
dation of oxymine by the xanthine/xanthine oxidase system. The results of this
enzymatic assay were reported in units (U) of SOD activity per ml (U/ml) of cells.
The activity of GSH-Px was assayed by quantifying the rate of oxidation of glutathi-
one to oxidised glutathione by H2O2. One unit of GSH-Px was defined at 412 nm as
the amount that reduced the level of GSH by 1 lmol/L in 1 min/mg protein (U/mg
protein).
2.6. Statistical analysis
The data in triplicate were subjected to analysis of variance (ANOVA) and ex-
pressed as mean ± standard deviation (n = 6). Analysis of variance and the differ-
ence among samples were determined by Duncan’s multiple-range test using the
Statistical Analysis System (SAS release 6.12) program. A level of p < 0.05 was used
as the criterion for statistical significance.
3. Results and discussion
3.1. Total flavonoid content
The total flavonoid content of TFPL was expressed as rutin
equivalents in mg/g of extracts. The TFPL extracts contained
192 ± 9.6 mg/g total flavonoids. Because flavonoids are responsible
for antioxidant activity, the high amount of total flavonoids in the
extract suggests that the extract possesses an antioxidant activity
in vitro (Lotito and Frei, 2004). Rutin is a flavonol glycoside plant
2691
metabolite with anti-oxidative, anti-inflammatory and anti-carcin-
ogenic effects. Rutin can also reduce the fragility of blood vessels
found in haemorrhagic disease and hypertension in humans
(Oomah et al., 1996). In this study, rutin was used as positive
control.
Flavonoids are very important constituents of plants because of
the scavenging ability conferred by their hydroxyl groups. The
flavonoids may contribute directly to anti-oxidative action. It is
known that polyphenolic compounds have inhibitory effects on
mutagenesis and carcinogenesis in humans when up to 1 g daily
is consumed from a diet rich in fruits and vegetables (Tanaka
et al., 1998). Flavonoid compounds from plants are known to be
good natural antioxidants. However, the activity of synthetic
antioxidants was often observed to be higher than that of natural
antioxidants (Ningappa et al., 2007). Flavonoid compounds at cer-
tain concentrations markedly slowed down the rate of conjugated
diene formation. Interest in phenolics is increasing in the food
industry because of their ability to retard oxidative degradation
of lipids, thereby improving the quality and nutritional value of
foods (Aneta et al., 2007).
3.2. Antioxidant activities
It has long been recognised that naturally occurring substances
in higher plants have antioxidant activity. Among those sub-
stances, the flavonoids that are widely distributed in plants have
the ability to scavenge free radicals, superoxide and hydroxyl rad-
icals by single-electron transfer (Havsteen, 2002; Le et al., 2007;
Leman’ska et al., 2001). An antioxidant exerts its antioxidant activ-
ity through various mechanisms, including chelating ferrous iron,
degrading peroxide, and scavenging free radicals. In our experi-
ments, the antioxidant and radical scavenging activity of TFPL
has been investigated by assessing their effect on TOAC, DPPH rad-
ical scavenging activity, superoxide anion removal, hydroxyl radi-
cal trapping potential, reducing power and metal chelating
ability. We speculate that the antioxidant profiles of TFPL and rutin
are similar, but may be quantitatively different, perhaps associated
with their similar but different chemical structures.
3.2.1. Total antioxidant activities
Total antioxidant activities reflect the capacity of a non-
enzymatic, antioxidant defence system. In the phosphomolybde-
num method, molybdenum VI (Mo6+) is reduced to form a green
phosphate/Mo5+ complex at acidic pHs. High absorbance values
indicate that the sample possesses significant antioxidant activity.
As shown in Fig. 1, total antioxidant activities of TFPL samples are
superior to rutin and the effects are concentration-dependent. The
TAOC of TFPL is close to that of rutin at a dose of 400 lg/mL.
3.2.2. Radical scavenging activity
It is well known that antioxidants can seize the free radical chain
of oxidation and form stable free radicals, which do not initiate or
propagate further oxidation. The DPPH radical was extensively
used to evaluate the free-radical scavenging capacity of antioxi-
dants (Halliwell and Gutteridge, 1999). Scavenging of the DPPH
radical is related to the inhibition of lipid peroxidation. DPPH rad-
ical involves a hydrogen atom transfer process (Kaviarasan et al.,
2007). Our results found that both TFPL and rutin were effective
at reducing the stable radical DPPH to the yellow-coloured diph-
enylpicrylhydrazine, indicating that these extracts are active in
DPPH radical scavenging (Fig. 2(A)). TFPL had significant scaveng-
ing effects with increasing concentrations in the range of 12.5–
500 lg/ml. However, the scavenging effect of TFPL was significantly
lower than that of rutin. At 200 lg/ml, TFPL and rutin exhibited
68.73% and 89.13% inhibition, respectively, and the EC50 values
were 96.36 and 41.52 lg/ml for TFPL and rutin, respectively (Table
2692 L. Sun et al. / Food and Chemical Toxicology 49 (2011) 2689–2696
Fig. 1. Total antioxidant activities of total flavonoids from persimmon leaves.
⁄
p < 0.05 and ⁄⁄p < 0.01 compared with rutin in the same concentration group.
Fig. 2. Radical-scavenging activity of total flavonoids from persimmon leaves
(TFPL). Radical-scavenging activities were assessed by measuring the DPPH
scavenging activities (A), superoxide anion scavenging activities (B), and hydroxyl
scavenging activities (C). ⁄p < 0.05 and ⁄⁄p < 0.01 compared with rutin in the same
concentration group.
1). The different concentrations of TFPL (12.5, 25, 50, 100, 200, 250
and 500 lg/ml) showed antioxidant activities in a dose dependent
manner (14.48%, 21.15%, 32.06%, 51.28% 68.73%, 75.62% and
91.59% inhibition, respectively) in the DPPH radical scavenging
Table 1
Effect of total flavonoids from persimmon leaves on different radical scavenging
activities.
Samples EC50 (lg/mL)a
DPPH radical Superoxide radical Hydroxyl radical
scavenging activity scavenging activity scavenging activity
TFPL 96.36 ± 2.63 41.58 ± 0.21 70.64 ± 3.22
Rutin 41.52 ± 1.96 130.58 ± 3.58 111.23 ± 4.37
a
EC50 is a measure of radical scavenging activity being the concentration
required to inhibit 50% free radical activity.
assay (Fig. 2(A)). A higher DPPH radical scavenging activity is asso-
ciated with a lower EC50 value. In this assay, the good antioxidant
activity of TFPL on the DPPH radical may be attributed to a direct
role in trapping free radicals by donating a hydrogen atom.
Superoxide anions play important roles in the formation of ROS
such as hydrogen peroxide, hydroxyl radical and singlet oxygen,
which induce oxidative damage in lipids, proteins and DNA (Pietta,
2000; Wickens, 2001). It was therefore proposed to measure the
comparative interceptive ability of the antioxidant extracts to scav-
enge the superoxide radical. In our study, superoxide-scavenging
activities of extracts were measured by auto-oxidation of hydroxyl-
amine in the presence of NBT (nitroblue tetrazolium). The reduction
of NBT in the presence of antioxidants was measured. A decrease in
absorbance at 560 nm indicates the consumption of superoxide an-
ion in the reaction mixture. As the data in Fig. 2(B) show, different
concentrations of TFPL (6.25–150 lg/ml) exhibited superoxide-
scavenging activity (13.21%, 32.14%, 41.70%, 54.42%, 76.87% and
87.56% inhibition, respectively). Aliquots of TFPL or rutin (100
lg/ml) exhibited 76.87% and 48.12% inhibition, respectively
(Fig. 2(B)). The EC50 value of TFPL on superoxide radical scavenging
activity was found to be 41.35 lg/ml, whereas the EC50 value of ru-
tin was found to be 130.58 lg/ml (Table 1). All of the extracts scav-
enged superoxide radicals in a dose dependent manner. Therefore,
when compared to rutin, the superoxide scavenging activity of the
extract was high. This could be due to the presence of reactive bio-
active constituents and the mixture of other nutrients in the ex-
tract. Results were statistically significant (p < 0.05). Based on our
results, it appears that TFPL scavenges superoxide radicals by com-
bining with superoxide radical ions to form stable radicals, thus ter-
minating the radical chain reaction (Wang et al., 2009).
Hydroxyl radicals are extremely reactive free radicals formed in
biological systems and have been implicated as a highly damaging
species in free radical pathology, capable of damaging almost every
molecule found in living cells (Gulcin, 2006). Hydroxyl radicals are
very strongly reactive oxygen species, and there is no specific en-
zyme to defend against them in humans (Liu et al., 2005). There-
fore, it is important to discover chemicals with good scavenging
capacity for these reactive oxygen species. The hydroxyl radical
scavenging capacity of an extract is directly related to its antioxi-
dant activity (Babu et al., 2001). The scavenging effect of TFPL
against hydroxyl radicals was investigated using the Fenton reac-
tion. The % inhibition of TFPL (25–125 lg/ml) on hydroxyl radical
scavenging was found to be 30.62%, 38.62%, 52.40%, 57.08% and
77.85%, respectively (Fig. 2(C)). All results showed antioxidant
activity in a dose dependent manner. The EC50 values of TFPL and
rutin were found to be 70.64 and 111.23 lg/ml (Table 1). The abil-
ity of the TFPL extracts to quench hydroxyl radicals seems to be di-
rectly related to the prevention of propagation of lipid
peroxidation; because TFPL seems to be a good scavenger of active
oxygen species, it will thus reduce the rate of the chain reaction.
Yen and Hsieh (1995) reported that xylose and lysine Maillard
reaction products had scavenging activity on hydroxyl radicals in
a dose response manner. This result may be attributed to the com-
bined effects of reducing power, the donation of hydrogen atoms
 L. Sun et al. / Food and Chemical Toxicology 49 (2011) 2689–2696
and the scavenging of active oxygen. Hagerman et al. (1998) have
also explained that high molecular weight and the proximity of
many aromatic rings and hydroxyl groups are more important
for the free radical scavenging activity of phenolics than are spe-
cific functional groups.
3.2.3. Reducing power
Reducing power is one mechanism of action of antioxidants and
may serve as a significant indicator of potential antioxidant activity
(Jayaprakasha et al., 2000). Several studies have indicated that the
antioxidant effect is related to the development of reductones (Yen
and Duh, 1993). Therefore, in this study, reducing activity was
determined based on the ability of extracts or rutin to reduce a
Fe3+/ferricyanide complex to form an Fe3+ ferrous complex. The
amount of Fe2+ was monitored by measuring the formation of
Perl’s Prussian blue at 700 nm (Yang et al., 2010). The dose–
response curve for the reducing activity of TFPL and rutin is shown
in Fig. 3. In the present study, TFPL showed a significantly higher
reducing power than rutin. The data suggest that TFPL has a good
ability to donate electrons to reactive free radicals, converting
them into more stable products and terminating the free radical
chain reaction.
3.2.4. Chelating activity on ferrous ions
In addition to initiating lipid peroxidation, metal ions possess-
ing catalytic ability have been correlated with arthritis and cancer
incidence (Halliwell et al., 1995). Additionally, ferrous ions are
thought to be effective pro-oxidants (Yamaguchi et al., 1998). Be-
cause EDTA is a strong chelator of metal, it is used as the stan-
dard metal chelator in this study. Ferrous ion chelating
activities of TFPL, rutin and EDTA are shown in Fig. 4. Ferrozine
can quantitatively form complexes with Fe2+. In the presence of
chelating agents, complex formation is disrupted, resulting in a
decrease in the red colour of the complex. Measurement of colour
reduction therefore allows an estimation of the metal chelating
activity. In this assay, TFPL and both standard compounds inter-
fered with the formation of ferrous and ferrozine complexes, sug-
gest that they have chelating activity and are able to capture
ferrous ion before ferrozine. The absorbance of Fe2+–ferrozine
complex was decreased by TFPL in a dose dependent fashion from
2.5 to 50 lg/mL. Although the iron chelating ability of TFPL was
moderate, it was significantly stronger than rutin. In addition,
the percentages of the metal chelating capacities of 50 lg/mL of
TFPL and 6.25 lg/mL of EDTA were 77.38% and 87.34%, respec-
tively. Chelating agents, which form r-bonds with metals, are
effective as secondary antioxidants because they reduce redox po-
tential, thereby stabilising the oxidised form of the metal ion.
Fig. 3. Reducing power of total flavonoid extract from persimmon leaves (TFPL).
⁄
p < 0.05 and ⁄⁄p < 0.01 compared with rutin in the same concentration group.
2693
Fig. 4. Chelating activity on ferrous ions of total flavonoids from persimmon leaves
(TFPL). EDTA was used as positive control. ⁄p < 0.05 and ⁄⁄p < 0.01 compared with
EDTA.
Extracts or compounds with chelating activity are believed to in-
hibit lipid peroxidation by stabilising transition metals. Accord-
ingly, it is suggested that the low-to-moderate chelating effect
of TFPL would be at least partially beneficial in protecting against
oxidative damage.
Rutin (quercetin-3-rutinoside) is one of the major flavonoids
found in a variety of plants. Rutin is receiving increasing attention
due to its various health beneficial biological activities, including
antioxidative, anti-inflammatory, and anti-hypertensive effects.
Yet practical limitations do exist regarding the incorporation and
effectiveness of flavonoids as antioxidants in many food systems,
possibly due to their poor water solubility (0.125 g/L) (Khalifa
et al., 1983). Total flavonoids from persimmon leaves possess a
good water-soluble. Therefore, TFPL possess higher antioxidant
activities that rutin in water as the solvent test system.
As potent antioxidants, flavonoids reportedly possess strong
free radical scavenging activities based on their ability to act as
hydrogen or electron donors. In addition, some flavonoids may also
react directly with radicals to form less reactive products, or else
chelate transition metals which could otherwise act as pro-
coxidants in the system (Le et al., 2007). Overall, it is the structural
elements making up the flavonoid or flavonoid esters which dictate
its antioxidative behaviour, including features like (i) degree and
position of hydroxylation, (ii) type and position of additional sub-
stituents, (iii) presence of double bonds or conjugation and (iv) gly-
cosylation (Leopoldini et al., 2011). Five flavonoid compounds,
kaempferol 3-O-h-D-galactopyranoside (TR), kaempferol 3-O-h-D-
glucopyranoside (AS), isorhamnetin 3-O-h-D-glucopyranoside (IS),
quercetin 3-O-h-D-galactopyranoside (HY) and quercetin 3-O-h-D-
glucopyranosyl-(6!1)-a-L-rhamnopyranoside (RU), isolated from
the leaves of persimmon have been reported (Saito et al., 1994;
Meng and Xu, 1998). Flavonols and flavones containing a catechol
group in ring B are found to be highly active, with flavonols more
potent than the corresponding flavones because of the presence of
the 3-hydroxyl group. On the contrary, rutin is a large 3-OH-substi-
tuted aromatic glycoside groups, so that the whole molecular
structure rearrangement, molecular energy is reduced, 30 -OH and
40 -OH activity has weakened more, and its antioxidant activity is
also greatly reduced. The presence of C-3 glycoside groups in ring
C of rutin diminishes the antioxidant activity (Leopoldini et al.,
2011). However, Kayoko et al.’s study shows the galloylated flavo-
nol glycoside mixture had an antioxidant activity approximately
twofold stronger than that of the nongalloylated flavonol glyco-
sides (Kawakami et al., 2011). This suggests that further studies
of these flavonoids on its pharmaceutical functions and three-
dimensional structure may be of help in the development of its
clinical application.
2694 L. Sun et al. / Food and Chemical Toxicology 49 (2011) 2689–2696

3.3. Effect of TFPL on MC3T3-E1 cells

3.3.1. Cell viability

We used the MTT assay to study the proliferative activity of
TFPL. MTT is reduced by mitochondrial dehydrogenase to produce
formazan, an insoluble purple compound. We measured cell prolif-
erative activity in terms of the intensity of the purple colour. As
Fig. 5 shows, increasing doses of TFPL are not toxic and do not in-
hibit the growth of MC3T3-E1 cells. Instead, TFPL promoted the
growth rate of the cells. At TFPL doses of 5, 10, and 20 lg/mL, cell
viabilities were 98.17%, 99.62%, and 100.32%, respectively, after
treatment for 24 h. The cell viability of MC3T3-E1 cells treated
with TFPL was significantly superior to the control group. The re-
sults indicated that TFPL was not harmful to MC3T3-E1 cells at
the tested concentration ranges and markedly facilitated the
growth rate of MC3T3-E1 cells at concentrations between 5 and
20 lg/ml. TFPL may act by facilitating glucose uptake and subse-
quently increasing pyruvate concentrations, leading to increased
cellular energy and proliferation. These findings in cells in vitro
may prove relevant to protecting against the loss of bone mass
and the development of osteoporosis in human subjects.

3.3.2. The levels of ROS and antioxidant enzymes

Reactive oxygen species are believed to play an important role
in cell death. Drugs that exhibit properties of an antioxidant by
regulating the levels of enzymes with antioxidant activity, such
as SOD, CAT and GSH, may serve as potential candidates for the
treatment of osteoporosis. To elucidate the effect of TFPL on
MC3T3-E1 cells, measures of oxidative stress, the ROS level, the
MDA level, the GSH level, and the activity of SOD and CAT were
measured. As shown in Fig. 6, TFPL had significant effects on ROS
levels, MDA content, SOD, CAT and GSH-Px activities in MC3T3-
E1 cells (p < 0.05). The SOD activities of cells treated with 10 and
20 lg/mL TFPL were significantly higher than the control
(p < 0.01). The activity of SOD was significantly higher in cells with
5, 10, and 20 lg/mL TFPL compared to controls (p < 0.05).
MDA is an end product of lipid peroxidation, and its content is
used to estimate the degree of oxidative damage in cells. Therefore,
antioxidant enzyme activities and MDA content are key indicators
of antioxidant capacity. It was reported that SOD converts superox-
ide radicals to hydrogen peroxide, which is subsequently con-
verted to water by CAT and GSH-Px (Wang et al., 2007). Thus,
SOD protects cells from the toxicity of superoxide radicals. The en-
zyme GSH plays an important role in regulating intracellular redox
status. An increase in GSH level protects cells against cell death
either by removing free radicals or by conjugating them with tox-
icants (Kong et al., 2009). Elevated GSH levels and SOD activities

Fig. 5. Effect of rutin and TFPL on cell viability in MC3T3-E1 cells. MC3T3-E1 cells Fig. 6. Effect of TFPL on the ROS levels (A), MDA levels (B), SOD activities (C), CAT
were incubated at 37 °C in the absence or presence of rutin and TFPL. Cell viability activities (D), and GSH levels (E) in MC3T3-E1 cells. MC3T3-E1 cells were incubated
was measured by MTT assay. ⁄p < 0.05 and ⁄⁄p < 0.01 compared with the control at 37 °C in the absence or presence of TFPL. ⁄p < 0.05 and ⁄⁄p < 0.01 compared with
group. the control group.
 L. Sun et al. / Food and Chemical Toxicology 49 (2011) 2689–2696
provide a repair mechanism for oxidised membrane components.
The results of this experiment indicated that SOD, CAT, and GSH-
Px activities in MC3T3-E1 cells treated with 1.25–20 lg/mL TFPL
were higher than those of controls. The MDA content of cells was
decreased by TFPL additions at the same concentrations. Thus, TFPL
in the range of 1.25–20 lg/mL could improve the antioxidant
capacity of MC3T3-E1 cells through increasing the activity of en-
zymes with antioxidant activity.
4. Conclusions
In conclusion, the present study has demonstrated that TFPL
possesses considerable amounts of flavonoids (192 lg catechin
equivalent/g of extract). Rutin, a lipid-soluble analogue of flavo-
noids, was used as a reference antioxidant compound. The data ob-
tained clearly indicate that the extract possesses potent total
antioxidant activity, scavenging superoxide anion and hydroxyl
radicals, reducing power and iron chelating activity, at levels supe-
rior to rutin. The TFPL was able to scavenge 1,1-diphenyl-2-pic-
rylhydrazyl, although less effectively than rutin. This study found
that the treatment of MC3T3-E1 cells with TFPL caused a marked
reduction in ROS and MDA production and a rise in GSH level,
SOD and CAT activities, suggesting that the protective effect of
TFPL may be related to its antioxidant ability. These antioxidant
activities could have contributed, at least partly, to the therapeutic
benefits of the certain traditional claims for persimmon leaves. In
view of the potential use of persimmon leaves in the functional
food industry, its therapeutic benefits and bioactive compounds
warrant further investigation.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was financed by the National Natural Science Foun-
dation of China (30801059, 10972177), by the Scientific and Tech-
nical Foundation of Xi’an City (2009, SF09031(3)), by a Project of
Basic Research of Xi’an Jiaotong University (08143011), and by a
Project of the National Undergraduate Student Innovative Experi-
ment of China. This work was partly supported by Institute of
Mitochondrial Biology and Medical, Xi’an Jiaotong University, Xi’an
China.
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