African Journal of Biotechnology Vol. 8 (10), pp. 2301-2309, 18 May, 2009

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DOI: 10.5897/AJB09.009

Full Length Research Paper

Antioxidant tannins from Syzygium cumini fruit

Liang Liang Zhang and Yi Ming Lin*

Department of Biology, School of Life Sciences, Xiamen University, Xiamen 361005, China.

Key Laboratory of Ministry of Education for Coastal and Wetland Ecosystems, Xiamen 361005, China.

Accepted 13 February, 2009

Hydrolysable and condensed tannins in the fruit of Syzygium cumini were identified using NMR, MALDI-

TOF  MS  and  HPLC  analyses.  Hydrolysable  tannins  were  identified  as  ellagitannins,  consisting  of  a

glucose core surrounded by gallic acid and ellagic acid units. Condensed tannins were identified as B-

type  oligomers  of  epiafzelechin  (propelargonidin)  with  a  degree  of  polymerization  up  to  eleven.  The

antioxidant  activity  were  measured  by  two  vitro  models:  1,1-diphenyl-2-picrylhydrazyl  (DPPH)  radical

scavenging  activity  and  ferric  reducing/antioxidant  power  (FRAP).  Tannins  extracted  from  S.  cumini

fruit  showed  a  very  good  DPPH  radical  scavenging  activity  and  ferric  reducing/antioxidant  power. The

results  are  promising  thus  indicating  the  utilization  of  the  fruit  of  S.  cumini  as  a  significant  source of

natural antioxidants.

Key words: Syzygium cumini, tannins, antioxidant activity, MALDI-TOF MS, HPLC.

INTRODUCTION

Tannins are significant plant secondary metabolites

subdivided into condensed and hydrolysable compounds

in vascular plants. Condensed tannins are also known as

proanthocyanidins (PAs), the oligomeric and polymeric

flavan-3-ols, which are linked through C4 - C8 or C4 - C6

linkages. The diversity of condensed tannins is given by

the structural variability of the monomer units (different

hydroxylation patterns of the aromatic rings A and B, and

different configurations at the chiral centers C2 and C3)

(Figure 1). The size of PAs molecules can be described

by their degree of polymerization (DP). The molecules

are water-soluble and are able to form complexes with

proteins

and

polysaccharides

(Haslam,

1998).

Hydrolysable tannins (HTs) represent a large group of

polyphenolic compounds that are widely distributed in the

plant kingdom. They are esters of a polyol (most often β-

D-glucose) with either gallic acid (gallotannins) or

hexahydroxydiphenic acid (ellagitannins). These ester

forms vary from simple compounds such as β-D-

glucogallin to compounds with M

values in excess of

2,500 (Haslam, 1992). The structural elucidation of poly-

meric tannins is difficult because of their heterogeneous

character. Due to this complexity and diversity, the cha-

*Corresponding author. E-mail: linym@xmu.edu.cn. Tel.: +86

592 2187657.

racterization of highly polymerized condensed tannins

thus remains very challenging, and less is known

regarding structure-activity relationships. Various techni-

ques including NMR and mass spectroscopy (MS) have

been used to characterize hydrolysable and condensed

tannins (Behrens et al., 2003; Chen and Hagerman,

2004; Vivas et al., 2004).

The fruits of Syzygium cumini (L.) Skeels are edible

and are reported to contain gallic acid, tannins,

anthocyanins and other components (Benherlal and

Arumughan, 2007). The juice of unripe fruits is used for

preparing vinegar that is considered to be a stomachic,

carminative and diuretic. The ripe fruits are used for

making preserves, squashes and jellies. The fruits are

astringent. A wine is prepared from the ripe fruits in Goa

(Wealth of India, 1976). Extract of seed, which is

traditionally used in diabetes, has a hypoglycaemic action

and antioxidant property in alloxan diabetic rats (Prince et

al., 1998) possibly due to tannins (Bhatia et al., 1971).

Tannins are antioxidants often characterized by reduc-

ing power (Mi-Yea et al., 2003) and scavenging activities

(Minussi et al., 2003). The antioxidant capabilities of

tannins depend on: (1) the extent of their colloidal state;

(2) the ease of interﬂavonoid bond cleavage or its

stereochemical structure; (3) the ease of the pyran ring

(C-ring) opening; and (4) the relative number of –OH

groups on A and B rings (Noferi et al., 1997). Compounds

with a trihydroxyl structure in the B-ring have the greatest

2302 Afr. J. Biotechnol.

= OH, afzelechin

= OH, epiafzelechin

G: Galloyl group

= OG, afzelechin gallate

= OG, epiafzelechin gallate

C4-C8 linkage

=H, G; propelargonidin

Figure 1. Chemical structures of flavan-3-ol monomers and polymers.

antioxidant activity (Rice-Evans et al., 1996).

The fruit of S. cumini has been shown to contain a

large range of hydrolysable tannins (Benherlal and

Arumughan, 2007). However, neither hydrolysable nor

condensed tannins have been characterized in S. cumini.

In this study we undertook the structural characterization

of tannins in S. cumini fruit using a combination of NMR,

MALDI-TOF MS and HPLC analyses. This would

contribute to a better understanding of the chemical

composition of S. cumini and the applicability of NMR,

MALDI-TOF MS and HPLC in the analyses of food

tannins. We also report the evaluation of free-radical

scavenging properties and ferric reducing/antioxidant

power of tannins extracted from S. cumini fruit.

MATERIALS AND METHODS

Plant materials and chemicals

Mature fruits of S. cumini were collected at the campus of Xiamen

University (Xiamen, China) and immediately freeze dried and

ground. The resulting powder was extracted with acetone:water

(7:3, v/v) and the organic solvent was eliminated by evaporation

under vacuum. The remaining crude tannin fraction was chromato-

graphed on an LH-20 column (Pharmacia Biotech, Uppsala,

Sweden) which was first eluted with methanol:water (50:50, v/v)

and then with acetone : water (7:3, v/v). The last fraction, containing

the polymeric tannins was freezed-dried and stored at -20°C till

further use.

1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-S-triazine

(TPTZ), potassium ferricyanide, butylated hydroxyanisole (BHA),

ascorbic acid, (+)-catechin, cesium chloride, gallic acid, ellagic acid

and tannic acid were purchased from Sigma Chemical Co. (St.

Louis, MO, USA). Reagents and solvents were of analytical or

HPLC grade. Deionised water was used throughout.

NMR analysis

C NMR spectra were recorded in CD

COCD

-D

O mixture with a

Varian Metcury-600 spectrometer (USA) at 150 MHz.

MALDI-TOF MS analysis

The tannin extract was mixed with a 1 M solution of dihydroxy-

benzoic acid (DHB) in 90% methanol in a 1:1 ratio, and 1 µl of the

mixture was spotted onto a ground stainless steel MALDI target for

MALDI analysis using the dry droplet method. Cesium chloride (1

mg/ml) was mixed with the analyte/matrix solution at the 1:3

volumetric ratio to promote the formation of a single type of ion

adduct (M + Cs

) (Xiang et al., 2006). A Bruker Reflex III MALDI-

TOF MS (Germany) equipped with an N

laser (337 nm) was used

in the MALDI analysis and all the data were obtained in a positive

ion reflectron TOF mode.

Acid hydrolysis

Acid hydrolysis of the ellagitannins was performed as described by

Oszmianski et al. (2007). The ellagitannins from S. cumini fruit

stone (25 mg) were hydrolyzed with 2 ml of 2 mol/l hydrochloric acid

in a boiling water bath for 1 h. After cooling, 2 ml of 2 mol/l NaOH

and then 6 ml methanol were added to the vial. The slurry was

sonicated for 20 min with occasional shaking. Further, the slurry

was centrifuged at 10,000 g and the supernatant was used for

HPLC analysis. The HPLC apparatus consisted of an Agilent 1100

diode array detector and a quaternary pump.

The samples were previously dissolved in a mobile phase and

then filtrated through a membrane filter with an aperture size of

0.45 µm. 10 ml of the clear supernatant was injected. Separation

was performed on a Hypersil ODS column (4.6 × 250 mm, 5 µm)

thermostatted at 30°C. The mobile phase was composed of solvent

A (0.1% v/v) trifluoroacetic acid (TFA) in water) and solvent B (0.1%

v/v) TFA in acetonitrile). The gradient condition was: 0~2

min

100% A, 2

~6

th

min 0~5% B, 6

~10

min 5% B, 10

~15

min

5~10% B, 15

~20

min 10% B, 20

~30

min 10~20% B, 30

~35

min 20% B and 35

~40

min 20~30% B. Other chromatographic

conditions were as follows: flow rate at 1 ml/min, detection at 280

and 254 nm, and scanning performed between 200 and 600 nm.

The identification of chromatographic peaks was made by

comparison of their relative retention times with those of external

standards, as well as by their UV–visible spectra. Ellagic acid

(detection 254 nm) and gallic acid (detection at 280 nm) was

quantified using the calibration curve established with ellagic and

gallic acid standards. Analysis was made in triplicate.

Free-radical scavenging activity

The free-radical scavenging activity was measured according to

Braca et al. (2001). A 100 µl of the sample at different concen-

trations (15 – 500 µg/ml) was added to 3 ml of DPPH solution (0.1

M methanolic solution). 30 min later, the absorbance was measured

at 517 nm. Lower absorbance of the reaction mixture indicates

higher free radical scavenging activity. The IC

value, defined as

the amount of antioxidant necessary to decrease the initial DPPH

concentration by 50%, was calculated from the results and used for

comparison. The capability to scavenge the DPPH radical was

calculated using the following equation:

DPPH scavenging effect (%) = [(A

– A

)/A

] × 100

where A

is the absorbance of the control reaction and A

is the

absorbance in the presence of the sample. BHA, (+)-catechin and

ascorbic acid were used as controls.

Ferric reducing/antioxidant power (FRAP) assay

FRAP assay is a simple and reliable colorimetric method commonly

used for measuring total antioxidant capacity (Benzie and Strain,

1996). Briefly, 3 ml of FRAP reagent, prepared freshly, was mixed

with 100 µl of the test sample, or methanol (for the reagent blank).

The FRAP reagent was prepared from 300 mM, pH 3.6, acetate

buffer, 20 mM ferric chloride and 10 mM 2,4,6-tripyridyl-S-triazine

made up in 40 mM hydrochloric acid. All three solutions were mixed

together in the ratio of 10:1:1 (v/v/v). The absorbance of reaction

mixture at 593 nm was measured spectrophotometrically after

incubation at 25°C for 10 min. The FRAP values, expressed in

mmol ascorbic acid equivalents (AAE)/g dried tannins, were derived

from a standard curve.

Statistical analyses

All measurements were replicated three times and one-way analy-

sis of variance (ANOVA) was used and the differences were

considered to be significant at P < 0.05. All statistical analyses were

performed with SPSS 11.0.

RESULTS AND DISCUSSION

NMR analysis

The signal assignment was made based on the publica-

tion of Czochanska et al. (1980). The spectrum shows

distinct signals at 157 ppm, which are assignable to C4’

propelargonidin

units

(afzelechin/epiafzelechin).

Indeed, procyanidin units (catechin/epicatechin) and

prodelphinidin

units

(gallocatechin/epigallocatechin)

generally showed a typical resonance at 144 - 145 and

145 - 146 ppm respectively (Czochanska et al., 1980;

Behrens et al., 2003). The absence of a clear signal with

such chemical shift in the spectra of the condensed

tannins from S. cumini fruit skin revealed that they are

only composed of propelargonidin units.

The region between 70 and 90 ppm is sensitive to the

stereochemistry of the C-ring. The determination of the

ratio of the 2,3-cis to 2,3-trans stereochemistries could

thus be achieved through distinct differences in their

respective C2 chemical shifts (Czochanska et al., 1980).

Whereas C3 of both cis and trans isomers occurs at 73

ppm, C2 gives a resonance at 76 ppm for the cis form

and at 84 ppm for the trans form. The absence of the

latter signal peak in the spectrum of the studied con-

Zhang and Lin 2303

densed tannin fraction indicated the presence of only

epiafzelechin subunits. The presence of a signal at 35.9

ppm was consistent with a C4 being shifted upfield by the

presence of a 3-O-gallate unit. This was further confirmed

by the observation of signals for ester carbonyl carbons

at 175.5 ppm (Gal-C7) and galloyl ring carbons at 114.0

ppm (Gal-C2, Gal-C6), 130.8 ppm (Gal-C1) and 143 -

145 ppm (Gal-C4). These results thus showed that the

polymeric propelargonidin of the studied S. cumini fruit

skin is predominantly constituted of propelargonidin with

(-)-epiafzelechin as the main constitutive monomer, some

with galloyl groups attached (Spencer et al., 2007).

As indicated above,

H and

C NMR spectroscopy

techniques were used to estimate the degree of polyme-

rization and the number-average molecular weight. The

C3 in terminal units generally have their chemical shift

around 67 ppm. Theoretically, its intensity relative to that

of the signal of the C3 in extension monomer units at 73

ppm could be used for elucidating the polymer chain

length. However, in the case of the spectra presented

here, the signal-to-noise ratio is too low to allow for such

quantification.

MALDI-TOF MS analyses

To obtain more detailed information on the chemical

structure of the condensed tannins and to overcome the

problems with determination of polymer chain lengths by

NMR spectroscopy, further characterization was conti-

nued by means of MALDI-TOF MS.

MALDI-TOF MS has

advantages over the other MS systems in terms of

sensitivity and mass range. The single ionization event

produced by MALDI-TOF MS allows the simultaneous

determination of masses in complex mixtures of low and

high molecular weight compounds. Several factors must

be optimized to develop MALDI-TOF MS techniques.

These factors include the selection of an appropriate

matrix, optimal mixing and optimal selection of cationi-

zation reagent. In our study, the Cs

was used as the

cationization ragent. This resulted in the best conditions

for their MALDI-TOF analysis and resulted in a relatively

simple MALDI-TOF spectrum.

Figure 2 shows the MALDI-TOF mass spectrum of the

studied polymeric mixture, recorded as Cs

adducts in the

positive-ion reflectron mode and showing a series of

repeating propelargonidin polymers. The polymeric

character is reflected by the periodic of peak series

representing different chain lengths. The results indicated

that S. cumini fruit skin tannins are characterized by

mass spectra with a series of peaks with distances of 272

Da corresponding to a mass difference of one afzelechin/

epiafzelechin between each polymer. Therefore, pro-

longation of condensed tannins is due to the addition of

afzelechin/epiafzelechin monomers. The spectrum show-

ed a series polyflavan-3-ols extending from the dimer

(m/z 679) to the undecamer (m/z 3129) that did not

contain ions with ∆2 amu lower than predicted in the

2304 Afr. J. Biotechnol.

Figure 2. MALDI-TOF positive reflectron mode mass spectrum of tannins from S. cumini fruit skin.

Masses represent the epiafzelechin homopolymer of the polyflavan-3-ol series [M+Cs]

Table 1. Observed and calculated masses

of heteropolyflavan-3-ols by

MALDI-TOF MS.

Mass calculations were based on the equation 274 + 272a + 152b + 133,

where 274 is the molecular weight of the terminal epiafzelechin unit, a is

the degree of polymerization (DP) contributed by the epiafzelechin

extending unit, b is the number of galloyl esters and 133 is the atomic

weight of cesium; N, no observed peaks corresponding to those

calculated ones.

positive-ion reflectron mode (Table 1).

On the basis of the structures described by Krueger et

al. (2003), an equation

was formulated to predict

heteropolyflavan-3-ols of a higher DP. The equation is

Polymer

Number of

galloylated esters

Calculated

[M+Cs]

+ a

Observed

[M+Cs]

+

Dimer

679

831

679

831

Trimer

951

1103

951

1103

Tetramer

1223

1375

1224

1375

Pentamer

1495

1647

1496

1648

Hexamer

1767

1919

1768

1921

Heptamer

2039

2191

2040

2192

Octamer

2311

2463

2312

2463

Nonamer

2583

2735

2584

2736

Decamer

2855

3007

2856

3008

Undecamer

3127

3279

3129

Zhang and Lin 2305

Figure 3. MALDI-TOF positive reflectron mode mass spectrum of olligomeric ellagitannins in S. cumini fruit stone. Lableled

masses are the molecular ions minus 1 proton plus Cs

274 + 272a + 152b + 133; where 274 is the molecular

weight of the terminal epiafzelechin unit, a is the degree

of polymerization (DP) contributed by the epiafzelechin

extending unit, b is the number of galloyl esters, 133 is

the atomic weight of cesium. Application of this equation

to the experimentally obtained data revealed the

presence of a series of condensed tannins consisting of

well-resolved oligomers. The broad peaks in these

spectra indicate, however, that there is large structural

heterogeneity within each DP.

For the condensed tannins indicated, each peak was

always followed by mass signals at a distance of 152 Da

corresponding to the addition of one galloyl group at the

heterocyclic C-ring. Thus, peak signals corresponding to

monogalloylated derivatives of various condensed tannin

oligomers were easily attributed. No propelargonidin

containing more than one galloyl group were detected.

Therefore, MALDI-TOF MS indicates the simultaneous

occurrence of a mixture of propelargonidin polymers,

monogalloylated derivatives of propelargonidin polymers.

This showed that there were a mixture of galloylated

propelargonidin and propelargonidin in S. cumini fruit skin

propelargonidin oligomers.

No series of compounds that are ∆2 amu multiples

lower than those described in the predictive equation for

heteropolyflavan-3-ols were detected. So there are no A-

type interflavan ether linkages occurring between

adjacent flavan-3-ol subunits. All compounds are linked

by B-type.

In the case of S. cumini fruit stone tannins (Figure 3), a

certain degree of regularity was observed in the MALDI-

TOF mass spectrum and notably these tannins possess

very similar mass distributions but different average

masses. In the spectra, four sets of peaks that are

separated by 152 Da are evident and have been assign-

ed to a unit of galloyl group using modeling correlations.

Structural assignment of these tannins advocates that S.

cumini

fruit stone is composed of gallic acid units centred

upon a core of glucose unit that is different from S. cumini

fruit skin. So, our study confirms the general classification

of the S. cumini fruit stone tannins as ellagitannins, that is

consisting of a glucose core surrounded by gallic and

ellagic acid units (Table 2). Almost superimposable mass

spectra were obtained for all the S. cumini fruit stone

ellagitannins analysed. Masses between 1500 and 5000

correspond to structures of oligomeric ellagitannins in

which two or more core glucose units are cross-linked by

dehydrodigalloyl or valoneoyl units. This is in agreement

with previously reported data concerning the same genus

plant Syzygium aromaticum (Tanaka et al., 1996) for

which two new ellagitannins were reported. These

different chemical groups are frequently composed of the

same building blocks but in different combinations and

numbers. For example, gallic acid occurs naturally but

can dimerize to form ellagic acid. Ellagic acid can

dimerize to form gallagic acid. Ellagic acid can combine

with glucose to form the unique compounds punicalagin

and punicalin. The different combinations and polymers

of the aforementioned form the large, diverse group of

compounds known as polyphenols, which show potent

antioxidant capacity and possible protective effects on

human health (Santos-Buelga and Scalbert, 2000).

These oligomers have been detected in S. cumini for the

first time in this study, although they are known to occur

in other plants (Quideau and Feldman, 1996) and further

study is required to elucidate their structure.

2306 Afr. J. Biotechnol.

Table 2. Calculated and observed masses for oligomeric ellagitannins in S. cumini fruit stone and possible

monomeric composition.

Mass + Cs

Observed

mass

Monomeric composition

Glucosyl

Gallagic

acid

Ellagic

acid

Gallic

acid

Dehydrodigallic

acid

Dimers

1551

1553

1701

1703

1853

1855

2003

2005

1

Trimers

2335

2336

2486

2488

2638

2788

2789

2

Tetramers

3120

3270

3272

3422

3574

3573

3

Pentamers

3905

3904

4055

4057

4207

4206

4209

4359

4358

Hexamer

4840

4839

4992

4991

Identification of hydrolytic products of ellagitannins

The ellagic acid in plants is present mainly in the form of

ellagitannins and is bound to glucose. Acid hydrolysis

transforms glucosylated and esteriﬁed ellagic acid into

their aglycones, and liberates the parent compound

ellagic acid and gallic acid (Daniel et al., 1989). Ellagic

and gallic acids are major products, as shown by a typical

HPLC chromatogram of the hydrolyzed products of

ellagitannins from S. cumini fruit stone (Figure 4) with

35.46 ± 3.00 mg/g dry tannins for ellagic acid and 19.73 ±

0.81 mg/g dry tannins for gallic acid.

Most quantitative evaluation of ellagitannins in fruit has

been on the hydrolyzed ellagitannins as ellagic acid

equivalents (Wada and Ou, 2002; Siriwoharn and

Wrolstad, 2004). This has significant problems in terms of

relating data to possible health effects, because there is

significant evidence that larger molecular mass tannins

(

＞1000 Da), including ellagitannins and procyanidins,

are not absorbed to any appreciable extent in their native

state (Cerda et al., 2004). Knowledge of ellagitannin

molecular structure, composition and quantity is needed

to understand their role in determining potential health

effects.

Radical-scavenging activities on 1,1-diphenyl-2-

picrylhydrazyl (DPPH)

Figure 5 shows the dose-response curve of DPPH radical

Zhang and Lin 2307

Figure 4. HPLC chromatograms of ellagitannins from S. cumini fruit stone after hydrolysis, detected by absorbance at 280

nm (a) and 254 nm (b). The peaks corresponding to gallic acid (1) and ellagic acid (2) are indicated on the chromatogram.

Other peaks are unidentified phenolics.

Antioxidant concentration (mg/ml)

100

200

300

400

500

600

100

fruit skin

fruit stone

(+)-catechin

BHA

ascorbic acid

(µg/ml)

Figure 5. Free radical-scavenging activities of tannins,

measured using ascorbic acid, BHA and (+)-catechin as DPPH

assay reference compounds.

scavenging activity of the tannin fractions from the S.

cumini

fruits, compared with (+)-catechin, BHA and

ascorbic acid. The tannins of the stone had a higher

activity than that of the skin. At a concentration of 0.25

mg/ml, the scavenging activity of tannins of the skin

reached 65.85%, while at the same concentration that of

the stone was 93.31%. IC

values were compared with

those of ascorbic acid and BHA in the system to assess

the antioxidant property of S. cumini fruit tannins (Table

3). A lower value of IC

indicates greater antioxidant

activity. IC

values of tannins from stone were superior

Table 3. Antioxidant activities of tannins of S. cumini fruit using the

(DPPH) free radical-scavenging assay and the (FRAP) ferric-

reducing antioxidant power assay.

The antioxidant activity was evaluated as the concentration of the

test sample required to decrease the absorbance at 517 nm by

50% in comparison to the control.

FRAP values are expressed in

mmol ascorbic acid equivalent (AAE)/g sample in dry weight.

Different letters on the same column show significant differences

from each other at P < 0.05.

to those of the reference ascorbic acid, (+)-catechin and

BHA. The effect of antioxidants on DPPH is thought to be

due to their hydrogen donating ability (Baumann et al.,

1979). Though the DPPH radical scavenging abilities of

tannins from S. cumini fruit skin were less than that of the

stone, the study showed that the tannins have proton-

donating ability and could serve as free radical inhibitors

or scavengers, acting possibly as primary antioxidants.

Ferric reducing antioxidant power

The reducing ability of the tannin fractions from S. cumini

fruit stone (6.21 mmol AAE/g) was higher than that of the

skin (3.02 mmol AAE/g) (Table 3). The antioxidant poten-

tial of the tannins from S. cumini fruit were estimated from

Sample

Antioxidant activity

IC

50/DPPH

(µg/ml)

a

FRAP (mmol AAE/g)

b

Fruit skin

165.05 ± 3.90a

3.02 ± 0.06d

Fruit stone

82.21 ± 0.77c

6.21 ± 0.19b

(+)-catechin

106.36 ± 4.28b

4.34 ± 0.07c

BHA

113.00 ± 4.28b

7.43 ± 0.14a

Ascorbic acid

85.68 ± 0.46c

2308 Afr. J. Biotechnol.

Antioxidant concentration (mg/ml)

100

200

300

400

500

600

0.0

1.0

1.2

1.4

1.6

1.8

fruit skin

fruit stone

(+)-catechin

BHA

(µg/ml)

Figure 6. The reducing power of tannins as compared to (+)-

catechin and BHA standards

their ability to reduce TPTZ-Fe

complex to TPTZ-Fe

The FRAP value of the skin tannins was significantly

lower than those of BHA and (+)-catechin. The FRAP

values for the stone tannins on the other hand were signi-

ficantly lower than that of BHA but higher than that of (+)-

catechin. Such potential reducing power activity might be

attributed due to the presence of hydrolysable tannins

present in the stone. Antioxidant activity increased pro-

portionally with tannins content, and all tannins showed

increased ferric reducing power with increasing concen-

tration (Figure 6). According to

Oktay et al. (2003), a

highly positive relationship between total phenols and

antioxidant activity appears to be the trend in many plant

species.

Conclusion

The results obtained showed that the condensed tannins

consisted of predominantly propelargonidin with 2,3-cis

stereochemistry. The mean degree of polymerization

determined through MALDI-TOF MS analysis was 5.0

and the number-average molecular weight was 1372.45

Da. Cationization by addition of Cs

allowed us to elimi-

nate the interference of the ∆16 mass differences

between Na

and K

with ∆16 mass differences that

results from pattern of hydroxylation. As a result of these

techniques, we have observed larger structural hetero-

geneity of oligomers than is generally appreciated in the

literature on plant tannins. The results from the appli-

cation of MALDI-TOF MS clearly demonstrate its power

as a tool to characterize the nature of tannins. Tannins

extracted from S. cumini fruit showed a very good DPPH

radical scavenging activity and ferric reducing/antioxidant

power.

ACKNOWLEDGEMENTS

Project (No. 30671646) was supported by the National

Natural Science Foundation of China, the Program for

New Century Excellent Talents in University (NCET-07-

0725) and the Program for Innovative Research Team in

Science and Technology in Fujian Province University.

REFERENCES

Baumann J, Wurn G, Bruchlausen FV (1979). Prostaglandin synthetase

inhibiting

-2

O radical scavenging properties of some ﬂavonoids and

phenolic

compounds.

Naunyn-Schmiedebergs

Arch

Pharmacol. 307: 1-77.

Behrens A, Maie N, Knicker H, Kogel-Knabner I (2003). MALDI-TOF

mass spectrometry and PSD fragmentation as means for the analysis

of condensed tannins in plant leaves and needles. Phytochem. 62:

1159-1170.

Benherlal PS, Arumughan C (2007). Chemical composition and in vitro

antioxidant studies on

Syzygium cumini

fruit. J. Sci. Food Agric. 87:

2560-2569.

Benzie IFF, Strain JJ (1996). The ferric reducing ability of plasma

(FRAP) as a measure of “antioxidant power”: The FRAP assay. Anal.

Biochem. 239: 70-76.

Bhatia IS, Bajaj KL, Ghangas GS (1971). Tannins in black plum seeds.

Phytochem. 10: 219-220.

Braca A, Tommasi ND, Bari LD, Pizza C, Politi M, Morelli I (2001).

Antioxidant principles from

Bauhinia terapotensis

. J. Nat. Prod. 64:

892-895.

Cerda B, Espin JC, Parra S, Martinez P, Tomas-Barberan FA (2004).

The potent in vitro antioxidant ellagitannins from pomegranate juice

are metabolised into bioavailable but poor antioxidant hydroxy-

dibenzopyran-6-one derivatives by the colonic microﬂora of healthy

humans. Eur. J. Nutr. 43: 205-220.

Chen Y, Hagerman AE (2004). Characterization of soluble non-covalent

complexes between bovine serum albumin and

β

-1,2,3,4,6-penta-

galloyl-

-glucopyranose by MALDI-TOF MS. J. Agric. Food Chem.

52: 4008-4011.

Czochanska Z, Foo LY, Newman RH, Porter LJ (1980). Polymeric

proanthocyanidins: Stereochemistry, structural units and molecular

weight. J. Chem. Soc. Perkin Trans. 1: 2278-2286.

Daniel EM, Krupnick A, Heur YH, Blinzler JA, Nims RW, Stoner GD

(1989). Extraction, stability and quantiﬁcation of ellagic acid in various

fruits and nuts. J. Food Comp. Anal. 2: 338-349.

Haslam E (1992). In: Plant Polyphenols; Hemingway, R.W., Laks, P.E.,

Eds.; Plenum Press: New York, p. 172.

Haslam E (1998). Practical Polyphenolics: From Structure to Molecular

Recognition and Physiological Action. Cambridge University Press:

Cambridge.

Krueger CG, Vestling MM, Reed JD (2003). Matrix-assisted laser

desorption/ionization

time-of-flight

mass

spectrometry

heteropolyflavan-3-ols

and

glucosylated

heteropolyflavans

sorghum. J. Agric. Food Chem. 51: 538-543.

Minussi RC, Rossi M, Bologna L, Cordi L, Rotilio D, Pastore GM, Duran

N (2003). Phenolic compounds and total antioxidant potential of

commercial wines. Food Chem. 82: 409-416.

Mi-Yea S, Tae-Hun K, Nak-ju S (2003). Antioxidants and free radical

scavenging

activity

of

Phellinus

baumii

(

Phallinus

of

Hymenochaetaceae

). Food Chem. 82: 593-597.

Noferi M, Masson E, Merlin A, Pizzi A, Deglise X (1997). Antioxidant

characteristics of hydrolysable and polyﬂavonoid tannins: An ESR

kinetics study. J. Appl. Polym. Sci

.

63: 475-482.

Oktay M, Gulcin I, Kufrevioglu QI (2003). Determination of in vitro

antioxidant activity of fennel (

Foeniculum vulgare

) seed extracts.

LWT-Food Sci. Tech. 36: 263-271.

Oszmianski J, Wojdylo A, Lamer-Zarawska E, Swiader K (2007).

Antioxidant tannins from Rosaceae plant roots. Food Chem. 100:

579-583.

Prince PS, Menon VP, Pari L (1998). Hypoglycaemic activity of

Syzygium cumini

seeds: Effect on lipid peroxidation in alloxan

diabetic rats. J. Ethnopharm. 61: 1-7.

Quideau S, Feldman K (1996). Ellagitannin chemistry. Chem. Rev. 96:

475-503.

Rice-Evans CA, Miller NJ, Paganga G (1996). Structure-antioxidant

activity relationships of ﬂavanoids and phenolic acids. Free Rad. Biol.

Med. 20: 933-956.

Santos-Buelga C, Scalbert A (2000). Proantocyanidins and tannin-like

compounds-nature, occurrence dietary intake and effects on nutrition

and health. J. Sci. Food Agric. 80: 1094-1117.

Siriwoharn T, Wrolstad RE (2004). Polyphenolic composition of marion

and evergreen blackberries. J. Agric. Food Chem. 69: 233-240.

Spencer P, Sivakumaran A, Fraser K, Foo LY, Lane GA, Edwards PJB,

Meagher LP (2007). Isolation and characterization of procyanidins

from

Rumex obtusifolius

. Phytochem. Anal. 18: 193-203.

Tanaka T, Orii Y, Nonaka GI, Nishioka I, Kouno I (1996).

Syzyginins

and B, two ellagitannins from

Syzygium aromaticum

. Phytochem. 43:

1345-1348.

Zhang and Lin 2309

Vivas N, Nonier MF, Gaulejac de NV, Absalon C, Bertrand A, Mirabel M

(2004). Differentiationof proanthocyanidin tannins from seeds, skins

and stems of grapes (

Vitis vinifera

) and heartwood of Quebracho

(

Schinopsis balansae

) by matrix-assisted laser desorption/ionization

time-of-flight

mass

spectrometry

and

thioacidolysis/liquid

chromatography/electrospray ionization mass spectrometry. Anal.

Chim. Acta. 513: 247-256.

Wada L, Ou B (2002). Antioxidant activity and phenolic content of

Oregon caneberries. J. Agric. Food Chem. 50: 3495-3500.

Wealth of India (1976). Raw Materials. New Delhi: CSIR, Vol X: 100-

104.

Xiang P, Lin YM, Lin P, Xiang C (2006). Effects of adduct ions on

matrix-assisted laser desorption/ionization time of flight mass spec-

trometry of condensed tannins: A prerequisite knowledge. Chinese J.

Anal. Chem. 34: 1019-1022.

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