ISSN-1996-918X

Pak. J. Anal. Environ. Chem. Vol. 17, No. 1 (2016) 43 – 49

doi: 10.21743/pjaec/2016.06.007

Methods for Extraction and Characterization of Tannins

from Some Acacia Species of Sudan
Isam Eldin Hussein Elgailani* and Christina Yacoub Ishak
Department of Chemistry, Faculty of Science, University of Khartoum, Khartoum, Sudan
*Corresponding Author Email: gailani23@hotmail.com
Received 29 September 2015, Revised 26 June 2016, Accepted 27 June 2016
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Abstract
The study is aimed to analyze and compare extraction methods of tannins from three common
Acacia species of Sudan. The Acacia species selected were Acacia nilotica, Acacia seyal and
Acacia senegal. Bark samples from bulk collections of the three Acacia species were extracted
with water, 80% methanol and 70% acetone. Two sets of extraction were made, one by boiling
and a second by shaking the samples in the respective solvents for eight hours at room
temperature. Although the amount of material extracted by these two procedures did not differ
greatly (P > 0.05), 70% acetone was a more efficient solvent than either water or 80% methanol.
The tannins of mature fruits extract of Acacia nilotica were identified by using Thin Layer
Chromatography (TLC), Ultraviolet and Infrared spectroscopy. Comparisons of absorption spectra
and TLC of the reference tannins and some phenolics with that of Acacia nilotica extracts
revealed the presence of both condensed and hydrolyzable tannins, since it consists of catechin,
tannic and gallic acids. Catechin considered to be the phenolic precursor of condensed tannins.
Hydrolysis of Acacia nilotica extract, tannic and gallic acids by butanolic-hydrochloric acid
yielded gallic acid which is considered to be a chemical precursor of hydrolyzable tannins.

Keywords: Tannins, Acacia species, Extraction, Characterization, Sudan

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Introduction

Tannins are polymeric phenolic compounds with Tannins complexes with sorghum proteins,
numerous hydroxyl groups and quite diverse in this complex is hard to digest by human and hence
chemical structure [1-2]. Hydrolysis of some of lower the protein value [8]. The tannin- protein
tannins yields the simple, seven-carbon gallic acid, precipitation behaviors confirmed complexity and
others give ellagic acid or other phenolic acids [3- differences in their nature and potentiality for
4]. Tannins are generally divided into the tanning or other uses [9]. Polyphenols and related
hydrolyzable and condensed tannins. Acacia structures are responsible for the antioxidant
species are found in different climate areas, there processes in the human body system [10]. Tannic
are even a few aquatic legumes [5]. In Sudan, acid with low levels affords protection against
Acacia species are widespread and are of polycyclic aromatic hydrocarbon-induced foresto-
medicinally and economically valuable [6]. Acacia mach and lung [11]. Tannins of Agrimonia
nilotica fruits have been used as one of the japonica has been used as antidiarrheic and a
traditional medicine and as an antimicrobial agent hemostatic in Japan and China [12].
in many countries around the world. Various parts
of the plants are selected especially roots, young Literature shows that tannins were
shoots and stem. The extract of Acacia nilotica extracted by different procedures and techniques.
leaves is play an important role in antibacterial In guava leaves, comparison for solvent extraction
processes [7]. for tannins by using ethanol and acetone which
resulted that ethanol 30% (v/v) is the best solvent
44 Pak. J. Anal. Environ. Chem. Vol. 17, No. 1 (2016)

[13]. Tannins also extracted from the bark of Pinus

oocarpa with sodium carbonate and sodium
bisulfite [14]. Tannins of Galium tunetanum were
extracted by two methods by ethanol 30% for 2
hours and the other by acetone for 24 hours [15].

The vegetable tannins may be classified

into two categories based on the nature of Leucocyanidin (III) Glucose core (IV)
combination to achieve the molecular size and
reactivity required. The first class is condensed
tannins which are not hydrolyzed in the presence
of acid, bases or appropriate enzymes, and the
second class of tannins is hydrolyzable tannins
which are hydrolyzed in the presence of acids,
bases or appropriate enzymes to give either gallic
acid or ellagic acid [3, 16-17]. Condensed tannins
contain only phenolic nuclei, most tannins of this
type are formed by the condensation of two or Gallic acid (V) Hexahydroxydiphenic acid (VI)
more of flavanols, such as catechin(I) and
epicatechin(II) and leucocyanidin(III), or can be
mixture of these [17-19].

Hydrolyzable tannins yield on hydrolysis

by acids, bases or appropriate enzymes glucose
core (IV) together with gallic acid (V) or its
congeners such as hexahydroxydiphenic acid (VI).
Also ellagic acid (VII) which is obtained by aerial Ellagic acid (VII)
oxidation of gallic acid [1, 16, 20]. Tannic acids
belong to the hydrolyzable group of tannins
[21- 22]. Material and Methods
Sampling
In this work, we compare the efficiency of
various solvents for extraction of tannins from bark Samples of bark of Acacia nilotica, Acacia
of the three common Acacia species of Sudan. The seyal and Acacia senegal from individual
effect of temperature and shaking on the extraction collections were used for the study extraction
efficiency is also compared. Thin Layer efficiency. Mature fruits of Acacia nilotica were
Chromatography, Infrared and Ultraviolet collected and used to identify the tannins. Bark
spectrometry were used for identification of was removed from wood before drying. Plant
compounds which are found in mature fruits Materials were taken from several trees in each
extract of Acacia nilotica and compared with the instance from the Sunt Industrial and Tourism
reference of tannins and related phenolics. Centre (Sunt Forest) at Khartoum and Debabat
Forest at South Kordofan State at West of Sudan .
Three individuals for each samples were used for
the analysis (n = 3).

Chemicals and reagents

The chemical materials used for the

Catechin (I) Epicatechin (II) analysis in this work were of high grade.
 Pak. J. Anal. Environ. Chem. Vol. 17, No. 1 (2016) 45

Extraction of bark samples acetic acid being used as developing solvent. The
extract was dissolved in acetone (100 ml) and
Air-dried bark samples (from bulk separated by TLC on cellulose, it gives three
collections) were ground in a Wiley mill (2 mm fractions when run with two dimensional TLC
screen). A portion (40 g) was extracted with water, (Fig. 1). Comparison of standards (Tables 2) and
another with 80% methanol, and a third with 70% the three isolated fractions gave the patterns shown
acetone (200 ml) by shaking at room temperature in Tables 3.
for 8 hours and another series by boiling for 10
minutes. The samples were filtered (Whatman 1 Degradation of tannins with alcoholic-
paper, 18.5 cm disc) and the residual material hydrochloric acid
rinsed with additional solvent (two portions each
of 50 ml). Extracts were transferred to a tared, In order to determine the composition of
round-bottomed flask and concentrated under tannins of Acacia nilotica whether it is
vacuum by rotary evaporator to form a thick hydrolyzable or not, the sample of mature fruits
extract. The sample extracts were then dried in a was hydrolyzed by alcoholic hydrochloric acid
vacuum oven at 60 0C until a solid material was according to the following procedure: 0.5 ml of
obtained. The amount of extract was determined by extract was heated for 2 hours at 95 0C with 5 ml
weight difference Table 1. 5% butanol-HCl [23], the product of acid
hydrolysis were characterized by TLC (Table 3).
Characterization of Acacia nilotica tannins
Detection Reagents
Samples of mature fruits from individual
After the development of the chromato-
collections of Acacia nilotica were used to
gram, tannins and related phenolics were detected
determine the tannins. Material was taken from
by iodine vapours. Tannins and related phenolics
several trees in each instance. Air-dried sample
appear as brown spot after exposure to iodine
was ground in a Wiley mill (2 mm screen). In
fumes in a closed tank.
order to determine the composition of tannins of
the sample, it was characterized by TLC, UV, and
Chemical tests of tannins extract
IR spectrophotometer and was compared with the
Test with ferric chloride solution
references (standards) of tannins and related
phenolics. To a 1 ml portion of the extract was taken
in a test tube and 5 drops of FeCl3 solution in
Identification of tannins by TLC methanol were added. A green to black precipitate
appears in the presence of tannins (Table 4).
Dried and powdered fruits of Acacia
nilotica (100 g) were shaken with water (500 ml) Test with gelatin solution
for 24 hours at 25 0C by using the mechanical
stirrer, the solution filtered through glass wool. 100 1 ml portion of the extract was taken in a
ml of extract were put into a 250 ml beaker, and test tube and added 1ml of gelatin (1%solution)
the pH adjusted to 6.2 by addition of ortho- and NaCl. The formation of a white precipitate will
potassium dihydrogen phosphate (10 ml) and show that tannins were exist [24] (Table 4).
sodium hydroxide solution (2N, approximately 5
ml) by using pH-meter before extraction with 50 Test with ferrous sulphate solution
ml of ethyl acetate for 10 times. Heat at 30 0C for
elimination of solvent, then a bright brown 1 ml portion of the extract was taken in a
amorphous powder will be obtained. test tube and 2 ml of 0.1% FeSO4 and 0.5% sodium
potassium tartrate were added. The appearance of
Thin-layer plates (size 20 cm length and violet colour indicates the presence of tannins.
20 cm width) and another (20 cm length 5 cm
width) were prepared with cellulose. 6% aqueous Chemical tests of tannins were applied for
the isolated fractions of Acacia nilotica mature
46 Pak. J. Anal. Environ. Chem. Vol. 17, No. 1 (2016)

fruits extract obtained from TLC for identification where x1 and x2 are the means, and t for t-value
of tannins (Table 4). ((at 95% confidence level, t-test table, P < 0.05).
The null hypothesis is accepted if the calculated
UV spectrophotometry of tannins and related value (left side) is less than the tabulated value
phenolics (right side) [25-26].
100 mg of the water extract, (dried) of Results and Discussions
mature fruits of Acacia nilotica were dissolved in
25 ml of methanol. Standards of tannins and Numerous studies have examined the
related phenolics were prepared by dissolving 10 solubility of tannins in solvents, but no solvent
mg in 25 ml of methanol. After that the solutions system has been found to be completely
were diluted with the same solvent (1:100), and the satisfactory. Solubility of tannins depend on many
spectrophotometric measurements were recorded factors including the structure of the tannins
as seen in (Table 5). On the other hand, the acid themselves. Aqueous acetone solutions are
hydrolyzed tannins was also determined generally most effective in removing both
spectrophotometrically as in (Table 6). condensed and hydrolyzable tannins. Pure solvents
were insufficient extraction media for the recovery
IR spectrophotometry of tannins and related of phenolics and particularly tannins.
phenolics
In this study, two sets of extractions were
About 1.0 g of the dried water extract of made, one by boiling and the other by shaking the
mature fruits of Acacia nilotica was dissolved in 5 samples in the respective solvents for 8 hours at
ml of methanol. Standards of tannins and related room temperature (Table 1). The solvents used for
phenolics were dissolved by the same manner. The extraction were distilled water, 80% methanol and
sample and standards were subjected to IR 70% acetone. Although the amount of material
measurements, (Table 7). On the hand, the sample extracted by these two procedures did not differ
was dissolved in acetone by the same manner as greatly (P > 0.05), 70% acetone was a more
previously described, (1.0 g in 5 ml), and subjected efficient solvent than either water or 80% methanol
to IR measurements and reveal the same IR spectra (Table 1) in terms of the weight of material
as in Table 7. extracted from a given weight of Acacia species
material and in the percentage of tannins in the
Statistical analysis phenolic materials extracted, and the percentage of
tannins exracted from the bark samples.
Each treatment was carried in replicates
and each sample was analyzed three time The Also in this work, the tannins of mature
results are expressed as mean (n = 3), by using fruits extract of Acacia nilotica were identified by
one-tail analysis of variance. Testing hypothesis TLC, UV and IR spectrometry. Comparison of the
for the comparison of the two procedures were absorption spectra and TLC chromatograms of the
carried out by using the following relations: reference tannins and some related phenolics with
( N 1  1)  S 2 ( N 2  1)
2 2
S that of Acacia nilotica extract revealed the
For S Pooled , S pooled  1
presence of both condensed and hydrolyzable
( N 1  1)  ( N 2  1)
tannins.
where S1 and S2 ar standard deviations, N1 and N2
are replicates (= 3 for each), and Spooled is pooled The results presented in our work, show
standard deviations. that when the extract was run with two
dimensional TLC, it give three fractions, (Fig. 1).
and for the comparison of the two procedures: Comparison of standards with the extract gave the
N1  N 2 pattern shown in Table 2 by TLC. The three
x1  x2  ts pooled fractions of the extract on TLC were scratched and
N1 N 2 taken separately, the composition of the pigment
 Pak. J. Anal. Environ. Chem. Vol. 17, No. 1 (2016) 47

fractions was investigated by TLC (Table 3). Some Table 4. Chemical Tests of the Isolated Fractions of Acacia nilotica
(Mature Fruits).
of the chemical tests were applied for each of the
three fractions in order to identify the nature or Property of Test Fraction (I) Fraction (II) Fraction (III)
property of the fractions, fraction I and III, 1. Ferric chloride negative test positive test positive test
(Table 4) did not show tannin properties. 2. Gelatin in the negative test positive test negative test
presence of sodium
chloride
Table 1. Total Extractives from Acacia Bark Samples with Water,
80% Methanol and 70% Acetone. 3. Ferrous sulphate in negative test positive test negative test
the presence of
sodium tartrate
Species Solvent Boiled % Unboiled %
(g) Extracted (g) Extracted
A. nilotia water 6.2 15.5 6.1 15.3
80% 10.2 25.5 10.0 25.0
MeOH
70% 10.5 26.3 11.2 28.0
acetone
Std. 0.2 0.1
dev. (S)
(Spooled) 0.1580
A. seyal water 7.7 19.3 8.1 20.3
80% 10.3 25.8 11.1 27.8
MeOH
70% 11.0 27.5 11.8 29.5
acetone
Std. 0.05 0.1 Figure 1. Diagram of The Two Dimensional (D1 & D2) TLC of
dev. (S) Acacia nilotica (Mature Fruits). Stationary phase: Cellulose.
Spooled 0.0791 Mobile phase: 6% Acetic acid. Detection reagent: Iodine vapours.
A. senegal water 3.4 8.5 3.7 9.3
The UV absorption spectra of the extract
80% 4.4 11.0 4.3 10.8
MeOH
and standards when using methanol as solvent,
70% 4.8 12.0 4.5 11.3 they have shown peak maxima at 280 nm,
acetone (Table 5), indicating the presence of catechin and
Std. 0.1 0.1 tannic acid. When the extract and the standards
dev. (S) were subjected to hydrolysis by alcoholic-HCl
(Spooled) 0.2270 acid, they showed maximum peaks at 272 nm,
* Values are the mean of three determinations (n=3), (std. dev. is the (Table 6) indicating the presence of gallic and
standard deviation),(P < 0.05). tannic acids. Catechin (Flavan-3-ols) is considered
to be a monomer of condensed tannins. Hydrolysis
Table 2. TLC of Some Standards of Tannins and Related
Phenolics. of Acacia nilotica extract, tannic and gallic acids
by butanolic-HCl acid yield gallic acid which is
Tannins and Related Retardation Factor (Rf) in 6% considered to be a chemical precursor of
Phenolics acetic acid as developing
system hydrolyzable tannins.
1. Gallic acid (standard) 0.47
Table 5. The UV Spectra of Standards of Tannins, Related
2. Tannic acid (standard) 0.47 Phenolics and the Sample Acacia nilotica (Mature Fruits) by
3. Catechin (standard) 0.47 Methanol as Solvent.
4. Catechol (standard) 0.73 Tannins and Related Phenolics Wavelength (nm)
Ethylenic Benzoic
5. m-hydroxybenzoic acid 0.71
Band Band
(standard)
(E2-band) (B-band)
Gallic acid (standard) 216 264
Table 3. TLC, "Cellulose", 6% Aqueous Acetic acid for the Three
Fractions Isolated from Acacia nilotica (Mature Fruits). Tannic acid (standard) 220 280
Catechin (standard) 208 280
Fration Retardation Factor (Rf) in 6% acetic acid as
Catechol (standard) 216 276
developing system
I 0.11 m-hydroxybenzoic acid(standard) 207 292
II 0.47
The sample of A. nilotica (Mature fruits) 216 280
III 0.71
48 Pak. J. Anal. Environ. Chem. Vol. 17, No. 1 (2016)

Table 6. The UV Spectra of Standards of Tannins, Related The IR absorption spectra of the extract,
Phenolics and the Sample Acacia nilotica (Mature Fruits) after
Hydrolysis by Butanolic-HCl. show the presence of hydroxyl group (OH-),
aromatic C-H stretch, carbonyl group C=O stretch,
Tannins and Related Phenolics Wavelength (nm) C=C ring stretch, C-O stretch and out-of-plane
Ethylenic Benzoic
Band Band
C-H bending when compared with standards,
(E2-band) (B-band) (Table 7).
Gallic acid (standard) 220 272
Tannic acid (standard) 220 272
Catechol (standard) - 277
Catechin (standard) - -
m-hydroxybenzoic acid(standard) - -
The sample of A. nilotica (Mature fruits) 220 272

Table 7. The IR Spectra of Standards of Tannins, Related Phenolics and the Sample Acacia nilotica (Mature Fruits).
Tannins and Related Groups (cm-1)
Phenolics
O-H stretch Aromatic C-H stretch C=O stretch C=C ring stretch C-O stretch Out-of-plane C-H bend
Gallic acid (standard) 3370 2950 1660 1660 1310 -
1525 1250
1415 1185
Tannic acid (standard) 3370 2750 1685 1660 1300 -
1515 1180
1435 -
Catechin (standard) 3370 2900 - 1595 1340 -
1500 1240
1435 1185
Catechol (standard) 3370 3040 - 1590 1355 730
1500 1235
1465 1185
m-hydroxy-benzoic acid 3370 2820 1670 1595 1300 750
(standard) 1500 1230
1455 1160
Observed Band of sample A. 3370 2920 1670 1595 1315 -
nilotica (Mature fruits) 1520 1190
1435 -
Literature Band (cm-1) 3200-3550 2880-3030 1650-1670 1500-1660 1080- 675-900
1300

Conclusions because of the importance and usefulness

of the tannins group and its applications in wide
This study has shown that the extraction range.
of three Acacia species using distilled water, 80%
methanol and 70% acetone has been Conflicts of interests
successfully made. The extraction was made
once by boiling and the other by shaking the The authors declare that there is no
samples in the respective solvent for 8 hours at conflict of interests regarding the publication of
room temperature. Results showed that the 70% this article.
acetone was the most efficient solvent among the
three solvent used. In addition, characterization Acknowledgments
study of Acacia nilotica extract revealed the
presence of some compounds of both condensed Special thanks to the Department of
and hydrolyzable tannins. Chemistry, University of Khartoum where this
evaluation and investigation have been carried out,
Recommendations that could be drawn from for laboratory facilities and valuable assistance in
this study are that further studies will be necessary the use of various equipments.
to identify the tannins in the other Acacia species
 Pak. J. Anal. Environ. Chem. Vol. 17, No. 1 (2016) 49

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