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Tannins of Tamarind Seed Husk: Preparation, Structural Characterization, and

Antioxidant Activities

Article  in  Natural Product Communications · June 2011

DOI: 10.1177/1934578X1100600619 · Source: PubMed

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 2011
NPC Natural Product Communications Vol. 6
No. 6
829-834
Tannins of Tamarind Seed Husk: Preparation, Structural
Characterization, and Antioxidant Activities
Puksiri Sinchaiyakita,*, Yohji Ezureb, Sarin Sripranga, Supakorn Pongbangphoc,
Nasapon Povichitb, and Maitree Suttajitc
a
Faculty of Science, Naresuan University, Pitsanuloke 6500,Thailand
b
Faculty of Pharmacy, Chiangmai University, Chiang Mai 50200, Thailand
c
Faculty of Medical Science, University of Phayao, Phayao 56000,Thailand

[email protected]

Received: November 8th, 2010; Accepted: January 31st, 2011

The high content (about 39%) of polymeric tannins in tamarind (Tamarindus indica L.) seed husk (TSH) was demonstrated, and an extract
(crude TSE) with a high content (about 94%) of polymeric tannins was prepared from TSH with a one pot extraction using ethanol/water
(3:2, v/v). The crude TSE was further purified with Sephadex LH20 to give one fraction (metTSE) eluted with methanol/water (3:2, v/v) and
another (acTSE) eluted with acetone/water (3:2, v/v). The tannins of acTSE were established as polymeric proanthocyanidins (PA) by 13C
NMR spectroscopy; this was further confirmed by IR and UV spectroscopy, n-BuOH/HCl and vanillin assays, and from HPLC pattern. The
ratio of procyanidins to prodelphinidins was 2:3, and the average degree of polymerization of acTSE was 7. Galloylated flavan-3-ols were
not detected in acTSE. The main ingredients of metTSE were confirmed to be polymeric PA by 13C NMR spectroscopy. The antioxidant
activities using DPPH and ABTS assays were investigated. The IC50 values of acTSE were 4.2±0.2 (DPPH assay) and 6.2±0.3g/mL (ABTS
assay).

Keywords: Tamarind seed husk, tannin, proanthocyanidin, antioxidant.

Tamarind (Tamarindus indica L.; family Fabaceae) is polymers from trimer to hexamer, were investigated in
widely distributed throughout the tropical and sub-tropical detail [5]. Recently, Povichit et al. [7] reported the high
regions from Africa to South Asia. The ripened fruit pulp content of polyphenolic compounds in TSH and their
is edible and a popular food. Tamarind seed (TS) is also of antioxidant activities, but did not specify the compounds.
important use. The polysaccharides, mainly consisting of Thus it seems that the polymers of polyphenolic
xyloglucans, prepared from tamarind seed kernel have compounds like polymeric tannins in TSH have not been
been used for cosmetics. Tamarind color extracted from fully investigated as yet.
the seed husk (TSH) has been commonly used as a food
pigment. The present study was designed to develop a simple and
efficient extraction method for manufacturing products of
Several studies on tamarind seed husk extract (TSE), its polymeric tannins with high contents of TSH and to study
preparation, the structural elucidation of its constituents, primarily the structural characterization and the
and antioxidant activities have been reported [1-7]. Only antioxidant activity of these tannins.
the reports of Tsuda et al. [1] and Sudjaroen et al. [5] deal
with the structural characterization of the compounds in Sephadex LH20 has been used for the purification of both
TSE. Tsuda et al. [1] used ethanol and ethyl acetate as condensed and hydrolysable tannins on a preparative scale.
extraction solvent and reported the isolation of simple The preparation and purification of polymeric tannins from
phenolic compounds such as (-)-epicatechin, 2-hydroxyl TSH were performed according to a slightly modified
3',4'-dihydroxyacetophenone, methyl 3,4- method of Peng et al. [8]. TSH was extracted with
dihydroxybenzoate, and 3,4-dihydroxyphenyl acetate. ethanol/water (3:2, v/v) at 60°C for 24 hours to give a
Sudjaroen et al. extracted tamarind seed and pericarp with crude extract of TSH. This was further purified using
methanol and acetone/methanol/water (70:29:0.5) and Sephadex LH20, which gave two fractions, metTSE eluted
showed that the content of polymeric tannins was with methanol/water (3:2, v/v) and acTSE, eluted with
remarkably high (77% of the total polyphenols). These, acetone/water (3:2, v/v).
however, were treated as unidentified and were not further
studied athough the other polyphenols, which were The samples were analyzed by HPLC according to the
procyanidin B2, epicatechin, procyanidin oligomer or method of Weber et al. [9]. The chromatogram of crude
830 Natural Product Communications Vol. 6 (6) 2011 Sinchaiyakit P et al.

R1

TSE showed the main peak around 60 min and a few peaks OH

of minor compounds, while that of acTSE showed only HO O

one peak. The peak area of crude TSE was about 94% that OH

of acTSE, suggesting a high content of acTSE in crude R2

R1
OH
TSE. The HPLC pattern of the main peak was similar to 6'
5'
4' OH

B n
that reported by Weber et al. [9], who considered this to be HO 7
8
8a O 2
1' 3'
OH

due to polymeric proanthocyanidins (PA), as reported 6

A
4a
C 2'
3 R2
5 R1
previously for grape seed extract (GSE) [8, 9]. This was OH
4

confirmed by 13C NMR and IR spectroscopy, and by other

OH

analytical methods. The n-butanol/HCl assay for acTSE HO O

OH

produced a strong red color, suggesting that acTSE OH

consisted of condensed tannins (polymeric PA). OH

R1 = H, procyanidin (PC)
R1 = OH, prodelphinidin (PD)
R2 = OH or galloyl
Grape seed (GS) is known as one of the most O

2"
representative natural resources for oligomeric and O
1" 3" OH

polymeric PA. A typical oligomeric PA is composed of 6"

5"
4"
OH

flavan-3-ol units linked together from C4 of one unit to OH

either C6 or C8 of the adjacent unit (B-type) and can Figure 1: General chemical structure of oligomeric and polymeric
coexist with an additional C2-O-C7 linkage (A-type) [10]. proanthocyanidins.
The further addition of flavan-3-ol units results in the
formation of polymeric PA (Figure 1). Therefore, acGSE, to the ratio of procyanidins to prodelphinidins (PC to PD),
polymeric PA from grape seed, was prepared according to the stereochemistry of the heterocyclic ring of the
the method described above (see experimental) for the monomer units, and the number-average molecular weight
purpose of comparing acTSE and acGSE. The HPLC of by 13C NMR spectroscopy, and investigated the polymeric
acGSE showed one peak, and the retention time was PA in different kinds of natural products [11]. Their
virtually identical with that of acTSE under the same method has been employed to study the chemical structure
analytical conditions. of polymeric PA of many natural resources, such as grape
skin [12], pear juice [13], and Lithocarpus glaber leaves
The calibration curve of acTSE in HPLC analysis was [14]. The 13C NMR spectra of the polymeric PA of these
obtained with high linearity (R2 >0.991): y = 1.37 × 107x natural resources showed similar patterns, which were
+ 3694501, where y = peak area and x = polymeric PA characteristic of polymeric PA.
(mg/mL). The calibration curve was used for the
determination of the content of polymeric PA of the Assignment of the 13C NMR spectra of acTSE and acGSE
samples. The calibration curve of acGSE was also was made based on the publications of Czochanska et al.
obtained: y =2.40 × 107 x – 2363707. [11] and Kennedy et al. [12, 15]. The results are
summarized in Table 1. The spectra of the polymeric PA
Czochanska et al. [11] reported that polymeric PA of acTSE and acGSE were in good agreement with those
consisting of procyanidin (PC) alone showed more reported in the past, representing all the necessary signals
intensive absorbance at 270-280 nm than the polymeric PA for polymeric tannins consisting of flavan-3-ols
consisting of prodelphinidins (PD) alone. The slope value (condensed tannins). Therefore, the polymeric tannins of
of the calibration curve of acTSE was smaller than that of acTSH were confirmed as condensed tannins.
acGSE, which suggested a lower content of procyanidin
(PC) chromophore in acTSE than that in acGSE. This was The detailed chemical structure of acTSE was further
also confirmed by 13C NMR spectroscopy. characterized by 13C NMR spectroscopy according to the
method of Czochanska et al. [11]. (1) The ratio of PC and
PD: The signals near 145 ppm are typical of the presence
Both acTSE and acGSE showed a UV max at 280 nm in
methanol, although that of acGSE was more intense than of PD units, and the ratio of PC to PD was estimated by
that of acTSE at the same concentration, which was the relative ratio of the peak areas at 144 ppm (C-3' and C-
4' of PC) and 145 ppm (C-3' and C-5' of PD). The PC/PD
attributed to the difference in PD/PC of acTSE and acGSE.
ratios were calculated using the expanded chart of 13C
The IR spectra of acTSE and acGSE were similar, though
NMR spectra (data not shown). The ratio of PC to PD of
that of acGSE showed a shoulder peak around 1700 cm-1,
suggesting the C=O stretch of galloyl ester (data not the polymeric PA of acTSE was estimated to be 2:3. In the
shown). case of the polymeric PA of acGSE, the estimation of PC
to PD was not exactly accurate because C-3" and C-5" of
the galloyl group overlapped the signals at 143-144 ppm
Further structural characterization of the polymeric tannins
[15], but the approximate value of PC/PD was calculated
in TSH was performed by comparing and analyzing the
13
C NMR spectra of acTSE and acGSE. Czochanska et al. to be 4:1. Accordingly, the higher content of PD units of
reported the elucidation of the chemical structure relating the polymeric PA of acTSE than that of acGSE was
confirmed. (2) Galloyl group: As for acGSE, the signals
Tannins of Tamarind Seed Husk Natural Product Communications Vol. 6 (6) 2011 831

Table 1: 13C NMR chemical shifts of polymeric proanthocyanidins. one pot extraction was calculated to be 92.6% by taking
Position Polymeric PA of acTSE ( ppm) three times repeated extraction as total polymeric PA.
Flavan-3-ol unit
C-2 77.1 After purification of crude TSE with Sephadex LH20, its
C-3(terminal unit) 67.0
C-3(extension unit) 71.8
composition was found to be 25.2±0.9% (w/w) of metTSE
C-4 36.5 and 73.9±1.5% (w/w) of acTSE from three different
C-4a 102.0 experiments. On the other hand, the content of polymeric
C-5 153.9-159.0 PA in crude TSE was about 88-95% (Table 2). These
C-6 96.7
C-7 153.9-159.0 results suggested that metTSE should contain a high
C-8 106.9 amount of polymeric PA. The content of polymeric PA in
C-8a 153.9-159 metTSE was calculated by HPLC analysis using acTSE as
C-1' 131.9
C-2' 114.5
a standard and found to be about 79% (w/w). The 13C
C-3' of PCa 144.6 NMR spectrum of metTSE was very similar to that of
C-3' of PDb 145.6 acTSE, showing all the carbons necessary for polymeric
C-4' of PC 144.6 PA. Therefore, the main ingredient of metTSE was
C-4' of PD 131.9
C-5' of PCc 116.2 confirmed to be condensed tannin similar to acTSE.
C-5' of PDd 145.6
C-6' 118.7 In summary, TSH was confirmed to contain a very large
a
PC = procyanidins, bPD = prodelphinidins, cnon-substituted aromatic amount of polymeric PA (about 39%, w/w). Therefore,
carbon, dsubstituted aromatic carbon with OH.
TSH is one of the best natural resources for polymeric PA
for the galloyl group were confirmed at 110 ppm (C-2" and production. Crude TSE with a very high content (about
94%, w/w) was simply and efficiently prepared by a one
C-6" of galloyl group) [12], and 138 ppm (C-4" of galloyl
pot extraction using aqueous ethanol. This method will be
group) [9]. These signals were not observed in the
very useful for manufacturing the polymeric PA products.
spectrum of acTSE, showing that the galloyl group was not
The polymeric tannins of TSH were confirmed as
present. (3) Degree of polymerization (DP): The DP of
acTSE was estimated to be about 7 from the ratio of polymeric PA (condensed tannin), which did not contain
galloylated derivatives with a higher content of PD in the
extension C-3 to terminal C-3. (4) The stereochemistry of
B ring than GSE. The average DP of acTSE was shown.
the heterocyclic ring of extension units: According to
However, the detailed chemical structure of the polymeric
Czochanska et al. [11], the 13C chemical shifts of C-2 (cis-
tannins from TSH should be elucidated by further studies
2,3) and C-2 (trans-2,3) should be 77 ppm and 84 ppm,
by gel permeation chromatography and phloroglucinol or
respectively. In our experiment, the trans-2,3 peak was not
observed in either acTSE or acGSE. Therefore, the thiolysis reaction to confirm the constituent units and
interflavonoid linkage. The potent antioxidant activity of
stereostructure (flavan C-2 and C-3 positions of extension
the polymeric PA of TSE was also shown.
units) of acTSE was considered to be all in the cis-2,3
form. Experimental
Chemicals: All chromatographic solvents were HPLC
The DP of acTSE by vanillin assay was 7.1±0.3, which is
grade (E. Merck, Germany, Darmstadt, Germany). (+)-
in good agreement with the value estimated by 13C NMR
Catechin, vanillin, potassium persulfate, 1, 1-diphenyl-2-
spectroscopy.
picryl hydrazyl (DPPH) and 2,2'-azino-bis(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS) were
The antioxidant activity of acTSE was measured by DPPH
purchased from Sigma-Aldrich (St. Louis, MO), and
and ABTS assays, both of which have been used as
Sephadex LH20 gel from Pharmacia Biotech AB (Uppsala,
standard methods for the estimation of the antioxidant
Sweden).
activity of foods and plants [16, 17]. The IC50 values of
acTSE were 4.2±0.2g/mL (DPPH assay) and 6.2±0.3
Preparation of crude TSE: The seeds of Tamarindus
g/mL (ABTS assay). indica Linn. were purchased in Maha Sarakham Province,
Thailand. To separate TSH easily from TS, TS (100 g) was
A one pot extraction from TSH was carried out and heated in a hot air oven at 100оC for 60 min, and then TSH
compared with the three times repeated extraction. TS was (28 g) was obtained by careful separation. The brown-red
heated to separate TSH easily, then the partly dried TSH seed husk was collected and ground into fine powder, part
was obtained and used for the extraction. The content of of which (10 g) was extracted with 150 mL of
polymeric PA in crude TSE was analyzed by HPLC and ethanol/water (3:2, v/v) at 60оC for 24 h. The insoluble
determined using acTSE as a standard. The results are materials were removed by filtration. Crude TSE (4.5 g)
summarized in Table 2. The content of polymeric PA in was obtained by lyophilizing the extracted solution after
fresh TSH reached about 39% (w/w), whereas in crude removing ethanol under reduced pressure at 60-70°C.
TSE prepared by one pot extraction with ethanol/water Extraction three times in the same manner was carried out
(3:2, v/v) at 60оC for 24 hours it was very high (about to compare with one pot extraction.
94%, w/w). The recovery of polymeric PA from TSH with
832 Natural Product Communications Vol. 6 (6) 2011 Sinchaiyakit P et al.

Table 2: Yield and content of polymeric PA in TSH and crude TSE.

Samples Number of extractions Yield a (%) Water content (%)b Content of polymeric PA (%)
Partly dried materialc Fresh material Extractd Fresh materiale
f
Crude TSE 1 43.5±2.0 - - 94.1±0.4 -
3g 47.0±1.8 - - 87.7±1.5 -
TSH - - 14.6±0.3 18.9±0.3 - 39.1
a
Percentage of the extract from the partly dried material (TSH ) using ethanol/water (3:2 v/v) at 60 °C for 24 h. bdry weight loss (dried to a constant weight at 110°C),
c
partly dried material (see the text for drying conditions), dcontent of polymeric PA in the extract, eestimated content of polymeric PA in the fresh TSH, fone pot
extraction, gthree times repeated extraction.

Preparation of crude GSE: Vitis vinifera cv. Ribier (Pok min. The column temperature was ambient, with an
Dum) was purchased at a market in Chiang Mai, Thailand injection volume of 20 L.
and stored at -40оC until used. The mature berries (1,050
g) were washed with tap water, pressed with a hand Calibration curves of polymeric PA: The HPLC analysis
squeezer, and the seeds and skins manually separated. The of acTSE and acGSE at concentrations of 1, 2, 3, 4 and 5
intact whole grape seeds (GS) (37.8 g) were dried at 75оC mg/mL dissolved in methanol/water (1:1, v/v) was
for 18 h in a hot air oven. The dried GS (23 g) were ground repeated 3 times at each concentration under the conditions
into fine powder and 19 g was extracted with ethanol/water described in quantitative HPLC analysis to give a 5 point
(3:2, v/v) at 60оC for 24 h at an extraction ratio of 15. The calibration curve. Calibration curves with high linearity
extraction was repeated 3 times in the same manner. The were obtained for acTSE and acGSE by plotting the
insoluble materials were removed by filtration. Crude GSE concentrations as a function of peak areas from HPLC
(2.6 g) was obtained by lyophilizing the extracted solution analysis of a 20 L injection volume. The samples were
after removing the ethanol under reduced pressure at 60- identified by comparing the retention time with those of
70оC. standards (acTSE and acGSE), and the concentrations of
the samples were determined from the calibration curve.
Purification of acTSE and acGSE with Sephadex LH20:
13
The crude TSE and crude GSE were further purified with C NMR spectroscopy: 13C NMR spectroscopy was
Sephadex LH20 by a method similar to that reported by performed according to a slightly modified method of
Peng et al. [8]. For crude TSE, Sephadex LH20 gel, pre- Kennedy et al. [12]. The samples (100 mg/mL, 1:2
swollen in methanol/water (3:2, v/v), was slurry-packed acetone-d6/D2O) were characterized by 13C NMR (100
into a glass column (2 cm i.d. x 14 cm), which was MHz, Bruker AVANCETM spectrometer), with chemical
equilibrated with methanol/water (3:2, v/v) prior to sample shifts in ppm referenced internally with acetone-d6. The
loading. The crude TSE (200 mg) was loaded onto the proton-decoupled, inverse-gated sequence, with 90о pulse
column, and metTSE was eluted with 5 column volumes of length (14s), 25125 Hz spectral width, 10K data points,
methanol/water (3:2, v/v) at a flow rate of 1.2 mL/min. 0.65 s acquisition time, relaxation delay of 4 s, 12 K scans,
The eluate was evaporated to dryness to give a powder and 5 Hz line broadening was carried out at a temperature
(metTSE, 49.8 mg). Polymeric PA was eluted with of 295.8 K.
acetone/water (3:2, v/v). The aqueous acetone fraction was
concentrated under reduced pressure at 60оC to remove UV and IR spectrometry: For UV spectrometry, the
acetone, and then lyophilized to give a powder (acTSE, samples were dissolved in methanol at concentrations of
148.2 mg). The polymeric PA from crude GSE was 20, 40, 60, 80, and 100g/mL for acTSE, and 10, 20, 30,
purified with SephadexLH20 in the same manner. The 40, and 50g/mL for acGSE. UV spectra ware recorded
crude GSE (200 mg) gave acGSE (73.6 mg) and metGSE on an UV-1700 spectrophotometer (Pharma Spec,
(123.7 mg). The acTSE and acGSE were used as standards Shimadzu, Japan). FT-IR spectra were recorded on a
for the determination of polymeric PA by HPLC/UV. Spectrum GX spectrophotometer (Perkin Elmer, USA;
KBr tablet method).
HPLC/UV analysis: The samples of TSE and GSE were
analyzed by reverse-phase HPLC according to the method Vanillin assay: Vanillin assay was carried out according to
of Weber et al. [9] using a Shimadzu SCL-10AVP system a slightly modified method of Butler et al. [18] using 70%
consisting of a LC-10ADvp pump, a SIL-10ADvp sulfuric acid as a solvent. Briefly, a 100 L aliquot of a
autosampler, and SPD-10AV detector. The column used freshly prepared solution of vanillin (1 g/100 mL) in 70%
was an Allima C18 5U column (250 mm x 4.6 mm I.D., 5
sulfuric acid was added to 50 L of acTSE dissolved in
m particle size, Altima, Mandel Scientific Company, 95% aqueous ethanol. After 15 min, the absorbance at 500
Canada). The mobile phase solvents A and B were 0.3% nm was measured. Measurements were carried out at
trifluoroacetic acid and acetonitrile. The linear gradient
concentrations of 0.5, 1.5, 2.5, 3.5 and 5 g/mL for (+)-
elution used was as follows: 10-15% B over 45 min; 15-
catechin, and 2.5, 7.5, 12.5, 17.5, and 25 g/mL for
60% B over 15 min, hold for 20 min; 60 to 10% B over 1
acTSE, and then calibration curves with high linearity
min; column equilibration at 10% B for 20 min. The flow
were obtained. DP was calculated from the slopes of (+)-
rate was 0.7 mL/min, and the data were collected for 80
catechin and the samples.
Tannins of Tamarind Seed Husk Natural Product Communications Vol. 6 (6) 2011 833

n-Butanol/HCl assay: n-Butanol/HCl assay was carried Briefly, 167 M DPPH in 95% ethanol solution (180 L)
out according to a slightly modified method of Vermerris was mixed with 20 L of the sample solution dissolved in
[19]. In brief, to 0.1 mL of samples was added 1 mL of an 95% ethanol. The mixture was incubated at 37оC for 15
acidic solution of ferrous sulfate [7.7 mg of FeSO4.7H2O min, and then the absorbance was measured at 540 nm
dissolved in 50 mL of conc. HCl- n-butanol (2:3, v/v)]. using a Multimode Detector (Model DTX 880, USA). The
The mixtures, loosely sealed in test tubes, were placed in a negative control without the sample was measured in the
water bath 95C for 15 min. same manner. All measurements were performed in
triplicate. The percentage of DPPH radical scavenging
ABTS assay: ABTS persulfate decolorization assay was activity of the sample was determined at 5 sample
carried out [20]. ABTS was dissolved in water to a 7 mM concentrations within the range of 10-90% reduction in
concentration. For producing ABTS radicals, the ABTS absorbance. The IC50 was estimated from the inhibition
solution was mixed to 2.45 mM potassium persulfate (final percent of DPPH radical-scavenging activity calculated by
concentration) at a ratio of ABTS/potassium persulfate of the following equation:
1: 0.5, M/M and allowing the mixture to stand in the dark Inhibition percent of DPPH radical-scavenging activity
at room temperature for 24 h before use. The ABTS = [(absorbance of control – absorbance of sample)/
solution was diluted with water to an absorbance of 0.7 ± absorbance of control] ×100, where 150.3 M DPPH
0.02 at 734 nm. To the diluted ABTS solutions (200 L), solution in 95% ethanol was used as a control.
which were put into 24 well-plates, were added the sample
solutions (2L) dissolved in 95% ethanol, and then the Acknowledgments - The authors wish to express their
absorbance at 734 nm was measured after 10 min grateful thanks to the Royal Golden Jubilee scholarship
incubation at room temperature. under the Thailand Research Fund for their financial
support throughout the Ph.D. program study of Miss
DPPH assay: The DPPH assay was carried out according Puksiri Sinchaiyakit. Our appreciation is conveyed to
to the method of Liu et al. [21], with a modification. Chiangmai and Naresuan University for their support.

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