Structural features and antioxidant activity of tannin from

persimmon pulp
Hai-Feng Gu
a
, Chun-Mei Li
a,
*
, Yu-juan Xu
a,b
, Wan-feng Hu
a
, Mei-hong Chen
a
,
Qiong-hong Wan
a
a
College of Food Science and Technology, Hua Zhong Agricultural University, Wuhan City, Hubei Province 430070, China
b
Sericulture and Farm Produce Processing Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou City,
Guangdong Province 510610, China
Received 24 August 2007; accepted 25 November 2007
Abstract
Phenolic compounds from persimmon pulp were extracted with methanol acidied with 1% HCl, and then puried on AB-8 macro-
porous resin. The tannic extracts obtained were fractionated by polysulfone ultraltration membrane with molecular weight cuto of
10,000 Da into two fractions: low molecular weight tannin (LMWT) and high molecular weight tannin (HMWT). HPLCMS analysis
showed that gallic acid was one of the main components of LMWT fraction. The molecular weight distribution of HMWT was deter-
mined to be in the range of 1.16 10
4
Da to 1.54 10
4
Da, with the molecular weight of 1.28 10
4
Da in M
n
and 1.39 10
4
Da in M
w
by GPC method. HPLCMS showed that the thiolysis degradation products of HMWT consist of (epi) gallocatechin, epigallocatechin-3-
O-gallate, epicatechin-3-O-gallate and an unknown monomer with the ratio of 1:7:3:1 by estimation of the peak area on HPLC. The
antioxidant properties of persimmon tannins were evaluated using the hydroxyl radical scavenging activities by 2-deoxyribose oxidation
system and salicylic acid system, superoxide anion scavenging activity, and linoleic acid lipid peroxidation inhibition activity, respec-
tively. HMWT exhibited excellent antioxidant activities in all tested systems in a dose-dependent manner. The antioxidant activity of
HMWT was signicantly stronger than that of LMWT and grape seeds proanthocyanidins (GSP), suggesting that high molecular weight
condensed tannins are the major antioxidant composition in persimmon pulp.
2007 Elsevier Ltd. All rights reserved.
Keywords: Persimmon (Diospyros kaki L.) pulp; Condensed tannin; Structural features; Antioxidant activity
1. Introduction
Persimmon (Diospyros kaki L.), which belongs to the
Ebenaceae family, is widespread in China, Japan and
Korea. In China, its fruits and leaves were traditionally
used for many medicinal purposes such as coughs, hyper-
tension, dyspnoea, paralysis, frostbite, burns and bleeding
(Mowat, 1990). Persimmon fruits were also reported to
exercise hypercholesterolemia, antioxidant and free radical
scavenging eects (Matsuo & Ito, 1978; Shela, Elzbieta,
Gustaw, Marina, & Simon, 1998). Persimmon fruit con-
tains abundant polyphenols, including condensed tannin
and other phenolic compounds, which are related to the
various physiological functions, including detoxication
eect on snake venom as well as toxic substances produced
by microorganisms (Okonogi, & Hattori, 1978; Okonogi,
Hattori, Ogiso, & Mitsui, 1979) the inhibitory eects on
human lymphoid leukemia cells (Achiwa, Hibasami,
Katsuzaki, Imai, & Komiya, 1997; Hibasami, Achiwa,
Fujikawa, & Komiya, 1996); the inhibitory eects on the
mutagenicity of C-nitro and C-nitroso compounds (Achi-
wa, Hibasami, Katsuzaki, Kada, & Komiya, 1996); and
the inhibition of the incidence of stroke and the extension
0963-9969/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2007.11.011
*
Corresponding author. Tel.: +86 13296599657.
E-mail addresses: lichmyl@126.com, lichmyl@mail.hzau.edu.cn (C.-M.
Li).
www.elsevier.com/locate/foodres
Available online at www.sciencedirect.com
Food Research International 41 (2008) 208217
of the life span of stroke-prone spontaneously hypertensive
rats (Ushida et al., 1995). However, due to the large molec-
ular structure and high absorbability, the separation and
determination of persimmon tannin is dicult and many
of the above research used crude persimmon extract or
persimmon peel and pulp as test materials. The crude plant
extract is a very complex mixture containing sometimes
hundreds of dierent compounds such as p-coumaric, gal-
lic acid, catechin, avonoids and condensed tannin existing
in persimmon (Mallavadhani, Panda, & Rao, 1998), but
not all the phenolic compounds from persimmon are of
physiological concern. One objective of this study is to
investigate whether high molecular weight condensed
tannin is the main antioxidant composition in persimmon
pulp.
Furthermore, it is meaningful to study the structural
characteristic of persimmon tannin to understand its antiox-
idant mechanism, but up to now, very few indication was
given about its structural characteristics. Only Matsuo and
Ito (1978) identied tannin in a Japanese persimmon and
found that the tannin consisted of catechin, catechin-gallate,
gallocatechin, gallocatechin-gallate and an unknown termi-
nal residue, and belonged to the proanthocyanidin B group
with carboncarbon interavan linkage between the C-4 of
one unit and the C-6 (or the C-8) of another unit (Itoo,
1986), and had the molecular weight of 1.38 10
4
Da in M
w
.
The structural characteristic of tannin may vary to a sig-
nicant extent due to the dierent breeds and producing
areas of persimmon. To some extent, thiolysis degradation
combined with HPLCMS could be the most useful tool
for characterizing the structure of tannin (Tanaka, Takah-
ashi, Kouno, & Nonaka, 1994). In the present work, mac-
roporous resin and ultraltration combined with GPC and
HPLCMSMS were used to separate, purify and charac-
terize tannin from persimmon pulp, and the antioxidant
activities were also evaluated.
2. Materials and methods
2.1. Chemicals
Methanol and acetonitrile were of HPLC grade (Fishers,
USA), polyvinyl alcohol standard, catechin, gallic acid, lin-
oleic acid, and 2-deoxyribose were purchased from Sigma
(USA), all solvents and reagents used in analysis were of
analytical grade from sinopharm chemical reagent factory
(Shanghai, China).
2.2. Plant material and sample preparation
Mature and fully colored fruits of the astringent persim-
mon were harvested in late October at the orchard in
Shanxi (China). After harvest, fruits were boiled in ve
volumes of distilled water at 100 C for about 10 min to
inactivate polyphenol oxidase. The persimmons were
peeled and the pulp was stored deep frozen at 20 C for
further use.
The pulp were cut into small pieces with a stainless knife
and 50 g of the pulp were extracted with 600 ml methanol
acidied with 1% HCl under reux conditions at 90 C
for 30 min. After ltration, the methanol solution was col-
lected. The procedure was repeated two more times and the
solution was combined. The content of total polyphenols in
the combined solution were measured by FolinDenis
method (Gahler, Otto, & Bohm, 2003). Briey, 1 ml of
extracts and 3 ml of FolinDenis reagent were mixed. After
5 min, 3 ml of 10% NaCO
3
were added and the mixture was
left at room temperature for 60 min. The absorbance was
measured with a spectrophotometer (Shima-dzu UV-
2200) at 760 nm. The determination was performed three
times. The amount of phenolics (expressed as mg gallic
acid/g fresh weight) was calculated from a standard curve
(20100 mg gallic acid/ml) prepared at the same time.
The content of condensed tannins in the combined solution
were vanillinHCl assay (Chavan, Shahidi, & Naczk,
2001). Briey, to 0.21 ml of the extract, 5 ml of 0.5% van-
illin reagent were added; a 5 ml of 4% concentrated HCl in
methanol was used as a blank. The absorbances of samples
and blank were read at 500 nm after standing for 20 min at
room temperature. Triplicate analyses were conducted and
the mean values were obtained. The amount of condensed
tannin (expressed as mg catechin/g fresh weight) was calcu-
lated from a standard curve (1050 mg catechin/ml) pre-
pared at the same time.
2.3. Separation and purication
The above methanol extracts were evaporated under
vacuum to remove the solvent. The concentrated extract
solution was applied onto a glass column (20 400 mm,
i.d.), packed with AB-8 macroporous resin (chemical plant
of Nankai University, Tianjin, China), washed rstly with
deionized water at a ow rate of 3 ml/min to remove sugar
and other soluble impurity. When practically no more col-
ored compounds and sugar were eluted and detected in the
outow, anhydrous methanol was used to elute the target
tannins at a ow rate of 2 ml/min. After eluting, solvent
was removed using a rotary evaporator under vacuum,
and the residue was lyophilized. The contents of total poly-
phenols and condensed tannins in the tannin extract were
measured by FolinDenis method and vanillinHCl assay,
respectively.
Three grams of the above tannin extract was diluted
with 3000 ml deionized water and then added onto the
layer of polysulfone hollow ber ultrane membrane with
molecular weight cuto of 10,000 Da (Tianjin, Tianfang,
China) to remove small molecules. With the operation
pressure of 0.05 MPa and ltration ux of 10 L/h, the
extract was separated into two parts; low molecular weight
tannin (LMWT) in the ltrate and high molecular weight
tannin (HMWT) stayed in the retentate. The obtained frac-
tions were evaporated and lyophilized for GPC, HPLC,
HPLCESIMS analysis and further antioxidant activity
evaluation. Total polyphenols and condensed tannin
H.-F. Gu et al. / Food Research International 41 (2008) 208217 209
contents of the two fractions were also detected as
previously.
2.4. HPLC analysis of dierent fractions of tannic extracts
from persimmon pulp and HPLCESIMS for low
molecular fraction
The separation of low molecules fraction was performed
using a HPLC series 1100 (Hewlett Packard, Waldbronn,
Germany) equipped with Chem.-Station software, a degas-
ser G1322A, a binary gradient pump G1312A, a thermo
autosampler G1329/1330A, and a diode array detector
G1315A. The column used was a 5 lm Hypersil ODS2
C
18
(4.60 mm 200 mm, i.d. Dalian Elite, China). The
mobile phase consisted of 2% acetic acid in water, eluent
A and acetonitrile, eluent B, at a ow rate of 0.8 ml/min.
The gradient program was as follows: 010 min, 010%B,
1015 min, 20%B, and 1530 min, 55%B. The injection vol-
ume for samples was 10 ll, monitoring was performed at
280 nm with the column oven set at 30 C, all samples were
ltered through a 0.45 lm syringe lter before analysis.
HPLCESIMS analysis were performed on a Agilent
1100 LCMS spectrometer (USA), the chromatographic
conditions were the same as described above except that
column: ZORBAX SC- C
18
(2.1 mm 150 mm, 5 lm, Agi-
lent, USA) and ow rate: 0.2 ml/min. A source tempera-
ture of 500 C, negative ion mode was used with a
sprayer needle voltage of 3.5 kV. The capillary temperature
was 350 C. The full scan mass spectra of the tannins from
m/z 100 to 700 were measured using 500 ms for collection
time and three micro scans were summed.
2.5. Gel permeation chromatography (GPC) and molecular
weight
GPC analysis of HMWT were carried out on Aglient
HPLC series 1100 at room temperature on an Aglient pl
aquagel OH 30 column (7.5 mm 300 mm, i.d., Aglient,
USA), particle size 8 lm. One milligram of HMWT was
dissolved in 1 ml of deionized water, and 50 ll was injected
to the column. Water was used as eluent at a ow rate of
1 ml/min with the column oven set at 25 C. The eluent
was monitored at 280 nm. Polyvinyl alcohol standard with
molecular weight of 106, 194, 620, 1470, 4120, 11,840,
26,000, respectively were used as standard markers for
the molecular weight. M
w
and M
n
express weight average
molecular weight and number average molecular weight,
respectively (Matsuo & Ito, 1978).
2.6. Thiolysis degradation
One milligram of HMWT was dissolved in 1 ml of meth-
anol, and 1 ml of thiolysis reagent (5% solution of benzyl
mercaptan in methanol containing 0.2 M HCl) was added
(Jean, Veronique, Franck, & Michel, 1996; Lingamallu,
Hiroshi, Hiroshi, Mayumi, & Mitsuru, 2004). After sealing,
the mixture was shaken and heated at 60 C for 2 h. The
products were then analyzed by HPLC under the following
conditions: solvent A, 0.5% acetic acid; solvent B,
methanol, elution with linear gradient: 05 min, 40
60%B, 515 min, 80%B, 1530 min, 85%B, the other condi-
tions were the same as used in Section 2.4 for low molecules
analysis.
2.7. Acid hydrolysis
Water solution and methanol solution of 2 mg/ml
HMWT were heated at 80 C for 5 h in 2 M HCl. These
two hydrolyzed products were rstly analyzed by HPLC
under the following conditions: solvent A, 0.5% acetic acid
in water; and solvent B, methanol, and the elution gradient
consisted of: 020 min, 525%B, 2030 min, 25%B. The
other conditions were the same as used in Section 2.4. Sec-
ondly, these two hydrolyzed solutions were analyzed for
anthocyanidins by another HPLC conditions as follows:
solvent A, 10% formic acid; solvent B, methanolwater
formic acid (45:45:10 v/v/v), elution with linear gradient
from 35% to 95% B in 20 min, detection at 540 nm, the
other conditions were not changed.
All acid hydrolysis solutions were scanned with Phar-
macspec UV-1700 spectrophotometer (Shimadzu, Japan)
from 800 to 230 nm, compared with the solution before
acid hydrolysis. All above products were analyzed by
HPLCESIMS as the method described in Section 2.4,
but for the anthocyanidin it was changed to positive ion
mode and the capillary temperature was at 325 C.
2.8. Hydroxyl radical scavenging activity
2.8.1. Hydroxyl radical scavenging activity in 2-deoxyribose
system
The hydroxyl radical scavenging activity of the two frac-
tions LMWT and HMWT were assayed by using the 2-
deoxyribose oxidation method (Halliwell, 1987) with slight
modication. The reaction mixture contained 0.4 ml of
0.2 M sodium phosphate buer (pH 7.4), 0.1 ml sample
solution of dierent concentrations, 0.1 ml of 1 mM
EDTA, 0.1 ml of 1 mM FeCl
3
, 0.1 ml of 12 mM hydrogen
peroxide, 0.1 ml of 60 mM 2-deoxyribose, and 0.1 ml of
ascorbic acid in a tube. After incubation at 37 C for 1 h,
the reaction was stopped by adding 1 ml of 2% trichloro-
acetic acid and 1 ml of 1.0% thiobarbituric acid. The mix-
ture was boiled for 15 min, cooled in ice and extracted by
n-butanol and the absorbance was measured at 532 nm
against n-butanol (as blank). The reaction mixture without
test sample was used as control, and grape seeds proanth-
ocyanidins (GSP) was used as positive control. All the
analyses were done in triplicates and average values were
taken. Hydroxyl radical scavenging ability was evaluated
as the inhibition rate (IR) of 2-deoxyribose oxidation by
hydroxyl radical, and expressed as follows:
IR% = [A
0
(A
1
A
2
)]/A
0
100%, where A
1
, A
0
, and
A
2
are the absorbance of the sample, the blank sample,
and the blank control, respectively.
210 H.-F. Gu et al. / Food Research International 41 (2008) 208217
2.8.2. Hydroxyl radical scavenging activity in salicylic acid
system
The eect of hydroxyl radical scavenging of the fraction
LMWT and HMWT were also assayed using the salicylic
acid system (Smirno & Cumbes, 1996) with slight modi-
cation. Firstly, 1 ml of sample solutions with dierent con-
centrations, 0.3 ml of 8 mM FeSO
4
and 0.25 ml of 20 mM
hydrogen peroxide were added into the centrifugal vial.
Then, 1 ml of 3 mM salicylic acid was put into the test tube
and incubated at 37 C for 30 min. Distilled water (0.45 ml)
was added to each vial to make a nal volume of 3 ml.
After centrifugation (2000 rpm, 10 min), the suspensions
were measured at 510 nm. The reaction mixture without
test sample was used as control, and grape seeds proanth-
ocyanidins (GSP) was used as positive control. All the
analyses were done in triplicates and average values were
taken. Hydroxyl radical scavenging ability was evaluated
as the inhibition rate (IR) of salicylic acid oxidation by
hydroxyl radical, and expressed as follows:
IR% = [A
0
(A
1
A
2
)]/A
0
100%, where A
1
, A
0
, and
A
2
are the absorbance of the sample, the blank sample
and the blank control, respectively.
2.9. Superoxide anion radical scavenging activity
The superoxide anion scavenging activity of the fraction
LMWT and HMWT were measured by the method of
autoxidation of 1,2,3-trihydroxybenzene (Wu, Zhang,
Miao, & Zhu, 2005). The reaction mixture consisted of
4.5 ml of 50 mM TrisHCl buer containing 2 mM EDTA
(pH 8.2), and 0.1 ml of sample with dierent concentra-
tions. The mixture solutions were preincubated at 25 C
for 10 min. The reaction was initiated by the addition of
0.2 ml of 5 mM 1,2,3-trihydroxybenzene (dissolved in
10 mM HCl). The absorbance at 320 nm was recorded
for 30 s. 10 mM HCl was served as the blank control for
adjusting to zero. As control, distilled water replaced the
sample solution was considered as the autoxidation rate
of 1,2,3-trihydroxybenzene, and GSP was used as positive
control. The scavenging activity on superoxide anion
(SASA) radicals was expressed as follows:
SASA% = (V
0
V
1
)/V
0
100%, where V
0
and V
1
are
the autoxidation rate of 1,2,3-trihydroxybenzene (DOD/
min), and the autoxidation rate of 1,2,3-trihydroxybenzene
in sample (DOD/min), respectively.
2.10. Linoleic acid lipid peroxidation inhibition activity
Lipid peroxidation of linoleic acid was measured by the
modied method described previously (Kishida, Toku-
maru, & Ishitani, 1993). Each reaction mixture consisted
of 4.1 ml of 2.5% linoleic acid in ethanol and 10 ml of
0.2 M phosphate buer (pH 7.4), 1 ml of 2.54 10
3
g/L
FeSO
4
were added as catalyzer. Dierent amounts of sam-
ples were added to the reaction mixture in a centrifugal
vial. The vial was placed in an oven at 40 C. After incuba-
tion for 18 h, the reaction was stopped by adding 1 ml of
25% trichloroacetic acid and 1 ml of 0.67% thiobarbituric
acid. The mixture was boiled for 15 min, cooled in ice
and the mixture were extracted with 4 ml n-butanol. After
centrifugation (8000 rpm, 8 min), the n-butanol phase was
measured at 532 nm. A control was performed with linoleic
acid but without the samples, and GSP was used as positive
control.
Inhibition% = [A
0
(A
1
A
2
)]/A
0
100%, where A
1
, A
0
,
and A
2
are the absorbance of the sample, the blank sample,
and the blank control, respectively.
3. Results and discussion
3.1. Purication and fractionation of tannic extract from
persimmon pulp
There are a large amount of low molecular weight sub-
stances such as sugar, gallic acid, and organic acid in crude
persimmon methanol extract. The removal of these inter-
fering components from the soluble persimmon extract is
an important pretreatment step for the analysis of persim-
mon tannin. Purication with macroporous adsorption
resin AB-8 is an eective way to remove sugar and other
low molecular weight polar substances, after purication,
the contents of total polyphenols and condensed tannins
in the crude extract reached 91.1% and 82.4% separately.
Matsuo and Ito (1978) reported that the molecular weight
of persimmon tannin was quite large. Therefore, further
purication with an appropriate lter device is desirable
to separate low molecular weight phenolic compounds
from high molecular weight tannin. In the present work,
ultraltration with polysulfone ultrane membrane with
molecular weight cuto of 10,000 Da (Tianjin, Tianfang,
China) was used to fractionate the partially puried tannic
extract of persimmon pulp. The content of condensed tan-
nins of the high molecular weight fraction (retentate)
reached 93.4% and no condensed tannin was detected in
the low molecular fraction (ltrate). The content of total
polyphenols in the ltrate was 87.4%.
HPLC analysis of the crude persimmon methanol
extract, the ltrate fraction with low molecular weight
and the retentate fraction with high molecular weight were
shown in Fig. 1. The HPLC chromatogram of the crude
persimmon methanol extract (Fig. 1a) showed a large
hump with many resolved small peaks on it, but after ultra-
ltration, a hump without any resolved small peaks on the
280 nm chromatographic prole of the retentate was
observed (Fig. 1c), suggesting that small molecular pheno-
lic compounds could be separated eectively from HMWT.
The four main peaks observed in the LMWT fraction
(Fig. 1b) were further analyzed by HPLCMS in the nega-
tive ion mode. Peak 1 showed an intense molecular ion
[MH]

at m/z 169, and was identied as gallic acid on

the basis of its retention time (t
R
), UVvisible and MS
spectra, compared with those of the commercial standard.
Both peak 2 and peak 3 showed the same molecular ion
[MH]

at m/z 461 and had a maximum absorbance at

H.-F. Gu et al. / Food Research International 41 (2008) 208217 211
275 nm and the diagnostic character of phenolic com-
pounds. Peak 4 showed an intense molecular ion [MH]

at m/z 609 and exhibited two major absorption peaks at

275 nm and 320 nm, respectively, which suggested that
peak 4 could be of avone compounds. Nevertheless, fur-
ther studies are still required in order to conrm the iden-
tities of the three peaks.
3.2. GPC analysis of the high molecular weight persimmon
tannin
Persimmon tannin appeared as a single peak with reten-
tion time of 5.244 min on the GPC chromatogram (Fig. 2).
This result was quite similar to that of Wu and Hwang
(2002) and Yonemori, Matsushima, and Sugiura (1983).
The molecular weight distribution of persimmon tannin
was determined to be in the range of 1.16 10
4
Da to
1.54 10
4
Da, with the molecular weight of
1.28 10
4
Da in M
n
and 1.39 10
4
Da in M
w
, in reference
to standard curve obtained with polyvinyl alcohol. The
regression equation of the standard curve was
y = 2235x + 25,010, and the R-squared value was 0.9928.
3.3. Thiolysis degradation
The reversed-phase HPLC chromatogram at 280 nm of
the high molecular weight persimmon tannin was charac-
terized by the presence of a broad hump eluting between
15 and 28 min. As observed in previous chromatographic
studies on polyphenolics from other fruits such as apple
(Bruno et al., 2000; Peng et al., 2001), such a hump in
the 280 nm chromatographic prole could be attributed
to polymeric polyphenols. A series of UVvisible spectra
registered on the whole width of the hump showed a single
absorption band at 278 nm and all these spectra were quite
similar to that of ()epicatechin or (+)-catechin, thus sug-
gesting that the corresponding phenolic molecules pre-
sented structural analogies with these avanols.
HPLC analysis of the reaction media allowed the assay
of the major products resulting from the thiolysis degrada-
tion of the HMWT fraction. The disappearance of the
aforementioned hump in the chromatographic prole after
thioacidolysis suggested that the degradation was com-
plete, and ve new peaks were detected distinctly (Fig. 3).
Peak 5 was identied as benzyl mercaptan by comparison
of the retention time (t
R
), UVvisible spectra and mass
spectra with those of the standard.
Identications of the other four degradation products
were performed on the basis of their UVvisible spectra
and HPLCMS/MS spectrometric determinations in the
negative mode. Fig. 4 showed the MS
2
spectra of the cor-
responding molecular ion peaks of the four thiolysis degra-
dation products of HMWT. Peak 1 (Fig. 4a), eluting at
9.6 min, showed a molecular ion [MH]

at m/z 427 with

a characteristic MS
2
fragment ion [(MPhCH
2
S)H]

at m/z 303, suggesting that it was stereoisomeric (epi)gallo-

catechin benzylthioethers. Peak 2 (Fig. 4b), eluting at
Fig. 1. HPLC chromatogram of crude extracts (a), LMWT (b) and
HMWT (c) (detected at 280 nm).
Fig. 2. GPC chromatogram of the high molecular weight persimmon
tannin (detected at 280 nm).
212 H.-F. Gu et al. / Food Research International 41 (2008) 208217
10.6 min, showed a molecular ion [MH]

at m/z 579 with

a characteristic MS
2
fragment ion [(MPhCH
2
S)H]

at m/z 455 and a major fragment ion at m/z 303, which cor-
responds to the further loss of galloyl residue
([(MPhCH
2
S)H152]

), A fragment ion at m/z

409 corresponding to the loss of O-galloyl, and gallic acid
at m/z 169 was also identied. Which suggested that peak
2 is a benzylthioether of epigallocatechin-3-O-gallate. Peak
3 (Fig. 4c), eluting at 12.0 min, showed a molecular ion
[MH]

at m/z 493 with characteristic fragment ions

[(MPhCH
2
S)H]

at m/z 369, and other two frag-

ment ions at m/z 433 and 309, respectively. Because there
was no more structural information, the certain structure
of Peak 3 is not clear. Peak 4 (Fig. 4d), eluting at
12.5 min showed [MH]

at m/z 563, with a characteristic

intense fragment ions [(MPhCH
2
S)H]

at m/z 439,
the intense fragment ions at m/z 411 resulting from the loss
of galloyl residue, and the intense fragment ions at m/z 287
corresponding to the loss of galloyl residue and continued
loss of a PhCH
2
S unit, suggesting that peak 4 was a
benzylthioethers of epicatechin-3-O-gallate. The ratios of
peak 1, peak 2, peak 3 and peak 4 were 1:7:1: 3 by the peak
area on HPLC.
3.4. Acid hydrolysis
The acid hydrolysis of the HMWT in both methanol and
water solution gave a deep red coloration, conrming the
presence of a signicant amount of condensed tannin with
proanthocyanidin structure. The UVvisible spectra of acid
hydrolysis solution in methanol showed a strong absor-
bance at 540 nm. However, the acid hydrolysis solution in
water showed absorbance at 450 nm and 540 nm in the
UVvisible spectra. HPLC analysis (Fig. 5) of the acid
hydrolysis products showed that there were four anthocya-
nins (a, b, c, d) in methanol acid hydrolysis solution with the
ratios of 5:2:3:1 by the peak area on HPLC. But only two of
them (a, c with the ratio of 3:2) were present in water acid
hydrolysis solution. HPLCESI/MS/MS analysis in posi-
Fig. 3. High performance liquid chromatogram of thiolysis products of
HMWT (detected at 280 nm).
Fig. 4. ESI (MSMS scan) spectra of thiolysis degradation products of
HMWT; (a) corresponding to the parent irons at m/z 427; (b) corre-
sponding to the parent irons at m/z 563; (c) corresponding to the parent
irons at m/z 493; (d) corresponding to the parent irons at m/z 579.
H.-F. Gu et al. / Food Research International 41 (2008) 208217 213
tive ion mode further identify the four acid hydrolysis prod-
ucts as (a) delphinidin (M
+
303), (b) an unknown com-
pound (M
+
319), (c) petunidin (M
+
317), and (d) cyanidin
(M
+
287). With HPLCESI/MS/MS analysis in negative
ion mode, gallic acid, EGCG and gallic acid derivatives
were determined to be present in methanol acid hydrolysis
solution at 280 nm. A molecular ion peak at m/z 184 with
characteristic MS
2
fragment ion [(MCH
3
)H]

at m/z
169 was identied as gallic acid methyl ester. And an
unknown compound showing [MH]

at m/z 317 with a

characteristic MS fragment ion [(MC
8
H
8
O
3
H]

at m/z
164 was also determined to be present in methanol acid
hydrolysis solution at 280 nm. The Chemical Structure of
thiolysis degradation and acid hydrolysis products of
HMWT were shown in Fig. 6.
3.5. Hydroxyl radical scavenging activities
Among the oxygen radicals, hydroxyl radical is the most
reactive and induces severe damages to the adjacent biomol-
ecules. The scavenging eect of HMWT against hydroxyl
radical was investigated by using two Fenton reactions.
Fig. 7 showed the hydroxyl radical scavenging eects of
HMWT by the 2-deoxyribose oxidation method, which
was indicated as the inhibition rate. The hydroxyl radical
scavenging activity of HMWT was increased in a dose-
dependent manner within the range of concentrations from
0.1 to 1.5 mg/ml. The highest hydroxyl radical scavenging
activity (90.5%) were found with 1.5 mg/ml of HMWT,
when compared with that of GSP (84.8%) and LMWT
(20.72%) at the same concentrations respectively (P < 0.05).
Fig. 8 showed the hydroxyl radical scavenging eects of
HMWT by using the salicylic acid method, the results of
which were also indicated as the inhibition rate. The hydro-
xyl radical scavenging activity was increased from 24.9% to
87.7% in a dose-dependent manner within the range of con-
centrations from 0.1 to 1.1 mg/ml of HMWT. The highest
(P < 0.05) hydroxyl radical scavenging activity (87.7%) was
found with 1.1 mg/ml of HMWT, when compared with
that of GSP (76.3%) and LMWT (24.4%) at the same con-
centrations respectively. The results obtained above using
two dierent methods indicated that the HMWT from per-
simmon pulp has very strong scavenging eects against
hydroxyl radical.
3.6. Superoxide anion radical scavenging activities
The scavenging activities of HMWT against superoxide
anion radical were measured using the 1,2,3-trihydroxy-
(1) R=OH, R =OH, R =H
(2) R=OH, R =H, R=O-galloyl
(3) Unknown
(4) R=H, R =H, R =O-galloyl
(1) R
1
=OH, R
2
=OH
(2) Unknown
(3) R
1
=H, R
2
=OCH
3

(4) R
1
=H, R
2
=OH
a
b
Fig. 5. Chemical structure of thiolysis degradation and acid hydrolysis
products of HMWT: (a): thiolysis degradation products, (b): acid
hydrolysis products.
Fig. 6. HPLC chromatogram of methanol solution acid hydrolysis
products (a), water solution acid hydrolysis products (b) absorbance at
540 nm is shown.
0
20
40
60
80
100
A B C D E F G H I J
Sample
I
n
h
i
b
i
t
o
n
%
Fig. 7. Hydroxyl radical scavenging activity of HMWT, LMWT and GSP
with 2-deoxyribose oxidation system. AH were dierent concentration of
HMWT (0.1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5 mg/mL); I, 1.5 mg/mL of GSP;
J, 1.5 mg/mL of LMWT. Values are means SD (n = 3).
0
20
40
60
80
100
A B C D E F G H
Sample
I
n
h
i
b
i
t
o
n
%
Fig. 8. Hydroxyl radical scavenging activity of HMWT, LMWT and GSP
with salicylic acid system. AF was dierent concentrations of HMWT
(0.1, 0.3, 0.5, 0.7, 0.9, 1.1 mg/mL); G. 1.1 mg/mL of GSP; H. 1.1 mg/mL
of LMWT. Values are means SD (n = 3).
214 H.-F. Gu et al. / Food Research International 41 (2008) 208217
benzene autoxidation system and the results were indicated
as the inhibition rate of HMWT on superoxide anion radi-
cal. As shown in Fig. 9, the superoxide anion radical scav-
enging activity was increased in a dose-dependent manner
within the range of concentrations from 0.5 to 5 mg/ml of
HMWT. The scavenging activity of HMWT at 5 mg/ml
against superoxide anion radical was found to be higher,
but not markedly, than that of GSP at the same concentra-
tion. Furthermore, the scavenging activity of LMWT at
5 mg/ml was found to be 9.82%, statistically signicant
lower when compared with HMWT and GSP. These results
showed that the HMWT have strong scavenging eects
against superoxide anion radicals.
3.7. Antioxidant activity in linoleic acid system
Lipid peroxidation is known to be one of the reactions
set into motion as a consequence of the formation of free
radicals in cells and tissues. Membrane lipids are abundant
in unsaturated fatty acids. Linoleic acid is especially the
target of lipid peroxidation. The antioxidant eects of
HMWT on the peroxidation of linoleic acid were investi-
gated and the results were presented in Fig. 10. HMWT
exhibited inhibitory activity on the peroxidation of linoleic
acid in a dose-dependent manner. With the concentration
of 0.10% in the nal reaction mixture, the inhibitory rate
of HMWT on linoleic acid peroxidation reached 66.93%,
much higher than that of GSP (37.33%). Quite dierently
from HMWT and GSP, LMWT was found to accelerate
lipid peroxidation as shown in Fig. 10.
4. Discussion
Generally, persimmon tannin was reported to be high
molecular weight compound. The separation and structural
determination of persimmon tannin are very dicult due to
the strong hydrogen bond-forming ability of this molecu-
lar, thus aggregating each other and rmly adsorbing to
various absorbents and other materials in various chroma-
tographies because of a surprisingly large number of phe-
nolic hydroxyl groups of persimmon tannin.
In the present study, macroporous adsorption resin
combined with ultraltration were successfully employed
to isolate and purify high molecular persimmon tannin
and low molecular phenolic compounds from crude
extract; and a considerable yield of HMWT (87%) was
obtained. After ultraltration, a hump without any
resolved small peaks on the 280 nm chromatographic pro-
le of the retentate was observed, suggesting that small
molecular phenolic compounds could be separated eec-
tively from HMWT by this simple, low cost and time-sav-
ing procedure.
GPC combined with HPLCESIMS determination was
developed for the analysis of high molecular weight tannin
from persimmon. GPC could directly provide information
on molecular weight range of HMWT (Schoeld, Mbugua,
& Pell, 2001). Final conrmation of the monomer unit con-
stitution of the structures was obtained from thiolysis deg-
radation and acid hydrolysis. When condensed tannins are
heated in the presence of acid and benzyl mercaptan, the
chain-ending unit at the bottom is released as an unsubsti-
tuted avanol, whereas all internal units appear as benzyl
thioethers (Kennedy, Matthews, & Waterhouse, 2000). In
principle, this would permit determination of both chain
length and composition of tannin when thiolysis products
are analyzed by HPLCMS. The thiolysis condition, at
60 C for 2 h, was suitably optimized after conducting at
various temperatures. It may concluded that the condensed
tannin from this Chinese persimmon consists of (epi) gallo-
catechin, epigallocatechin-3-O-gallate, epicatechin-3-O-gal-
late, and an unknown monomer unit, with the molecular
weight of 1.28 10
4
Da in M
n
and 1.39 10
4
Da in M
w
.
The result is dierent from that of Matsuo and Ito (1978).
Many diseases including cancer, cataracts and cardio-
vascular diseases et al. are associated with oxidative dam-
ages from hydroxyl radical, superoxide anion radical and
lipid peroxidation. Recently, polyphenols in plant tissues
such as grape seeds, apple pulp, hawthorn and persimmon
leaf have shown signicantly strong antioxidant activities.
It has been reported that persimmon seeds extracts have
a stronger radical scavenging activity than grape seeds
0
10
20
30
40
50
60
70
80
A B C E F D G H
Sample
I
n
h
i
b
i
t
o
n
%
Fig. 9. Superoxide anion radical scavenging activities of HMWT, LMWT
and GSP. AF was dierent concentration of HMWT (0.5, 1.0, 1.5, 2.0,
2.5, 5 mg/ml); G, 5 mg/mL of GSP; H, 5 mg/mL of LMWT. Values are
means SD (n = 3).
-10
0
10
20
30
40
50
60
70
80
A B C D E F
Sample
I
n
h
i
b
i
t
i
o
n
%
Fig. 10. Inhibition eect on Linoleic acid lipid peroxidation of HMWT,
LMWT and GSP. AD were dierent concentration of HMWT (0.02%,
0.06%, 0.08%, 0.10%) in reaction system; E, 0.10% of GSP in reaction
system; F, 0.10% of LMWT in reaction system. Values are means SD
(n = 3).
H.-F. Gu et al. / Food Research International 41 (2008) 208217 215
extracts (Ahn et al., 2002). Some researches investigated
the radical scavenging activity of methanol extracts of per-
simmon folium, Laminaria japonica, and Undaria pinnati-
da against DPPH (1,1-diphenyl-2-picrylhydrazyl) and
found the persimmon extracts was the most potent (Han
& et al., 2002). However, recent investigations have shown
some dierences between dierent test systems for the
determination of antioxidant activity (Schlesier, Harwat,
Bohm, & Bitsch, 2002). Therefore, it was recommended
to use at least two test systems to evaluate the ecacy of
antioxidation. In the present study, we used four dierent
systems to investigate whether high molecular weight
condensed tannin are the main antioxidants in persimmon
pulp. HMWT from persimmon showed strong antioxidant
activity in all of the test systems and was found to be
more potent antioxidant than LMWT from persimmon
and oligomeric proanthocyanidins from grape seeds
(GSP), the former of which was even found to accelerate
lipid peroxidation. Hagerman and et al. (1998) suggested
that tannin, or polymeric polyphenolics, may be much
more potent antioxidant than are simple monomeric phen-
olics duo to the high molecular weight and the proximity of
many aromatic rings and hydroxyl groups. The results
obtained here were in agreement with the suggestion and
further indicated that the high molecular weight condensed
tannins were the major antioxidants in persimmon pulp.
Acknowledgments
This study was partially supported by the Guangdong
Agricultural Science and technology program
(2007A020200003-2) and we are grateful to Erning Yang
for his support in HPLCMS and Xiaoman Gu for her
help in GPC.
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