Acta Universitatis Cibiniensis

Seria F Chemia 8(2005-2):21-32

21










Method for Quantitative Determination of Polyphenolic
Compounds and Tannins from Vegetal Products

I. Palici
1
, B. Tia
1*
, L. Urica
1
and D. Tia
1

Abstract

The presence of polyphenolic compounds, tannoids and nontannoids, in vegetal
products affects their therapeutic properties.
In order to perform their quantitative determination, a method has been conceived
which includes a series of chemical reactions, followed by a phase of spectrophotometric
determinations and the processing of experimental data using a mathematical algorithm.
The determination of total polyphenolic has been made using solutions of
phosphowolframic acid and sodium carbonate as reactants.
The percent of tannins is calculated after a preliminary quantitative determination
of non-tannoids polyphenols, being equal to the difference between total polyphenols and
non-tannoid polyphenols. The content in tannins has been expressed as tannic acid and
pyrogallol [1, 2].
This work presents the analytical method and results obtained in analyzing 4
vegetal products. However, the method has been applied successfully in numerous other
cases.

Keywords: polyphenolic compound, tannin, vegetal product.


1
Faculty of Pharmacy ,Victor Babe University of Medicine and Pharmacy, P-a .E Murgu No.2,
300041, Timisoara, Romania
*
To whom the correspondence should be addressed.

I. Palici, B. Tia, L. Urica and D. Tia
22
I. Introduction

Tannins are present in vegetal products as complex mixtures in which predominate
esters of some polyphenolic acids with glucids, in the case of hydrolysable tannins, or
products of cathechin condensation, able to give steady, characteristic bounds, with the
aminoacids from proteins structure [1].
In plants, macromolecular tannins are accumulated in ligneous tissues, barks and
roots. In vegetal organs with intense physiological activity, tannins are found as smaller
molecules. Tannins are present either dissolved in vacuolar sap of some parenchymatic
cells as cortical parenchyma, secondary xylem and pith, or in specialised cells like
idioblastes. Their biosynthesis is produced through the shikimic acid pathway [3].
Numerous vegetal products are appreciated for their antiseptic, hemostatic,
antidiarrhoeic, antihemorroidal properties. Some tannins manifest P vitamin-like effects,
regulating the resistance and the permeability of capillary vessels. For mint species it is
estimated that tannins are responsible for antiseptic and antidiarrhoeic effects while non-
tannoid polyphenols are for the antispasmodic one [4]. Some polyphenolcarboxilic
compounds have a role in the inhibition the biosynthesis of B
4
leucotrien (LTB
4
), a fact
which offers a serious reason for their utilisation in the treatment of allergic inflamations
and asthma [5].
The determination of tannins from vegetal products is of special importance, in
view their contribution to desired therapeutic actions. Numerous methods for the
determination of tannins are known. The most important are the following [3]:
a gravimetric method based on the precipitation of tannins with copper acetate
colorimetric method Ionescu Popescu which consists of the treatment of a
vegetal extract with a solution of tungsto-phosphoric acid followed by the
discoloration of the resultant solution with potassium ferricyanide in a quantity to
the tannin entered in the reaction
skin powder method, based on a preliminary determination of the polyphenolic
total, followed by adsorption of tannins on skin powder. Subsequently, the
polyphenols remaining in solution are quantitatively determined. Tannins are
determined by difference.
The method proposed in this work represents a modified variant of skin powder
method and consists of an indirect determination of tannins. In the first stage the
polyphenolic total is determined. In the second stage, after the adsorption of tannins on
standard skin powder, the nontannic polyphenols are determined in the filtered solution.

II. Experimental

Quantitative determination of tannins has been made using a spectrophotometric
method. The spectrophotometer used was a JENWAY 6100. As reagents, tannic acid,
tungstophosphoric acid, pyrogallol and natrium carbonate have been used. Since tannins
are present in the vegetal products as complex mixtures of chemical structures we have
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Seria F Chemia 8(2005-2):21-32
23
used as reference two substances with tannoid properties, namely tannic acid and
pyrogallol. These were used consecutively to construct the standard curves.
In order to draw the standard straight line, ten solutions of tannic acid have been
prepared with concentrations within the interval 0.001 % and 0.01 %, in steps of 0.001 %.
0.2 g of tannic acid have been weighed and completed with water to 20 g. A solution of
0.01 % tannic acid in water has resulted, which has been diluted according to Table 1.

Table 1. Table of dilution for obtaining the standard concentrations of tannic acid.
Solution number
Volume of initial
solution of tannic acid
0,01 %
[ml]
Volume of water used
for dilution
[ml]
Concentration (m/m) in
tannic acid of resulted
solution [%]
0 standard 0.00 4.00 0.000
1 0.40 3.60 0.001
2 0.80 3.20 0.002
3 1.20 2.80 0.003
4 1.60 2.40 0.004
5 2.00 2.00 0.005
6 2.40 1.60 0.006
7 2.80 1.20 0.007
8 3.20 0.80 0.008
9 3.60 0.40 0.009
10 4.00 0.00 0.010

Solutions with concentrations between 0.001 and 0.01 percent, at intervals of 0.001 have
been obtained and worked out as follows. 2 ml from each solution have been treated with
1 ml tungstophosphoric acid and 17 ml of natrium carbonate 50 % solution. After 120
seconds the absorbance at wavelength of 750 nm has been read. The absorbance has been
determined using water as the standard. Values are presented in Table 2.
A statistical evaluation of the values of absorbance was done, in order to eliminate
values considered statistically unacceptable and also to identify and remove the terminal
segments of the curve, which do not correspond to the criteria of application for Lambert
Beers law. Hence, the angular coefficients for each segment of the curve have been
calculated. Standard deviation for the row of values obtained has been determined and the
elimination criterium for each value has been verified.
I. Palici, B. Tia, L. Urica and D. Tia
24
Table 2. Values of absorbance for standard straight line, used for the determination
of polyphenolic compounds expressed in tannic acid.
Current number Concentration in tannic acid [%] Absorbance
0 standard 0.000 0.000
1 0.001 0.017
2 0.002 0.027
3 0.003 0.035
4 0.004 0.046
5 0.005 0.061
6 0.006 0.070
7 0.007 0.082
8 0.008 0.099
9 0.009 0.103
10 0.010 0.105

Individual values for each angular coefficient are calculated as follows:
1
1

=
i i
i i
i
A A
C C
X
where C
i
and A
i
represent individual values for concentration and absorbance.
They are presented in Table 3.

Table 3. Individual values of angular coefficients for the standard curve, used for the
determination of polyphenolic compounds expressed in tannic acid.
Current number
1
1

=
i i
i i
i
A A
C C
X
1 0,0588
2 0,100
3 0,125
4 0,0909
5 0,0667
6 0,111
7 0,0833
8 0,0588
9 0,250
10 0,500

Acta Universitatis Cibiniensis
Seria F Chemia 8(2005-2):21-32
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The formula for standard deviation is the following:

1
) (
1
2

n
x x
s
n
i


where s = standard deviation, x
i
= individual values for angular coefficients,
i
x =
average of angular coefficients and n = number of elements in the studied row [6].

After the application of the elimination criteria, the last two values of the angular
coefficient have been removed because (d
i
= 0.355) > (s
*
x/s = 0.268), respectively (d
i
=
0.145) > (s
*
x/s = 0.113).
Therefore the standard straight line has the shape presented in Fig. 1.
y = 11,54x
0
0,01
0,02
0,03
0,04
0,05
0,06
0,07
0,08
0,09
0 0,002 0,004 0,006 0,008
Concentration in polyphenolic compounds [%]
A
b
s
o
r
b
a
n
c
e

Fig. 1. Standard straight line for quantitative determination of the polyphenolic
compounds expressed in tannic acid.

For the preparation of the analytical solution, 0.5 g of finely pulverized vegetal
product has been put in a balloon flask of 250 ml, together with 150 ml water and boiled
for 30 minutes under an ascending condenser. The mixture was then cooled under a water
stream, diluted with water to 250 ml and filtered through a filter paper. 5 ml of filtrate has
been diluted with water to 25 ml. 2 ml of the resultant solution has been treated with 1 ml
tungsto-phosphoric acid (R) and 17 ml of natrium carbonate 50 % solution. After 120
seconds, the absorbance has been read at wavelength 750 nm, using water as a standard
[7].
In this case, the quantitative determination of the polyphenolic compounds in dry
vegetal products has taken into account the following:
I. Palici, B. Tia, L. Urica and D. Tia
26
5 ml filtered solution corresponds to (0.5/250) * 5 = 0.01 g dry vegetal product
after completion with water of 5 ml filtered solution to 25 ml, 1 ml of assay
solution corresponds to 0.01/25 = 0.0004 g dry vegetal product, having a
concentration of 0.04 %
after the quantitative determination of the polyphenolic compounds in assay
solutions, by extrapolation from the regression straight line, the content of the
polyphenolic compounds in dry vegetal product, expressed in tannic acid, has been
calculated applying the following formula:
0.04 * 11.54
abs * 100
% C
1
TT
=


where
C
TT
% is the total polyphenolic percent in the dry vegetal product,
expressed in tannic acid, and
abs
1
is the absorbance indicated by spectrophotometer for the quantitative
determination of the total polyphenolic compounds.

According to the previous formula, the function of absorbance obtained for the
solution corresponding to each vegetal product, the percent of polyphenolic compounds,
expressed in tannic acid, in dry the vegetal product has been calculated [8, 9].
The content of polyphenolic compounds can be expressed also in pyrogallol. In
order to perform this operation, a standard curve has been drawn by determining the
absorbances for a series of standard solutions of pyrogallol, with concentration between
0.001 % to 0.01 % in steps of 0.001 %. These solutions have been obtained after the
dilution with water of a solution of 0.01 % pyrogallol according to Table 4.

Table 4. Table of dilution for obtaining standard concentrations of pyrogallol.
Solution number
Volume of initial
solution of pyrogallol
0,01 %
[ml]
Volume of water used
for dilution
[ml]
Concentration (m/m) in
pyrogallol of resulted
solution [%]
0 standard 0.00 4.00 0.000
1 0.40 3.60 0.001
2 0.80 3.20 0.002
3 1.20 2.80 0.003
4 1.60 2.40 0.004
5 2.00 2.00 0.005
6 2.40 1.60 0.006
7 2.80 1.20 0.007
8 3.20 0.80 0.008
9 3.60 0.40 0.009
10 4.00 0.00 0.010
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The values of the absorbance read for the series of standard solutions are presented
in Table 5.

Table 5. Values of absorbance for standard straight line used at the determination of
polyphenoliccompounds expressed in pyrogallol.
Current number Concentration [%] Absorbance
1 0.001 0.009
2 0.002 0.019
3 0.003 0.028
4 0.004 0.039
5 0.005 0.049
6 0.006 0.061
7 0.007 0.069
8 0.008 0.080
9 0.009 0.087
10 0.010 0.091

A statistic evaluation has been performed in order to eliminate values considered
statistically unacceptable, and also to identify and remove the terminal segments of the
curve which do not correspond to the criteria of application for Lambert Beers law. The
individual values for each angular coefficient have been determined. They are presented in
Table 6.

Table 6. Individual values of angular coefficients for the standard curve used for the
determination of polyphenolic compounds expressed in pyrogallol.
Current number
1
1

=
i i
i i
i
A A
C C
X
1 0,111
2 0,100
3 0,111
4 0,0909
5 0,100
6 0,0833
7 0,125
8 0,0909
9 0,143
10 0,250
I. Palici, B. Tia, L. Urica and D. Tia
28

After the application of the elimination criteria, the last two values of the angular
coefficient must be removed because (d
i
= 0.130) > (s
*
x/s = 0.0957), respectively (d
i
=
0.0367) > (s
*
x/s = 0.0360).
Therefore the standard straight line has the shape presented in Fig. 2.
y = 10,19x
0
0,01
0,02
0,03
0,04
0,05
0,06
0,07
0,08
0 0,002 0,004 0,006 0,008
Concentration in polyphenolic compounds [%]
A
b
s
o
r
b
a
n
c
e

Fig. 2. Standard straight line for quantitative determination of polyphenolic compounds
expressed in pyrogallol.

Applying the previous reasoning, the concentration of the polyphenolic
compounds, expressed in pyrogallol, has been calculated using the formula:

0.04 * 10.19
abs * 100
% C
1
TP
=

where

C
TP
% is the total polyphenolic percent in the dry vegetal product, expressed in
pyrogallol, and
abs
1
is the absorbance indicated by spectrophotometer for the quantitative
determination of the total polyphenolic compounds.

According to the previous formula, depending on the absorbance obtained for the
solution corresponding to each vegetal product, the percent of the polyphenolic
compounds, expressed in pyrogallol, has been calculated for the dry vegetal product [8, 9].
The concentration in tannins has been established, after a preliminary quantitative
determination of the nontannic polyphenols, according to the following algorithm.
10 ml of assay solution filtered previously has been stirred together with standard
skin powder for 60 minutes. Subsequently the mixture has been filtered. In this case, only
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29
nontannic compounds remained in solution, while tannins have been absorbed on the skin
powder. 5 ml of filtrate has been diluted with water to 25 ml. 2 ml of resulted solution has
been treated with 1 ml tungstophosphoric acid (R) and 17 ml of natrium carbonate 50 %
solution. After 120 seconds the absorbance has been read at wavelength 750 nm using
water as a standard.
Having in view the standard straight lines, presented in figures 1 and 2, in this
stage, the two concentrations of nontannic polyphenols have been determined, expressed in
tannic acid respectively in pyrogallol, using the following formulas:

0.04 * 11.54
abs * 100
% C
2
NT
=


where
C
NT
% is the total polyphenolic percent in dry vegetal product, expressed in tannic
acid, and
abs
2
is the absorbance indicated by spectrophotometer for the quantitative
determination of the nontannic polyphenols

respective
0.04 * 10.19
abs * 100
% C
2
NP
=


where
C
NP
% is the total polyphenolic percent in dry vegetal product, expressed in
pyrogallol, and
abs
2
is the absorbance indicated by spectrophotometer for the quantitative
determination of the nontannic polyphenols.
Finally, the percentage of tannins in the vegetal products has been calculated by
difference.
Percentage of tannins in dry vegetal product, expressed in tannic acid is:

C
T
%= C
TT
%- C
NT
%

Percent of tannins in dry vegetal product, expressed in pyrogallol is:

C
P
%= C
TP
%- C
NP
%



I. Palici, B. Tia, L. Urica and D. Tia
30
III. Results and discussions

The percentage of total polyphenols, nontannic polyphenols and tannins in the
vegetal products Herba, from species Ajuga reptans (bugleweed) and Mentha pulegium
(pennyroyal) and Folium from species Mentha x piperita ( peppermint) and Mentha
longifolia ( horse mint) has been determined [10]. The results are presented in Table 7.
The values obtained for the quantitative composition of the polyphenolic
compounds in the analysed products are close to those mentioned by other authors for the
species from Lamiaceae. In this work, the quantitative determinations have shown a
content in tannins from 4.85 to 11.70 percent, while in other scientific works the percent
values of the tannins are between 7 %, in the vegetal product Herba from Ajuga reptans,
and 20 % for the same product from species Glechoma hederacea. For the vegetal product
Folium from Mentha x piperita, a percent of 12 % tannins is mentioned in the literature. It
is important to mention that this variant of the skin powder method is specific for tannins
with a relatively high degree of condensation, while the smaller molecules like chlorogenic
and rosmarinic acid present a reduced affinity to the proteins from skin powder. The
structures named generically tannins are chemically very heterogenous. Depending on
their chemism, on the method of analysis, and on other factors like harvesting period,
location, climatic conditions, there is a possibility that the value for the content of the
reported tannin might be different from one scientific work to another. In this work, the
values obtained are close to those reported by other authors [2, 11, 12, 13].
Acta Universitatis Cibiniensis
Seria F Chemia 8(2005-2):21-32
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Table 7. Percentage of total polyphenols, nontannic polyphenols and tannins of the vegetal products Herba, from
species Ajuga reptans and Mentha pulegium, and Folium, from species Mentha x piperita and Mentha longifolia
I. Palici, B. Tia, L. Urica and D. Tia
32
IV. Conclusions

The method proposed for investigation proved to be facile and reliable.
Determination of tannins requires a minimal set of materials and devices. The matemathic
model used for the interpretation of the results is a verified one, very efficient and easy to
apply. Based on the above mentioned facts and taking into account the concordance of
obtained results with those reported in the scientific literature, the conclusion is that this
work represents a step forward in the laborious activity involved in the elucidation of the
chemical composition of vegetal products.

V. References

1. Popescu, H., Grecu, L., Cioac, C., Roca, M., Analiza chimic a produselor naturale
medicinale, Lito IMF Cluj-Napoca, 1981.
2. Palici, I., Cercetri preliminare asupra unor grupe de compui chimici din lamiacee
medicinale, Referat doctorat, 1998.
3. Ciulei, I., Grigorescu, E., Stnescu, U., Plante medicinale, fitochimie si fitoterapie, vol.
1, Editura Medical Bucureti, 1993.
4. Wagner, H., Pharmazeutische Biologie, Drogen und ihre Inhalts-stoffe, Gustav-Fischer
Vlg., Stuttgart-New York, 1985.
5. Kimura, Y., Okula, H. , J. Nat. Prod., 1987, 50 (3), 392.
6. Tia, D., Chimie analitic cantitativ. Titrimetrie, Ed. Mirton, Timioara, 1998.
7.* * * Dozarea taninurilor din produsele vegetale, Farmacopeea roman, Ed. X-a, Editura
Medical, Bucureti, 1993.
8. Palici, I. , Lamiacee medicinale din flora Banatului, Ph. D. Thesis, 2000.
9. Grimm, H., Analysis of regressions, Ed. Pergamo Press, 1973.
10. Ciocarlan, V., Flora Ilustrat a Romniei, Ed. Ceres, Bucureti, 2000.
11. Hoppe, H., Drogenkunde Walter de Gruyter,Berlin - New York, 1975.
12. Cannas, A., Tannins: Fascinating but Sometimes Dangerous Molecules, Cornell
University, 1996.
13. Craciun, F., Bojor, O., Alexan, M., Farmacia naturii, vol. 2, Ed. Ceres, Bucureti,
1977.