J Sci Food Agric 1993, 61, 161-165

Gravimetric Determination of Tannins and

their Correlations with Chemical and Protein
Precipitation Methods
Harinder P S Makkar, Michael Blummel, Norbert K Borowy and Klaus Becker*
Institute for Animal Production in the Tropics and Subtropics, University of Hohenheim, Box 700562,
7000 Stuttgart 70, Germany
(Received 3 September 1992; revised version received 13 October 1992; accepted 30 October 1992)

Abstract. A method for gravimetric determination of tannins based on binding

with insoluble polyvinylpyrrolidone (PVP) is presented. The gravimetric method
gives the absolute amount of tannins and avoids problems of standards associated
with spectrophotometric methods. The method was applied to nine browse and
tree leaves. The values obtained correlate significantly with tannins determined
spectrophotometrically, protein precipitation capacities and protein precipitable
phenolics. This method together with other tannin assays will be useful in
nutritional studies. The present study also demonstrates the different behaviour of
tannic acids from different commercial sources towards PVP suggesting the
presence of different moieties in tannic acids from different commercial sources
and even among batches from the same source thereby affecting the results
obtained using the spectrophotometric methods. Use of well-defined tannic acid as
a standard in spectrophotometric methods is suggested which will allow
meaningful comparison of values obtained from different laboratories.

Key words : tannins, polyvinylpyrrolidone, gravimetric method, chemical methods,

protein precipitation methods.

INTRODUCTION The present method to quantify tannins gravi-

Tannins are complex polyphenolic compounds with
great structural diversity and wide phylogenetic dis-
tribution. Several methods are available for quantitation
of tannins (see the reviews of Tempe1 1982; Deshpande
et a1 1986; Makkar 1989). None of the available methods
determines tannins in absolute terms but measures
relative to one or the other standard, namely, tannic or
gallic acids in methods based on the oxidation-reduction
principle, catechin or quebracho tannins in methods of
condensed tannin determination, and bovine serum
albumin (BSA) as a standard protein in precipitation
methods. Gravimetric methods do not suffer from
disadvantages associated with colorimetric methods
(Reed et a1 1985). Gravimetric methods are not necess-
arily perfect-these have generally lower sensitivity than
colorimetric methods.
metrically originated in order to overcome the possible
overestimation of dry matter digestibilities in tannin-rich
feeds when determined gravimetrically as in the methods
of Orskov et a1 (1980), Tilley and Terry (1963) or in a
modified Hohenheim gas test (Bliimmel et a1 1990).
Under such experimental conditions tannins are solubil-
ised but might be indigestible (Chesson 1981; Orskov
1991) or partially digested (Makkar, H P S, unpub-
lished). The present method is based on weighing the
tannin extract before and after treatment with insoluble
polyvinylpyrrolidone (PVP) and removal of the PVP by
centrifugation. The PVP binds to tannins (Loomis and
Battaile 1966) and therefore the difference in weight is
due to the tannins. The colorimetric methods for tannin
determination based on binding with the PVP are
available (Laurent 1975; Watterson and Butler, 1983).

* To whom correspondence should be addressed.

161
J Sci Food Agric 0022-5142/93/$06.00 0 1993 SCI. Printed in Great Britain
162 H P S Makkar, A 4 Blummel, N K Borowy, K Becker

TABLE 1
Tannin levels using the gravimetric and various chemical methods"

Name Tannins TP Tannins" CTd Proanthocyanidins

(g kg-', (g & - I ) (g kg-') (g kg-') (A550,", g-l)
gravimetrically)

Panicum maximum 0 11.1 f 0 4 2.2 f0.3 1.O 0.04 13.04 f 1.43

Dialium guineense 52-4 53.4 f2.5 39.1 f2.4 26.5 f0.2 1346f70
Acioa barterii 85-0 96.6f 1.1 82.0f 1.1 72.7 f3.1 2686f 110
Quercus incana 69.5 71.1 f 3 . 6 56.4 f3.6 12.3 f 1.1 29 1.2 f 5.55
Cassia sieberiana 21.0 40.5 f3.6 26.5 f3.0 6.6 f0.6 142.6 f2.9 1
Piliostigma reticulatum 60.7 50.2 f2.3 40.5 f 1.9 20.7 f 1.4 778 f 20
Guiera senegalensis 60.6 60.1 f 1.6 40.5 f2.3 9.9 f0.6 136.9 f4.76
Cajanus cajan 48.2 50.4 f2.2 34.4 f 1.2 22.0 f0.5 394 f5.0 1
Dichostachys cinerea 58.7 89.8 f 1.6 65.5 f2.6 38.5 f0.5 612.7 f 7.66

a The data are expressed as mean fSD ( n = 3 or 4) and are on dry weight basis.
Difference of TP before and after PVP treatment using Fohn Ciocalteu method.
Tannic acid equivalent.
Catechin equivalent.

EXPERIMENTAL 15 min at about 4"C, centrifuged and supernatant

Materials
Air-dried samples of various leaves were used in the
study (Table 1). PVP, BSA and catechin were obtained
from Sigma (Deisenhofen, Germany). Tannic acids (TA)
were from Merck (Darmstadt, Germany), lots
016K13540873 and 108K14678573, termed as I and 11,
respectively; and Sigma lot 120H3387, termed as 111. All
collected. Two disposable aluminium weighing dishes,
one for the PVP treated and other for the untreated
extracts were weighed. Aliquots (10 ml of untreated and
20 ml of the PVP treated extracts) were pipetted into the
respective dishes and dried to a constant weight (about
30min for the untreated and 60min for the treated
extracts) on a hot plate or in an oven at 100°C. The
dishes were transferred to a desiccator and weighed when
room temperature was achieved. This procedure of

aluminium dishes (10 cm in diameter, weighing 2.5 g)

were from Sartorius (Goettingen, Germany).
-
other chemicals were of analytical grade. The disposable
Methods
Preparation of tannin extract
Dried leaf (200 mg) ground to pass through an 80 mesh
screen was suspended in 10ml aqueous acetone
(acetone: water, 7 : 3 v) in a centrifuge tube. Ice-cold
suspensions were subjected to Ultra-turrax (Janke and
Kunkel, IKA-Labortechnik, Heitersheim, Germany)
treatment at 20000 rpm at 4°C (tubes kept in ice) for a
total period of 6 min (content of total phenol (TP) in the
supernatant as determined by Folin Ciocalteu (FC)
reagent (see below) reached plateau after 5 min of the
treatment) with cooling after every 2 min of the treat-
ment. The tubes were centrifuged at about 2000 x g for
20min and the supernatant collected was kept on ice
until analysed (generally within 4 h).
Gravimetric determination of tannins
About 110 ml of the extract prepared as described above
was divided into two portions of 65 ml and 45 ml. The
PVP (6.5 g, 50 mg ml-') was added to the former after
diluting 1 : 1 with distilled water, the mixture stirred for
drying the aliquots and weighing was repeated three
times to obtain four values for each treated and untreated
extract. The values observed did not deviate by more
than 5 % from their mean. The average weight cor-
responded to 10 ml of the undiluted extracts. Thus, the
difference in weight for the treated and untreated extracts
gave the weight of tannins in 10ml of the extract or
200mg of the leaves. The results were expressed as
percent tannins on dry matter basis.
Other analytical procedures
The FC method (Julkunen-Tiitto 1985) for TP was used
for optimisation of the conditions for the gravimetric
method. The difference in the TP values before and after
the PVP treatment (unbound) represent the tannin levels
as TA (Merck) equivalent.
The vanillin-HC1 method of Broadhurst and Jones
(1978) was used for determination of condensed tannins
(CT) with some modifications which allow determination
of CT in presence of acetone (Makkar and Becker 1993).
The results are expressed as catechin equivalent. The CT
were also determined by the proanthocyanidin assay
using butanol-HC1-Fe3' reagent (Porter et a1 1986).
Protein precipitation capacity (PPC), specific activity,
protein precipitable phenolics (PPP) and percentage PPP
were determined by the method of Makkar et a1 (1988).
Gravimetric determination of tannins
The PPC was also determined as described by Asquith
and Butler (1985) except that the reaction volume was
3 ml. To the BSA-dye (2 ml) containing 2 mg ml-' BSA
was added 50 OO/ methanol and then increasing amount of
tannin-containing extract to make 3 ml. The slope of the
BSA precipitation curves represents mg BSA precipitated
per mg leaf. The extracts for the determination of PPC
and PPP were in 50% aqueous methanol and not in
aqueous acetone, as acetone interferes with the formation
of the protein-tannin complex.
The PVP was purified using 10 O h v/HCl as described
by Loomis (1974).
RESULTS AND DISCUSSION
Effect of increasing amount of PVP
Different quantities of the PVP were added to the
solutions of TA I and I11 (2 mg ml-') prepared in water,
50 % v/aqueous methanol and 70 YOv/aqueous acetone
and stirred for 15 min at about 4°C. The TA as TP was
determined using the FC method in the supernatant
obtained after centrifuging the contents. In case of TA I,
the PVP bound TA to a greater extent in aqueous
methanol or aqueous acetone than in water, whereas on
using TA 111 the binding was higher in water followed by
aqueous methanol and aqueous acetone (Fig 1). PVP is
known to bind tannins mainly through hydrogen bonds.
Organic solvents, such as alcohols or acetone decreased
the affinity of PVP towards tannins (Loomis and Battaile
1966). Higher binding of TA I to the PVP in aqueous
acetone or aqueous methanol is therefore surprising. The
TA I was at least 2 years old, but recently obtained
Tannic acid IU Tannic acid I
40
I
III
IIJI
III I 1 I l l 1
0 20 40 60 80 100 C 20 40 60
PVP (rng rnP)
Fig 1. Extent of binding of tannic acid (2 mg ml-l) (m)
water,
( 0 )70% v/aqueous acetone, and (A)50% methanol from
two different sources on insoluble polyvinylpyrrolidone (PVP).
(0) Using HCI-treated PVP in 70 % v/aqueous acetone. TA I
and 111 were from Merck and Sigma, respectively.
163
tannic acid (TA 11) also gave similar results except that
the extent of binding to PVP was 1-10% higher for the
new batch. Another point worth noting is the higher
degree of binding of TA 111 to the PVP (Fig 1). These
results suggest the presence of some moities in TA I of
non-tannin nature but with positive reaction in the FC
reagent, and these moities could differ from batch to
batch. Hagerman et a1 (1992) also noticed different
components in different preparations of TA. In 70%
aqueous acetone, addition of the PVP beyond 50 mg ml-'
did not increase the binding of TA substantially (Fig 1).
Therefore, 50mg PVPml-' was added initially for
binding the tannins of the leaf extracts with TP content
< 2 mg TA equivalent ml-'. As binding efficiency of leaf
tannins to the PVP could differ from that of the TA, the
PVP at a level of 50mgml-' was also added to 1 : 1
diluted (with 70% aqueous acetone) extracts, which is
equivalent to 100 mg ml-l of the undiluted extract. The
levels of TP in the supernatant after the PVP treatment
were lower in the diluted extracts of all the leaves studied,
which gave higher values of tannin by 190% and 84%
than that observed using undiluted extract for Acioa
barterii and Dichostachys cinerea, respectively, and 1 4 '10
for others. Further increase in the PVP did not increase
the apparent tannin level substantially. In colorimetric
methods, some workers (Loomis and Battaile 1966;
Watterson and Butler 1983) have used the PVP after
treatment with 10% HCl. After the HCl treatment and
drying, clumps of PVP were formed. Loomis and Battaile
(1966) have suggested not breaking the clumps, as it
could lead to the formation of soluble PVP. The efficiency
of HC1-treated PVP when used without breaking the
clumps was poorer compared to untreated PVP (Fig. 1).
However, when the clumps were broken by grinding,
there was no difference in the binding efficiencies of the
treated and untreated PVP, suggesting that poorer
binding of HC1-treated PVP in the clump form was due
to lesser surface area available for binding to TA.
Tannin levels by gravimetric, chemical and protein
precipitation methods
It is interesting to compare the tannin values obtained by
the present method with the FC method (Table 1). For
Cassia sieberiana and D cinerea, the values obtained by
the gravimetric method were lower than those by the
colorimetric method. Tannins in Panicum maximum were
too low to be measured by the gravimetric method
(Table 1). Phenolic compounds of different structures
give different response to the FC reagent. The difference
I I , ,
in values obtained by these two methods may be due to
80 100 the different nature of tannin of the leaves and the TA.
Each g of tannins from different sources has different
reducing powers varying from 0.75 to 1.26 g TA
equivalent (ratio of colorimetric to gravimetric values ;
Table l), and also different protein binding/precipitating
capacities (Table 2), suggesting different biological
164 H P S Makkar, M Bliimmel, N K Borowy, K Becker

TABLE 2
Tannin levels by protein precipitation methods"

Name PPC (mg BSA precipitated per mg leaf) PPP Specific PPP
(pg TA equivalent activityb (YO)
Dye-binding Makkar et a1 per mg lean
method (1988), x Y

Panicum maximum nd' nd nd - ~

Dialium guineense 0.161 f 0 0 0 2 0.106 f0.002 8.00 k0-55 13.25 63.14

Acioa barterii 0.604 f0.007 0.634 f0.008 34.44 f0.66 18.41 89.02
Quercus incana 0.338 f0.007 0.124 f0.006 19.47 f 1-49 6.37 49.87
Cassia sieberiana 0.067 f0006 0.043 f0.003 6.10f0.59 6.97 38.34
Piliostigma reticulatum 0.185 f0.004 0.086 f0.002 12.24f0.33 7.03 61.79
Guiera senegalensis 0.161 f0.008 0.078 f0.002 8.91 f 1.05 8.75 29.04
Cajanus cajan 0.170 0.0 12 0.060 f0.004 9.05 f0.27 6.63 58.12
Dichostachys cinerea 0.507 k0.006 0.287 & 0.006 32.10 f0-53 8.94 57.49

a The data are expressed as meankSD (n = 3 or 4) and are on dry weight basis.
mg BSA pptd/mg TA equivalent ( x / y , y in mg TA equivalent/mg leaf).
nd-not detected.
PPC, protein precipitation capacity; PPP, protein precipitable phenolics.

TABLE 3
Correlations between tannin levels obtained by different methods"

TP Tannins-C CT PAS PPC-dye PPC-Makkar PPP

binding et a1 (1988)
Tannins-G 0.88** 091*** 0.69* 0.63 0.79* 0.67* 0.75*
TP 0-99*** 0.80** 0.63 0.95*** 0.81** 0.94***
Tannins-C 0.84** 0.70* 0.96*** 0.85** 0.95***
CT 0.93 * * * 0.87** 0.96*** 0.84**
PAS 0.70* 088** 0.65
PPC-dye binding 0.90*** 0.99***
PPC-Makkar et a1 (1988) 0.88**

P < 0.05; ** P < 0.01; *** P < 0.001.

Tannins-G, Tannins-C, tannins measured gravimetrically and colorimetrically respectively; TP, total phenols; CT, condensed
tannins; PAS, proanthocyanidins.

responses of different tannins even at the same level in
animal diets. This emphasizes the need to study struc-
ture-activity relationship of polyphenols. Insoluble PVP
also binds to simple phenols (Loomis 1974). However,
contribution of simple phenols towards observed tannin
levels would be negligible, as strength of binding of
phenols to the PVP is in proportion to the number of
phenolic hydroxyl groups (Clifford 1974) and presence
of organic solvents decrease the extent of binding
(Anderson and Sowers 1968). The values in Table 1 for
the tannin levels (as tannic acid equivalent) are obtained
from the calibration curve using TA I. The ratio of slopes
of the curves (amount of tannic acid, x-axis versus
absorbance at 725 nm, y-axis) for the TA 1:TA I11 was
1.07 (moisture content of both TA I and I11 was similar),
which would give higher values for tannins by 7 % on
using TA I11 as a standard. The PPC of TA I11 was also
substantially lower than of TA I (Makkar, H PS,
unpublished). These observations together with different
affinities of different TAs towards the PVP (see above)
suggests the need to use well-defined standards. Recently,
Hagerman et a1 (1992) have fractionated TA into
different fractions using HPLC. It is suggested that the
chemical companies should produce well-characterised
tannic acid (analogous to fraction V of BSA used as a
standard in protein assays). Use of such a standard
would ensure reproducibility between and within lab-
oratories, and would allow meaningful comparison of
values obtained from different laboratories.
There was wide variation in the tannin levels in the
leaves studied (Tables 1 and 2). Tannin levels were
negligible in P maximum and high in A barterii. About
89% of the TP bind protein, and the protein-binding
affinity of phenols as evident from the specific activity
was also highest in A barterii (Table 2). The PPC by the
dye-binding method (Asquith and Butler 1985) were
Gravimetric determination of tannins
invariably higher than those observed by the method of
Makkar et a1 (1988), although the correlation between
the values obtained by these methods o r with PPP were
highly significant (Table 3). Higher values from the dye-
binding method are due to higher amount of the protein
present in the assay (4 mg per 3 ml versus 2 mg per 3 ml
in the method of Makkar et a1 1988) (Hagerman and
Robbins 1987).
Correlations between gravimetric and other methods
The best correlation of values obtained gravimetrically
was with tannins (r = 0.91, P < 0.001), followed by TP
( r = 0.88, P < O.Ol), PPC-dye binding ( r = 0.79,
P < 0.05), CT ( r = 0.69, P < 0.05) and PPC-Makkar
et a1 (1988) (r = 067, P < 0.05). The PPC values are the
slopes of the curves which do not represent the true
values as these d o not account for the intercept. Poorer
correlation with PPC methods could be due to the
inherent disadvantage of the BSA precipitation curves
generated by increasing the tannin-containing extract in
an assay containing fixed amount of protein.
The gravimetric method in combination with other
methods may be useful in providing better insight into
the effect of tannins o n biological systems. It would also
help in the proper interpretation of dry matter digesti-
bilities for tannin-rich feeds obtained gravimetrically by
in-sacco and in-vitro methods.
ACKNOWLEDGEMENT
One of the authors (HPSM) is thankful to the Alexander
von Humboldt Foundation, Bonn for awarding the
AvH-doctoral Research Fellowship under which this
work was undertaken. The authors are thankful to D r H.
Steingass, Institute for Animal Nutrition and Dr M.
Yuonan for supplying the leaves.
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