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thesis entitled

PINTO BEAN HULLS, BUT NOT CONDENSED TANNINS,
ALTER COPPER BIOAVAILABILITY IN RATS

presented by

Su-Fen Weng

has been accepted towards fulfillment
of the requirements for

M.S. Human Nutrition

degree in

 

 

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DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PINTO BEAN BULLS, BUT NOT CONDENSED TANNINS,
ALTER COPPER BIOAVAILABILITY IN RATS

BY

Su-Fen Weng

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1994

ABSTRACT

PINTO BEAN BULLS, BUT NOT CONDENSED TANNINS,
ALTER COPPER BIOAVAILABILITY IN RATS

BY

Su-Fen Weng

Bioavailability of copper in diets containing condensed
tannins was determined. Male rats were fed: control, 1.6 %
catechin or 25 % pinto bean hulls diet in Expt I, control or
25% hulls in Expt II. The level of catechin was based on the
amount of condensed tannins in the hulls diet. No significant
differences were found in weight gain and food intake. Liver
relative weight was significantly lower in hull-fed rats.
Catechin—fed rats had a higher mean tibia weight. Tissue
copper concentration were similar in all groups. Hull-fed
rats had significantly elevated plasma copper and
ceruloplasmin activity but decreased total liver copper.
However, condensed tannins fed as catechin had no effect on
cooper bioavailability at the levels fed.in this studyu Thus,
these results suggest that in rats the consumption of bean
hulls, but not condensed tannins, is associated with apparent

mobilization of copper from the liver to the circulation.

For my family

iii

ACKNOWLEDGMENTS

I wish to express my gratitude to Dr. Wanda L. Chenoweth
for her encouragement and understanding throughout my graduate
studies. I would also like thank Dr. Maurice R. Bennink and
Dr. Dale R. Romsos for their commitment on my research and
personal growth, Dr. Pao K. Ku, Department of Animal Science,
for the technical assistance during my experiment time, Dr.
Greg Fink for his help with statistical analysis.

The friendship and support through the best and worst of
times of Dr. Rachel A. Schemmel, Ritu Ummadi, Pervaiz Akhtar,
Michael Gonzalez, Chau Trans, Chingju Lin, Liang-Ting Chang,
Ping-Chao Tsai (beans peeler), Tsui-Ying Liu, and especially
Mary Schneider have also been appreciated.

Finally, I want to thank every member of my family for

their confidence in my abilities and their continual interest

in my graduate studies.

iv

TABLE OF CONTENTS

Page

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . viii
LIST OF IIGURES . . . . . . . . . . . . . . . . . . . . . ix
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEI . . . . . . . . . . . . . . . . . . . . 4
CHEMISTRY OF TANNINS . . . . . . . . . . . . . . . . 4
TANNINS IN BEANS . . . . . . . . . . . . . . . . . . 10
TANNINS ANALYSIS . . . . . . . . . . . . . . . . . . 13
Colorimetric Methods . . . . . . . . . . . . . . 15

Protein Precipitating Methods . . . . . . . . . . 17

Other Methods . . . . . . . . . . . . . . . . . . 18
PHYSIOLOGICAL EFFECTS OF TANNINS . . . . . . . . . . 20
Effects on Food Intake and Mortality . . . . . . 21
Interactions with Protein and Starch . . . . . . 22

Effects of Tannins on Mineral Metabolism . . . . 25

Iron . . . . . . . . . . . . . . . . . . . . . 25

Zinc . . . . . . . . . . . . . . . . . . . . . 29

copper . . . . . . . . . . . . . . . . . . . . 29

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . 37
MATERIALS . . . . . . . . . . . . . . . . . . . . . 37

RESEARCH DESIGN . . . . . . . . . . . . . . . . . . 38

Preliminary Analyses . . . . . .
Animal Studies . . . . . . . . .
EXperiment I . . . . . . . . .
Experiment II . . . . . . . . .
PREPARATION OF BEANS . . . . . . . .
ANIMALS . . . . . . . . . . . . . .
DIETS . . . . . . . . . . . . . . .
ANALYTICAL METHODS . . . . . . . . .
Tannins . . . . . . . . . . . . .
Mineral Analyses . . . . . . . .
Bean and rat diets . . . .

Plasma . . . . . . . . . .

Tissues . . . . . . . . .

NEST standards . . . . . .
Ceruloplasmin Activity . . . . .
Hemoglobin . . . . . . . . . . .
Statistical Analyses . . . . . .
EXperiment I . . . . . . .
EXperiment II . . . . . .

RESULTS . . . . . . . . . . . . . . . . .
PRELIMINARY ANALYSES . . . . . . . .
ANIMAL STUDIES . . . . . . . . . . .
Food Intake, Body Weight and Food
Organ Weights . . . . . . . . . .
Copper . . . . . . . . . . . . .

Zinc O O O O O O O O O O O O O 0

vi

0 O O O O
O O O O O
O O O O O
O O O O O
C O O O O
C O O C C
O O O I O
O O O O O
O O O O C
O O O O O
O O O O O
O O O O O
0 O O O O
O O O O O
O O O O O
O O O O O
O O O O O
O O O O O
C O O O O
O O O O O
O O O O O
O O O O O

Efficiency

38

38

38

39

39

4O

41

41

41

45

45

47

47

48

48

48

49

48

49

50

50

53

53

55

55

60

Iron

DISCUSSION

PRELIMINARY ANALYSES

ANIMAL STUDIES .

CONCLUSIONS

RECOMMENDATIONS .

REFERENCES

vii

60

63

63

67

74

76

78

LIST OF TABLES

TABLE Page
1 Analytical methods for tannin . . . . . . . . . . . 14

2 Some effects of condensed tannins
in rats, chickens, and pigs . . . . . . . . . . . . 23

3 Composition of diets in Experiment I . . . . . . . 42
4 Composition of diets in Experiment II . . . . . . . 43

5 Analyzed condensed tannin, copper,
zinc and iron content of diets . . . . . . . . . . 46

6 Tannin concentration of ten different dry beans . . 51

7 Relative distribution of tannin and mineral
content in four brands of pinto beans . . . . . . . 52

8 Body weight, food intake and
food efficiency ratios . . . . . . . . . . . . . . 54

9 Copper, zinc and iron intake . . . . . . . . . . . 56
10 Organ wet weights and relative organ weights . . . 57
11 Organ dry weights . . . . . . . . . . . . . . . . . 58

12 Ceruloplasmin activity, plasma copper,
zinc, iron, hematocrit and hemoglobin . . . . . . . 59

13 Concentration and total content
of copper in tissues . . . . . . . . . . . . . . . 61

14 Concentration and total content
of zinc in tissues . . . . . . . . . . . . . . . . 62

viii

Figure

Structure
Structure
Principle
Principle

Procedure

LIST OF FIGURES

of gallotannin and ellagitannin

of condensed tannin .

and example of vanillin assay

of mineral metabolism .

of vanillin assay .

ix

INTRODUCTION

Legumes provide a valuable source of food energy to
people, especially in developing countries. They are also a
good source of protein, dietary fiber, vitamins (Bl, niacin,
pantothenic acid, and biotin) and certain minerals (iron,
copper, and zinc). Increased use of legumes in western diets
would bring these diets closer to recommended dietary goals in
terms of complex carbohydrates and dietary fiber (Walker,
1982). However, it is also well recognized that mineral
bioavailability from plant sources is generally low. The
factor or factors responsible for this low bioavailability
have not been clearly identified.

In the past years, much attention has been given to
destruction of antinutritional factors such as trypsin/
chymotrypsin inhibitors, phytohemagglutinin (lectins) , and
phytates by appropriate processing. Recently tannins
(polyphenols) in many edible plant products have received
increasing attention as a result of their possible influence
on the nutritional and aesthetic qualities of foods,
biochemical and physiological functions and pharmacological

implications (Reddy et a1., 1985 Garcia-Lopez et a1., 1990).

2

Phenolic compounds occur naturally in many plants
including coffee, tea, and legumes. Tannins are known to
function as natural defense agents in plants against insects,
fungi, and preharvest seed germination (Salunkhe et a1.,
1990). The presence of phenolics in certain colored legumes
may improve their acceptability over white beans, perhaps due
to taste.

If present in appreciable quantities, tannins lower the
nutritional value and biological availability of dietary
proteins. Experiments with a variety of laboratory animals
have shown that dietary tannins decrease the growth rate, feed
efficiency, and egg production by laying hens (Butler et a1.,
1986) . Tannins have been shown to cause leg abnormalities and
ultrastructural changes in the livers of chicks and affect the
physical or sexual maturity' and even. death in Ihamsters
(Jansman, 1994). The major antinutritional effect of dietary
tannins is the formation of stable complexes with dietary
proteins and.perhaps with digestive enzymes, which ultimately
results in poor digestion and absorption of dietary nutrients. ‘
Tannins have also been reported to form complexes with starch
and reduce its digestion.

Tannins also are known to act as an inhibitory factor on
iron absorption when tea and coffee are consumed with various
diets (Disler et a1., 1975; Morck et a1., 1983; Brune et a1.,
1989). Conflicting results have been reported for the effect

of tannins in legumes on iron bioavailability (House and Van

3
Campen, 1994). The effect of legume tannins on copper has
received little attention. Therefore the focus of the present
study is on the effect of bean hull tannins on copper

bioavailability.

LITERATURE REVIEI

CHEMISTRY OF TANN INS

Originally, the term "tannin" was used by Seguin (1796)
to describe substances in ‘vegetable extracts which are
responsible for converting animal skins into stable product
leather (Salunkhe et a1., 1990). The substances essential in
the tanning process were later identified as polyphenolic
compounds with various molecular size and complexity. Tannins
are present in a large number of products of vegetable origin
used as human foods or animal feeds. 'The form of the phenolic
compounds vary from small monomeric phenolic acid (e.g. ,
gallic acid) to large polymerized polyphenols (e.g., tannins).

Bate-Smith and Swain (1962) defined plant tannins as
naturally occurring water-soluble phenolic compounds with a
molecular weight between 500 and 3000, containing a
sufficiently large number of phenolic hydroxyl or other
suitable groups which are capable of precipitating alkaloids
as well as gelatin and other proteins from aqueous solution
(Gupta and Haslam, 1980). From this definition it is clear
that tannins are chemically not well-defined substances but
rather a group of substances with some common properties.
Polyphenols referred to as tannins have a considerable number

of phenolic groups. They are capable of forming effective

5

cross-links with other molecules as hydrophilic or hydrophobic
complexes. According to White (1957) , phenolic compounds with
a low molecular weight (<500) do not form stable cross-links
with other molecules. However, compounds with a much higher
molecular weight (>3000) do not show tanning properties
because they appear to be too large to penetrate into the
collagen fibrils in hides (Swanson, 1993).

Tannins have been classified by Freudenberg (1920) into
hydrolyzable and non-hydrolyzable or condensed tannins which
are differentiated by their structure and reactivity toward
hydrolytic agents (Gupta and Haslam, 1980) . Hydrolyzable
tannins are classified as gallotannins (so-call tannic acid)
or ellagitannins on the basis of 'tannic acid structure
relative to gallic or ellagic acids. Figure 1 shdws the
structure of gallotannin and ellagitannin, respectively.
Tannic acid is a well-known gallotannin and contains 8 to 10
moles of gallic acid per mole of glucose. Ellagitannin is a
hexahydroxydiphenic acid linked with glucose as a diester in
addition to gallic acid. Hydrolyzable tannin can be acid,
alkaloid, or enzymatically hydrolyzed to yield glucose or some
other polyhydroxy alcohol and gallic acid or some phenolic
acids related to it (Swanson, 1993) . Although many low
molecular weight phenols are metabolized in the bodies of
higher animals, the metabolic fate of these phenolic compounds
is not yet clearly known (Salunkhe et a1., 1990). Jansman

(1994) suggested that in animals tannic acid is degraded into

Gallic acid Ellagic acid 0
H o g— 0 OH
no} no @ o 0.,
H0 H0 0—
ii
0
l) Gallotannin ('l‘annic acid)
01-!
n o
H C—O OH
mo cw, “( © 0.,
“‘ 0 0 @ OH 01-]
H0 \ o
'0 0 0 @ OH
H 0
OH OH

 

HO @ COO
o 0..

2) Ellagitannin
H0

110 @ c— ocn2
on
0

il

11 o
o— c OH
H0 0 P)
on
no 0

ii)

no
on on

Figure 1 Structure of gallotannin and ellagitannin

GOO

7

gallic acid derivatives, mainly 4-methoxy gallate (4-O-methyl
gallic acid). Oral administration of tannic acid to chickens
resulted in some:gallic acid excretion in the urine but.not in
the feces. Sources of hydrolyzable tannin are seed pods,
bark, woods, fruits and leaves of plants belonging to the
family Leguminosae, Fabaceae, Combretaceae, Anaacadiaceae
(Salunkhe et a1., 1990).

A second category of polymeric flavonoids, the condensed
tannins are resistant to hydrolysis in the gut, but on severe
acid or alkaline treatment yield less-soluble polymeric
phlobaphanes or monomeric f lavonoids such as catechin or
epicatechin. Condensed tannins contain linkages between the
4 '-position of one catechin residue and the 6'- or 8'-position
of another flavonoid. They are mainly oligomers of flavan-3-
ols (e.g., catechin) and flavan-3,4-diols. Figure 2 shows
examples of condensed tannins. The full chemical nature and
exact metabolic routes of condensed tannins still have not
been elucidated (Gupta and Haslam, 1980; Jansman, 1994) .
Condensed tannins are also :referred. to as flavolans or
procyanidins. Flavan-3-ols with a molecular weight below 3000‘
are soluble compounds. Higher polymerized procyanidins become
insoluble and are often.more closely linked to the structural
tissue of the plant (Salunkhe et a1., 1990) . Flavan-3,4-diols
belong to the class of leucoanthocyanidins because they
polymerize upon heating in acid solutions not only to

phlobaphene-like products (tannin reds), as flavan-3-ols do,

A. Flavan-3-ols
Catechin
o ..
on OH
OH
Gallocatechin OH
HO 0
g Q on
0H 0H
on
B. Flavan-3,4-diols
Leucocyanidin on
o on
OH
OH OH

Leucoanthocyanidin

 

Figure 2 Structures of condensed tannins

but also to anthocyanidin.

Condensed tannins are the predominant class of
polyphenols in green vegetables, fruits, cereals, and legumes
(Salunkhe et a1., 1990) . The concentration of tannins in
plant tissue used commercially as sources of food may vary
from 5 to 50% of the material's dry weight (Singleton, 1981).
The pigmented, varieties of cereals and legumes contain 2 to 4%
condensed tannins, although values of up to 7 to 8% are
reported for red high-tannin sorghum varieties (Salunkhe et
a1., 1990). Other food and beverages, such as cider, cocoa,
and red wines, contain considerable amounts of condensed
tannins. Strong preparations of these beverages may have as
much as a gram of tannin per liter. An intake of dimeric
flavans as high as 400 mg/day in the American diet from a
variety of sources has been reported (Reddy et a1., 1985).
The total dietary tannin intakes in such cases would be
somewhat higher than dimeric flavans. However, this may
depend on the composition of the diet and diet patterns. High
bean-based diets may supply higher levels of dietary tannins.
For example, analyses of diets consumed in different regions
of India indicated that daily intake of tannins varies from

1500 to 2500 mg (Rao and Prabhavati, 1982).

10

TANNINS IN BEANS

Tannins in legume seeds appear to play a role in the
crop's resistance to being eaten by birds. They also play a
role in their susceptibility to attack by fungi and pests and
in the incidence of preharvest germination. Tannins may form
a physical barrier, which prevents water imbibition necessary
for germination (Salunkhe et a1., 1990).

With regard to the legume seeds, tannins have been found
in dry beans (Phaseolus vulgaris) , peas (Pisum sativum),
chickpeas (Cicer arietinum L.) and lentils (Lens culinaris).
In most grain legumes tannins are present as condensed tannins
(Salunkhe et a1., 1990; Jansman, 1994).

Tannin content in beans is unevenly distributed in the
seed and varies with seed color (Martin-Tanguy et a1., 1977;
Deshpande et a1., 1982) . White beans have been shown to
contain a low amount of tannin, whereas colored seeds
contained high concentrations irrespective of the method of
estimation. Cabrera and Martin (1986) found a clear
correlation between color of the flower, seed color and tannin
content of faba beans. White-flowering varieties, with no
pigments in the flowers, yielded white and.grey seeds with low
tannin contents. Colored-flowering varieties yielded seeds of
different color with the amounts of tannins increasing
progressively in seeds having green, red, beige or brown

colors.

11

Condensed tannins are mainly located in the hulls of the
colored seeds, with low or negligible amounts in the
cotyledons. Desphande and Cheryan (1985) reported that the
seed coats contained approximately 0.5 to 7% tannins on a dry
weight basis. Rao and Prabhavathi (1982) reported that after
decorticating there was very little tannin detectable in
cotyledons, indicating that almost all the tannins of the seed
are present in the seed coat. Deshpande and Salunkhe (1982)
also found that removal of seed coats lowered the assayable
tannin content of beans by 68 to 95% and dehulled pinto beans
had a 94.6% reduction of assayable tannin content expressed as
mg catechin equivalents. Later, Laurena et a1. (1984) also
detected 2 to 3 times greater concentration of assayable
tannins in eight varieties of cowpeas when tannins were
determined in seed coats alone. Deshpande and Cheryan (1985)
showed that when expressed on total seed weight basis, the
concentration of assayable tannins in seed coats was 1.1 to
2.5 times compared to the concentration in whole bean flours.

Soaking beans before cooking is a common practice used to
soften the texture and hasten the cooking process. Reduction
in ‘tannin concentration. of several beans by soaking in
different solutions was reported by Deshpande and Cheryan
(1985). The leaching of bean tannins increased with the time
of soaking. Soaking winged beans and cowpeas for 24 hr
reduced tannin concentration by 50% and over 50%,

respectively, as reported by Sathe and Salunkhe (1981) and

12

Laurena et al. (1986) . since tannins are present in the
outermost layers of the seed during soaking, they may diffuse
into the seed and bind with proteins to form insoluble
complexes. Deshpande and Cheryan (1985) suggested that the
apparent loss of tannin after soaking was caused by the
formation of such complexes which were not assayable by the
method used to determine tannin content.

A reduction in tannin concentration of beans can be seen
during cooking, another common process used for softening
beans. Cooking whole seeds and discarding the cooking water
caused a reduction of 37.5 to 77% in the tannin concentration
in beans (Salunkhe et al., 1990). According to Bressani et
al. (1982), cooking resulted in a 61 to 98% decrease in the
tannin concentration of beans and a 17.8 to 50.9% decrease
when taking into consideration the tannin concentration in the
cooking waters. Price et a1. (1980) reported that moist heat
cooking of grains reduced the assayable tannin from 2.6 to
0.4%; however, this apparent reduction in tannins was not
accompanied by a proportional improvement in weight gain or
food efficiency in a rat feeding trial. A decrease in the
tannin concentration of mung beans, pigeon beans and cowpeas
during cooking has also been reported (Gahlawat and Sehgal,
1992). Bressani et al. (1982) suggested that the decrease in
tannin concentration may be not due to an actual decrease in
tannins but due to their binding to other organic substances

and protein thus reducing their extractability, since

13
compounds that have reacted with amino groups of proteins are
not extracted by methanol (Reddy et a1., 1985; Gahlawat and
Sehgal, 1992) . Alterations in the chemical structure of
tannin as a result of heating have never been shown (Jansman,
1994).

Condensed tannins constitute the major portion of dry
bean tannin concentration. Relatively few studies have been
reported on the chemical nature of legumes tannins. Martin-
Tanguy et al. (1977) reported that seed coats of colored-
flowering varieties of faba beans (Vicia faba L.) contain
condensed tannins of the proanthocyanadin type. They found
the polymers consist of molecules of flavan-3-ols (catechin
and gallocatechin) and flavan-3,4-diols (leucocyanidin and
leucodelphinidin) and are joined together by a carbon-carbon
linkage between C4 of one unit and C6 or C8 of other units.
Chains were linearly linked with a flavan-3-ol at the terminal

end.

TANNINS ANALYSIS

According to Deshpande and Cheryan (1987) , one of the
major difficulties encountered in polyphenol research is the
lack of a standard quantitative method for the analysis of
phenolics that would be suitable for a wide range of seeds,
forage crops and food products under varying experimental

conditions. Several different methods have been developed for

14
the measurement of tannins in plants, based on different
principles and measuring different structural groups in these
compounds, either all phenols as a group apart from nonphenols
or specific individual substances or classes of phenols.
However, these assays, due to the complexity and heterogeneity
of tannins, do not provide completely satisfactory results.
They can, nevertheless, be categorized into three groups:
colorimetric methods, protein precipitating methods and other
methods. TABLE 1 gives the standard and compounds measured in

these methods.

 

TABLE 1

Analytical methods for tannins

 

 

Method Standard What is measured?
Vanillin Catechin Leucoanthocyanidins
Proanthocyanidins
Folin-Denis Catechin Total phenols
Prussian Blue Tannic acid All reducing compounds
Protein Precipitating Tannic acid Proanthocyanidins

 

Adapted from: Salunkhe et al. (1990), p 43.

15

W

The methods most frequently used in the earlier
experiments on beans are the vanillin-hydrochloric acid assay,
expressed as catechin equivalents (Swain and Hillis, 1959;
Burns, 1971; Price et al., 1978); the Folin Denis assay,
expressed as tannic acid (Gupta and.Haslam, 1980; Bressani et
a1., 1983; Deshpande and Cheryan, 1987) and Prussian blue
assay, expressed as tannic acid (Price and Butler, 1977).

The vanillin-HCl assay is widely employed as a method for
the quantitative determination of condensed tannins in fruits,
sorghum and forage legumes. The assay determines more
specifically the polymeric phenols, especially flavan-3-ols,
dihydrochalcones and proanthocylanidins. The basic reaction
is based on the substitution of aldehyde groups in vanillin
for the resorcinol group in flavanols and favanoids, yielding
a red colored condensation product which is measured
spectrophotometrically at 550 nm. Figure 3 summarizes the
principle and shows an example of the condensation reaction.
For convenience, catechin, a monomeric flavan-3-ol unit, is
used as the standard substance and.the assay gives no reaction
with substances such as gallic acid and tannic acid without
resorcinol groups (Brune et al., 1991).

Polyphenols of cereals and legumes are predominantly
condensed tannins (Thompson et al., 1983). Bressani et a1
(1983) , therefore, stressed a need to have more specific

methodology to determine tannins and their chemical nature in

16

 

l) Principle of vanillin assay OH

Plain—1.3.. ‘T: R—i:—@-ou

2) Example of vanillin assay

on
c1130 *
HO 0
o on
W— n 0 @ cu=01r
+
on
on on on
Vanillin radical Leucocyanidin

ou
01,0 no 0
on
on
on on

Red colored condensation product

Figure 3 Principle and example of vanillin assay

‘

Adapted from: Salunkhe et al. (1990). p 80.

17

beans. Recently Deshpande and Cheryan (1985) have critically
reevaluated the vanillin reaction for the detection of tannins
in legume. They modified the 0.5% vanillin assay as described
by Price et al. (1978) , by using appropriate sample blanks to
eliminate variation due to the background color caused by
anthocyanin-like pigments. The inherent color variation
within a given sample can be minimized by the careful
selection of at least three representative samples and
grinding them separately prior to the analysis.

The Folin-Denis method is nonspecific and detects all
phenolic groups in the extract, including those found in
extractable proteins, as well as reacting with reducing
substances such as ascorbic acid (Thompson et al., 1983) .
Since not all phenolics are of nutritional concern, the "total
phenol" content of foods as routinely expressed may not be a
true index of the nutritional quality of foods. The use of
tannic acid as a "reference standard" is also questionable

since its biological properties differ from those of tannins

of flavonoid origin.

We

Protein precipitating assays can be used to determine
either the tannin content of a sample or the biological
activity of tannins (Hagerman and Butler, 1978) . Different
Proteins such as gelatin, casein, bovine serum albumin,
hemoglobin and different enzymes have been used in this assay

by various investigators (Salunkhe et al., 1990) . Each

l7

beans. Recently Deshpande and Cheryan (1985) have critically
reevaluated the vanillin reaction for the detection of tannins
in legume. They modified the 0.5% vanillin assay as described
by Price et al. (1978) , by using appropriate sample blanks to
eliminate variation due to the background color caused by
anthocyanin-like pigments. The inherent color variation
within a given sample can be minimized by the careful
selection of at least three representative samples and
grinding them separately prior to the analysis.

The Folin-Denis method is nonspecific and detects all
phenolic groups in the extract, including those found in
extractable proteins, as well as reacting with reducing
substances such as ascorbic acid (Thompson et al., 1983) .
Since not all phenolics are of nutritional concern, the "total
phenol" content of foods as routinely expressed may not be a
true index of the nutritional quality of foods. The use of
tannic acid as a "reference standard" is also questionable

since its biological properties differ from those of tannins

of flavonoid origin.
WW

Protein precipitating assays can be used to determine
either the tannin content of a sample or the biological
activity of tannins (Hagerman and Butler, 1978) . Different
Prateins such as gelatin, casein, bovine serum albumin,
hemoglobin and different enzymes have been used in this assay

by various investigators (Salunkhe et a1., 1990) . Each

18

protein precipitating assay gives a different response with
tannins of different sources. Many factors that may be
responsible for the differences have been suggested by
Hagerman and Butler (1978) such as the characteristics of the
tannins (molecular weight, structural heterogeneity), the
protein source (degree of glycosylation, amino acid
composition and molecular weight) and reaction conditions (pH,
temperature, reaction time, relative concentrations of the
reactants). Tannins tend to complex with proteins such as
gelatin or specific proline-rich proteins that have a high
content of proline resulting in a protein with a loose
structure (Asquith and Butler, 1986).

M1199;

To understand the biochemistry of physiologically active
tannins, it is necessary to separate and purify tannins in an
unmodified state. Qualitative assays with thin-layer
chromatography (TLC) have evolved to quantitative assays with
high-performance liquid chromatography (HPLC) . More detailed
information on the structure and nature tannins can be
obtained by mass spectral analysis, droplet countercurrent
chromatography, centrifugal partition chromatography (Okuda et
81- , 1989) , nuclear magnetic resonance (NMR) and circular
dichroism (CD) spectroscopy (Jansman, 1994) .

Some attempts have been made to separate various tannin
eAttracts chromatographically on gel permeation columns. The

InOat successful results were obtained with columns of

19
hydroxypropylated dextran gel such as Sephadex LH-20 by using
alcoholic solvents, aqueous methanol or ethanol. According to
Okuda et al. (1989), tannins can be separated on the basis of
differences in adsorptivity of polyphenolic compounds on the
gel rather than on the basis of gel filtration. In a first
step using 95% ethanol as eluent, non-tannin phenolic
compounds were separated from the tannin-containing compounds.
Subsequent elution of the tannin fractions from two sorghum
varieties in aqueous acetone (50/50 v/v) yielded different
chromatographic patterns. Marquardt et al. (1977) used this
procedure to purify and fractionate the tannins from the hulls
of faba beans into four fractions. The fractions differed in
their degree of polymerization which can be attributed to
their solubility in methanol and ether. Fraction A was highly
polymerized, while fraction B and C were intermediate, and
fraction D was found least polymerized. Condensed tannins of
faba bean hulls yielded 2 fractions (A and B) on separation
‘with Sephadex LH-20 column. Fraction A contained at least 15
low molecular weight polyphenolics and B, the major fraction,
contained soluble condensed tannins. When applying the same
chromatography to extracts of a white flowering variety of
faba bean, low molecular weight compounds were still found,
but no peaks were found in the chromatograms which could be

identified as condensed tannins (Marquardt et al., 1977).

20

PHYSIOLOGICAL EFFECTS OF TANNINS

Most of the effects of dietary tannins in animals are
considered antinutritional (Jansman, 1994; Salunkhe et al.,
1990). A few beneficial effects related to stimulation of
estrogen (Jansman, 1994) and production of a flat blood
glucose response (Thompson, 1988) have been attributed to
consumption of specific polyphenolic compounds. Singleton
(1981) stated that dietary tannins at appropriate levels may
have a general antibiotic effect by suppressing the growth of
detrimental flora in the alimentary tract. However, it is
still questionable whether preventive or pharmacological
effects can be expected from tannins occurring in foods and
feedstuff.

The influence of tannins on the overall acceptability and
utilization of food/feed, including nutritional parameters
such as feed intake, weight gain, and utilization of minerals,
has been studied by some investigators. Reports on the
nutritional consequences of dietary tannins are based mostly
on either in vitro studies or experiments with nonruminants.
Some of them have been carried out with isolated tannins from
feedstuffs or with 'standards' of commercial tannins, such as
tannic acid and catechin. Most studies, however, were carried
out with raw or fractionated feedstuffs (e.g. , hulls of legume
seeds) of the same plant species containing different levels

of tannins as analyzed by- one of the available methods. In

21
these studies the effects or differences found were fully or
partly related to the differences in tannin level in the

experimental diet.

WWII!

Conflicting reports have been published on the effect of
dietary tannins on feed intake. Because tannins are known to
have a bitter or astringent taste, a depression of food intake
might be expected (Glick and Joslyn, 1970; Reddy et al.,
1985) . In contrast, it has been suggested that a slightly
astringent taste increases the palatability of feed and
stimulates feed intake (Gupta and Haslam, 1980) . An increased
feed intake was found in chicks fed sal seed (Shores robusta) ,
a seed that contains high levels of hydrolyzable tannins
(Zomabade et al., 1979) . An opposite effect, however, was
found in cockerels by Ahmed et al. (1991) .

The physical basis for astringency may be that tannins
bind and perhaps precipitate salivary mucoprotein. This
effect would reduce the lubricating property of saliva, give
the mouth the feeling of dryness and affect the ability to
swallow the food (Mole, 1989) . Direct binding of tannins to
taste receptors may be a second more direct mechanism by which
tannins affect palatability of food. Glick (1981) , however,
reported the effect of tannin (especially gallic acid) on food
jJ'l‘l:ake is not mediated entirely through taste aversion or

through other gastrointestinal factors, since a continuous

22
daily infusion of a gallic acid solution (2%) resulted in a
significant reduction of food intake.

TABLE 2 summarizes some effects of dietary tannins in
several feedstuffs on the performance of rats, poultry and
pigs. The level and type of tannins as well as differences
among animal species may explain the conflicting results with
respect to the effect of tannins on feed intake (Jansman,
1994).

A high mortality rate was reported in chickens and rats
due to supplementation with 5% tannic acid (Click and Joslyn,
1970; Vohra et al., 1966) , but no mortalities occurred at a 5%
level of catechin (Click and Joslyn, 1970). Similar effects
have been observed with the addition of tannins to swine
diets (Salunkhe et al., 1990). Glick (1981) and Reddy et al.
(1985) reported that the ingestion of certain polyphenols by
chicks and rats reduced growth. Marquardt et a1. (1977)
reported that chicks fed a diet containing 3.9% of condensed
tannins extracted from faba beans had markedly depressed
growth rate. Martin-Tanguy et al. (1977) found that high
tannin content horse beans reduced laying rates of hens.
Feeding of tannin-rich faba beans in a longer trial increased

mortality (Lindgren, 1975).

Tannin consumption in animals causes a decrease in

Protein digestibility (Jansman et al., 1993) and possibly

23

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24

also in humans due to formation of insoluble protein-tannin
precipitates in the gastrointestinal tract (Butler et a1. ,
1989). Tannins, by definition, are able to form complexes
with proteins. Hydrogen bonds and hydrophobic interactions
appear to be the principal linkages involved (Artz et al.,
1987). Casein, bovine serum albumin, 61 protein from beans,
and carob pod proteins resist proteolytic digestion when
complexed.with tannins (Desphande and Damodaran, 1989). Such
complexes may not be dissociated in the range of pH existing
in the gastrointestinal tract and thus may be excreted in the
feces (Reddy et al., 1985).

Using a competitive binding assay, Hagerman and Butler
(1981) clearly showed differences in binding affinities
between proteins and tannins. Asquith and Butler (1986)
confirmed the earlier reports in studies of quebrancho, wattle
and pinto bean tannins. They concluded that condensed tannins
from either sorghum or pinto bean had a particularly high
affinity for proline-rich proteins.

Griffiths (1981) found that removal of tannin-containing
hulls had a significant positive effect on the solubility of
faba bean proteins. This effect on protein solubility was not
found in low-tannin varieties of faba beans.

Tannins are also known to interact with carbohydrates,
particularly starch, although their affinity seems to be less
than for proteins. Deshpande and Salunkhe (1982) studied the

interaction of tannic acid and catechin with starches of

25
different legumes. Processed amorphous amylopectin and
amylose associated more with phenolic compounds than did
native starch. The in vitro digestibility of starches
associated with tannic acid or catechin was reduced by 9-17%

compared with control group.

WW
Knowledge about the effect of tannins on mineral

utilization is limited. Tannins are known to form complexes
with divalent metal ions, rendering them less available for
absorption (Salunkhe et a1., 1990). Among the minerals, the
relationship between polyphenol compounds and iron has been
studied the most. Interactions of tannins with other minerals
such as copper and zinc has received little attention

(Jansman, 1994).

Iron

Tannins from different species have variable effects on
iron utilization and tannins did not significantly depress
iron absorption in some studies (House and Van Campen, 1994).

The inhibition of iron absorption observed when tea
(Disler et al., 1975; Hallberg and Rossnader, 1982; Brune et.
al., 1989), some wines (Bezowda et a1., 1985) or coffee
(Hallberg and Rossander, 1982; Morck et a1., 1983; Brune et
a1., 1989; Wang and Kies, 1991) are drunk with a test meal has

been ascribed to the content of phenolic compounds in these

26

beverages. Epidemiological relationships have also been shown
between coffee intake and low' serum. ferritin values in
menstruating women (Soustre et al. , 1987) , between coffee
intake and low maternal and infant hemoglobin in pregnant
women (Munoz et al., 1988), and between tea drinking and a
higher incidence of anemia in children (Merhav et al., 1985).
Certain vegetables have been reported to decrease iron
absorption in proportion to their content of phenolic
compounds (Gillooly et al., 1983; Tuntawiroon et al., 1991).
All of the recent investigations have demonstrated poor
bioavailability of iron contained in legumes but the factors
responsible are poorly understood. Torrance et al . (1982) and
Rao and Prabhavathi (1982) have suggested that tannins are
responsible for the low bioavailability of iron in legumes.
Merhav et al. (1985) suggested the mechanism is related to a
chelating effect of the tannins on metallic iron. Griffiths
(1982) found a high iron-binding capacity of extracts from
seed coats of colored-flowering varieties of faba beans while
white-flowering varieties did not show this property.

House and Van Campen (1994) conducted a series of studies
to assess the effects of tannins extracted from hulls of
Ibeans. They reported that tannins in single meals did not
affect 59Fe absorption by moderately anemic, growing rats. In
&a long term feeding trial, they observed some impairment in
.iron absorption in growing rats resulting from consumption of

‘tannin-containing diets at 0.5% level. In contrast, tannins

27

did not affect 59Fe absorption by mature rats. They concluded
that bean tannins consumed as part of typical diets probably
have little adverse effect on iron absorption. In a human
study using a bean soup test meal, Lynch et al. (1984) showed
that iron absorption was uniformly low, but not statistically
significant from a reference meal. Garcia-Lopez et al. (1990)
found that in rats iron absorption from legume-containing test
meals was reduced only when legumes were fed before and after
the test meal but was not reduced when casein was the only
protein source in the habitual diet. They suggested that
tannins per se did not have an inhibitory effect on iron
absorption but habitual feeding of diets with a high tannin
content may lead to changes in the intestine which in turn may
alter the absorption of iron.

A series of studies were conducted to explain the
different effects of phenolic compounds on mineral absorption
observed with coffee/tea and legumes. Using a weanling rat
model, Greger and Lyle (1988) reported that ingestion of 1.17%
catechin did not affect iron metabolism. However, Gillooly et
a1. (1983) found that addition of tannic acid to a broccoli
meal dramatically reduced iron absorption from 29.7 to 1.5
percent in human subjects. Similarly, Siegenberg et al.
(1991) reported the addition of tannic acid to white-bread
test meals resulted in a dose-related inhibition of iron
absorption. The maximal inhibitory effect was achieved

between 12, 26, and 55 mg tannic acid/ 80 g bread. The

28
dose-response curve then leveled off, even with very large
doses (263 and 833 mg) of tannic acid reducing absorption to
about one-fifth. This finding implies that the interference
of tannic acid on iron absorption is not related to its
protein-precipitating effect, since white bread has a low
content of protein.

In human subjects, Brune et a1. (1989) studied the
relationship between the phenolic structure of tannins and
inhibitory effect on iron absorption. They found the
inhibition of iron absorption by tannic acid was strongly
dose-related. The smallest amount (5 mg) inhibited absorption
by 20%, 25 mg by 67% and 100 mg by 88%. Gallic acid inhibited
iron absorption to the same extent as tannic acid, per mol
galloyl groups. However, no inhibition on iron absorption was
observed when catechin was added to the test meal. These
results showed that the galloyl groups may be a major
determinant of the inhibitory effect of phenolic compounds on
iron absorption; however, catechin, a phenolic catechol group,
seems to be of minor importance in iron absorption.
Furthermore, Brune et a1 . (1989) suggested that the inhibitory
effect of galloyl groups is due a direct complex formation
between the phenolic hydroxyl and the iron ions. They
reported that molecules with aromatic rings bearing two
hydroxyl (catechol group) or three hydroxyl (galloyl group)
positioned at adjacent carbon atoms have iron-binding

properties in vitro. The monomer gallic acid and its polymer

29
tannic‘ acid have ten galloyl groups, and these substances
readily yield blackish precipitates when added to an iron
solution, Catechin which has one resorcinol (meta-dihydroxy)
group in the A-ring and one catechol (ortho-dihydroxy) group

in the B-ring does not form complexes with iron.

zinc

The effects of tannins on zinc utilization have received
little attention. Greger and Lyle (1988) reported that rats
fed diets with added 1.17% catechin showed increased zinc
concentrations in the tibia but not kidney or liver. Greger
and Emery (1987) observed that rats fed coffee and
decaffeinated coffee that contained 5% polyphenols had
elevated tibia zinc concentration. The effect of various
forms of tea with different tannin profiles on tissue content
of zinc was conflicting; IRats fed tea (desiccated.and liquid)
tended to have higher tibia zinc; the difference was
significant for those given liquid tea (Greger and Lyle,
1988). In contrast, in the same study chronic ingestion of
tea was associated with lowered zinc concentrations in kidneys

and decreased apparent absorption of zinc.

Copper

Knowledge of copper requirements, absorption, and
transport and of the biological roles of copper has

accumulated over the past 60 years, but remains frustratingly

30
incomplete today (Danks, 1988). Much remains to be learned
about the transport of copper to and within body cells. Thus,
reviewing some basic concepts of copper metabolism will be
presented before discussing the effects of tannins on copper
bioavailability.

The adult (70 kg) human body contains 80 mg copper, but
reported values range from 50 to 120 mg (O'Dell, 1990). The
tissue copper concentration is age related, with the newborn
and very young values normally being higher than adults. The
highest tissue concentration in most animals occurs in liver
with variably lesser amounts in the heart, kidney, spleen and
brain (Hunt and Groff, 1990). The essentiality of copper is
due to its participation as an enzyme activator or inhibitor
in critical reactions. The U.S. adult recommended daily safe
and adequate range of copper is 1.5 to 3.0 mg, but the average
American consumes 1.2 to 0.9 mg copper/day, respectively, from
1982 to 1986 (NRC, 1989). The homeostatic regulation of
copper is, therefore, of prime importance.

A steady level of copper in the body depends on.a balance
between intestinal absorption and biliary excretion, with
small additional losses in sweat and skin (Danks, 1988). The
short half-life of the copper isotopes (5‘Cu: 12 hr and.57Cu:
61 hr), the lack of their availability at specific time
periods, and the limited use of gamma emitters in humans
dictated by human subjects have hampered studies of copper

absorption in terms of its incorporation into and release from

31
copper enzymes (Lonnerdal et al., 1985; Sandstrom et al.,
1993). Figure 5 shows the principles of mineral metabolism
(Sandstrom et a1., 1993). Absorption of copper is maximal in
the duodenum with both active and passive components to the
system.(O'Dell, 1990). Research has shown that up to 12 ug of
64Cu administered orally to rats is absorbed rapidly with.peak
absorption at 30 minutes, and 30% of the dose is absorbed by
8 hr (O'Dell, 1990). Within the intestinal mucosal cells
copper can interact with metallothionein (MT). MT is an
unusual protein that contains 61 amino acids, 20 of which are
cysteine. Many experimental findings suggest that MT acts to
bind and detoxify excess copper especially in kidney and liver
(Danks, 1988). O'Dell (1990) suggested that intestinal MT
serve primarily as a negative rather than a positive modulator
of copper absorption. A role of MT in transport of copper
within.the liver cells, however, has been suggested since some
studies have shown.57Cu to bind to MT as soon as it is taken
into liver cells (Danks, 1988).

Newly absorbed copper is bound primary to albumin as the
transport form in the portal blood (Cousins, 1985) . The
conventional view has been that copper is taken up by the
liver and incorporated into ceruloplasmin (Cp) , a glycoprotein
containing six atoms of copper per molecule. Cp is released
into the blood where it normally constitutes 90% of the copper
present in plasma (O'Dell, 1990). Cp carries copper to

nonhepatic tissues for synthesis of cuproenzymes such as

32

 

 

 

 

 

 

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(“kinda

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it u ii u

 

 

 

 

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El

 

Adapted from: Sandstrom et al. (1993). p 79.

33

cytochrome c oxidase, superoxide dismutase and lysyl oxidase
(Cousins, 1985) . However, the mechanism(s) by which Cp copper
is made available to cells is unclear (O'Dell, 1990). Cp has
a ferroxidase activity that has been considered important in
the release of iron from stores. Cp is believed to be
necessary for the attachment of iron to transferrin for
transport in the plasma by catalyzing the oxidation of ferrous
iron to ferric iron (Linder, 1985). It also has amine oxidase
activity against a range of biologically active amines.

Copper is primarily excreted via the gastrointestinal
tract with < 3% of the intake appearing in the urine. Biliary
excretion represents the primary route by which gut-absorbed
copper is excreted (Aoyagi and Baker, 1993) , although a
significant percentage is probably lost through intestinal
secretion and sloughing off of cells (Linder, 1985). Biliary
excretion increased substantially with excess copper intake
(Danks, 1988) . The mechanism by which copper eventually
enters the bile and is excreted is very poorly understood.
There is not even consensus about the molecular form.of copper
present in the bile (Aoyagi and Baker, 1993).

Factors that influence copper bioavailability have been
revealed recently. Amount and type of protein may have an
effect on the availability of copper in neonates (Sandstead,
1982). Cow's milk and dairy products contain little copper
and that present.is poorly absorbable (Lonnerdal et al., 1985;

Danks, 1988). Since the digestibility of cow's milk casein

34

may be low in infants, the bioavailability of copper bound to
cow's milk casein may also be low (Lonnerdal et al., 1985).
A high dietary intake of ascorbic acid decreases copper
bioavailability, impairs copper absorption from ligated
intestinal segments of the rat, and lowers blood and liver
copper concentrations in guinea pigs and human (O'Dell, 1990;
Finley and Cerklewski, 1983). Compared.with starch, fructose
and sucrose aggravate the signs of deficiency, suggesting a
negative relationship between fructose and copper status
(Johnson and Flagg, 1986; Fields et al., 1986). Danks (1988)
stated that fructose increases the requirement for copper,
rather than interfering with its absorption. O'Dell (1990)
suggested that both fructose and ascorbic acid are strong
reducing compounds and may convert copper to the cuprous
state, thereby impairing utilization as well as absorption.

Little has been published on the effects of tannins on
copper metabolism (Jansman, 1994). Kies and Dmoren (1989)
reported in human studies that feeding 21 g of high (red)
tannin wheat brans for 14 days had a greater negative effect
on apparent copper utilization than low (white) tannin wheat
brani They also observed that after consumption of 8 grams of
black tea infusion daily for ten days fecal copper losses in
human subjects were significantly increased. Kies and Umoren
(1989) thus demonstrated that tannins in wheat bran and tea

may have contributed to decreased copper utilization.

35

In contrast, Greger and Emery (1987) showed that rats fed
either coffee or decaffeinated coffee, which has been
estimated to contain 5% of tannins (Brune et al., 1989), had
significantly higher concentrations of copper in their livers
‘than the control group. Plasma copper and ceruloplasmin and
kidney copper levels tended to be greater in rats fed coffee
although the differences were not statistically significant.
Similarly, Greger and Lyle (1988) showed that in rats
ingestion of diets containing more than 1.17% black tea
enhanced copper absorption leading to elevated liver copper
level. Ingestion of black tea by anemic rats elevate plasma
ceruloplasmin activity and tended to elevated plasma copper
compared to those of pair-fed control animals. However, when
rats were fed the same level of catechin, similar results were
not seen, thus suggesting that catechin in black tea was
unlikely responsible for this effect. DiSilvestro and Harris
(1983) observed that in normal chicks injection of catechin
stimulated lysyl oxidase activity in aorta tissue. Lysyl
oxidase is a copper-containing enzyme that aids in the
crosslinking of elastin and collagen, a role that is integral
to connective tissue and blood vessel maintenance. Copper is
the major regulator of lysyl oxidase in copper-deficient
animals. O'Dell (1990) stated that in copper deficient
animals both ceruloplasmin and lysyl oxidase activities
respond rapidly and correlatively to copper repletion.

DiSilvestro and Harris (1983) suggested that catechin may make

36
more Of the absorbed copper available to the enzyme thus
producing the large increase in copper-induced activation of
lysyl oxidase in catechin-treated chicks.

Based on the limited reports in the literature, the need
for additional research to clarify the effect of tannins on
copper utilization is apparent. Experiments by Greger and
Emery (1987) and Greger and Lyle (1988), which included only
tea, coffee and catechin gave inconsistent results.
DiSilverstro and Harris (1983) injected rather than fed
catechin and did not measure tissue copper. Furthermore, the
mechanism(s) by which catechin from various sources (chemical
form, coffee, tea infusion, other'plants, or beans) may affect
copper metabolism in vivo is unclear. As Salunkhe et al.
(1990) stated : "...... more research is needed to ascertain
the harmful and beneficial effects of dietary tannins, and fix
the threshold levels which will again vary with the type or
source of tannins and the consumers.". The purpose of the
present research was to test the hypothesis that tannins in
pinto bean hulls had a inhibitory effect on copper
bioavailability.

MATERIALS AND METHODS

MATERIALS

Ten kinds of dry beans including black eye peas, domino
black, faba, lentil, lupin, kidney dark, kidney light, pinks,
pinto and small white from Morrice Grain and Bean Company
(Morrice, MI) were kindly provided by Dr. Uebersax. The
following brands of pinto beans were purchased from local
sources: Spartan, Sysco and Brown's Best. Catechin-(+) ,
vanillin (vanilaldehyde), O-dianisidine dihydrochloride,
glycerol and the hemoglobin kit (Catalog No. 525-a) were
purchased from Sigma Chemical (St. Louis, MO). Methanol, 30%
hydrogen peroxide (H202) , sodium acetate-3H20, concentrated
sulfuric acid (H2804), copper, zinc, and iron standard
solutions (1000 ppm) were purchased from J.T. Baker Chemical
(Phillipsburg, NJ). Bovine liver (No 1557a) and wheat flour
(No 1567a) were purchased from the National Institute of
Standards and Technology (NIST) (Gaithersburg, MD). Metofane
(methoxyflurane) was from Pitman-Moore (Mundelein, IL),
hydrochloric acid (HCl) and glacial acetic acid were from
Mallinckrodt (Paris, Kentucky), ultrapure concentrated nitric

acid (HNO3) was from Fischer Scientific (Pittsburgh, PA).

37

38

RESEARCH DESIGN
222W

Preliminary experiments were done to determine the
quantities of condensed tannins in various beans having
different seed coat colors using the 0.5% vanillin-HCl assay
described by Deshpande and Cheryan (1985) . Experiments were
done to determine the influence of extraction time on tannin
values. Relative distribution of tannins in seed coat and
cotyledon was determined in beans from different sources.
Mineral content (copper, zinc and iron) of dry beans including
whole beans, cotyledon, and hull was determined using atomic
absorption spectrophotometry (AAS) (Perkin-Elmer 2380,

Norwalk, CT) .

AnimaLSfudiee
Experiment I: The effect of feeding commercial catechin

or condensed tannins in pinto bean hull on copper utilization
was determined in weanling Sprague-Dawley male rats (Harland,
Indianapolis, IN). After 14 days of adaptation the rats were
assigned to three groups (eight rats per group) based on body
weight. Rats were fed ad libitum for 21 days one of the
three dietary treatments: 1) control diet without bean hulls,
2) control diet with 1.6% catechin, 3) control diet with 25%
Pinto bean hulls.

At the end of three weeks, all rats were anesthetized

With metofane and then killed by open cardiac puncture. Blood

39
samples collected from the heart were centrifuged at 1500 x g
for 20 minutes; plasma was removed and frozen. Livers,
tibias, and kidneys were rinsed in saline, cleansed of
adhering matter and frozen.

EXperiment II: To further clarify the effect of pinto
bean hulls on copper utilization, a second experiment was done
comparing diets with and without pinto bean hulls. After
three days of adaptation Sprague-Dawley male rats were
assigned to two groups (eight rats per group) based on body
weight. Rats were fed ad libitum for 15 days one of the two
dietary treatments : 1) control diet without bean hulls, 2)
control diets with 25% pinto bean hulls.

Rats in Experiment II consumed more food and grew more
rapidly than rats in Experiment. I. In order to have
comparable body weights in both experiments, rats in
Experiment II were killed after 15 days of dietary treatment.
Procedures for collection of samples were the same as those
used in Experiment I. .

Care and treatment of rats were approved by the All
University Committee on Animal Use and Care at Michigan State

University.
PREPARATION OF BEANS

Whole bean samples used for tannin analyses were ground

to obtain flours of 820 micron particle size (100% of the

40
samples passed through 20-mesh screen) using a micro-mill
(Chemical Rubber company, Cleveland, OH). In order to obtain
the hulls and cotyledon, dry pinto beans were soaked in
deionized water (4:1 water:bean) for 4-6 or 17-19 hr; the seed
coats were then manually removed from the cotyledon and air
dried. Dried.hulls and cotyledon were then ground separately
into flours of 820 micron particle size using a coffee mill
(Robert Krups company, Denver, CO). Samples used for mineral
analyses were prepared in the same way as described for the
tannin analyses. Pinto beans (Brown's Best) for the rat
experiment were soaked for 4 hr and hulls were prepared

following the same procedures described above.

ANIMALS

Rats were housed individually in suspended stainless
steel cages in a temperature- and humidity-regulated room with
alternating 12-hour light/dark cycle with lights on at 7:00
am. Temperature was maintained at 20-22° C, humidity at 68-
70%. Food consumption and body weights were measured twice
weekly. Deionized water was supplied ad libitum throughout
the studies using water bottles with plastic caps and

stainless steel sipper tubes.

41

DIETS

The composition of the diets was formulated in accordance
with general guidelines prepared by the American Institute of
Nutrition (AIN, 1976; Reeves et al., 1993) with modifications
in the amounts of cornstarch, fiber, copper, zinc and iron
contents (TABLE 3 and 4). The levels of copper, zinc and iron
in the diets were adjusted according to the levels present in
the pinto bean hull diets used in each study. In each dietary
treatment, 25 g/ 100 g diet of cornstarch was substituted
either by the same amount of pinto bean hulls in the hull
diets or by the same amount of cellulose in both control and
catechin diets. The 25% cellulose was added to the control
and catechin diets in order to compensate approximately for
the fiber present in hulls diet (Srisuma, 1989). In
Experiment I, the catechin and hulls diets were formulated to

contain a similar amount (1.6%) of condensed tannins.

ANALYTICAL METHODS

_ Tannins

The tannin content of beans was determined using the 0.5%
vanillin assay of Deshpande and Cheryan (1985) (Figure 5).
The procedure was as follows: 200 mg of dried ground beans or
1.0 g of diets was extracted with 10 ml absolute methanol for

20 minutes with mechanical shaking. In a separate experiment

42

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44

 

Ground sample 200 mg

 

10 ml methanol I _

 

Extraction 20 min
centrifuge 1500 g x 10 min

 

Residue discarded 1 ml Supernatant

 

 

 

8%HCl/MeOH —} 5"“

1.1 Reagent 5 ml
° 4 % HCI lMeOH
1% Vanillin] MeOH J i

‘1

 

 

 

 

 

 

 

w "
Sample Mix Blank Mix

20min 20min

A 500 Sample A500 Blank

T

Asoo

Figure 5 Procedure for 0.5 % vanillin assay

 

Adapted from: Salunkhe et al. (1990). p 82.

45

extraction times of 20 min and 24 hr were compared. Samples
were then centrifuged at 1500 x g for 10 min and 1 m1 of the
supernatant was added immediately to 5 m1 of the color reagent
which was composed of a 1:1 mixture of 8% HCl/methanol and 1%
vanillin/methanol. After 20 min, the absorbance was read at
500 nm against a sample blank in which the vanillin was
deleted from the color reagent. The tannin concentration was
calculated from a standard curve using catechin-(+) standards
of 5, 10, 20, 30, 40, 50, 60 and 70 mg catechin/50 ml
methanol. The tannin content of the samples was expressed in
terms of mg catechin equivalents on a dry weight basis.

All the extractions were carried out at least in
duplicate; tannin assays were run in triplicate. All the

assays were conducted at room temperature (2511°C).

Wires;

Bean and rat diets: Triplicate 1 9 samples of ground
beans were weighed and placed in 25 ml Erlenmeyer flasks with
10 ml concentrated HNO3 and 2 to 3 glass beads. The flasks
were placed on a hot plate at low heat for 1 to 2 days and
then allowed to go to dryness. Five ml of 30% 8202 was added.

If a white ash was not obtained, more H§02(5 to 10 ml) was
added. When digestion was complete, samples were diluted with
0.1 N HCl (copper, 5 m1; zinc/iron, 25 m1) prior to analyzing
by AAS. TABLE 5 summaries the analyzed values for mineral

content of the diets used in Experiment I and II.

46

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47

Plasma: In Experiment I, plasma samples were diluted 1:7
and 1:21 with deionized water for analysis of copper and zinc,
respectively, by atomic absorption] emission spectrophotometry
(AAES) (Smith-Hieftje 4000, Thermo Jarrell Ash company,
Franklin, MA). To determine total plasma iron, samples were
deproteinized with 20% trichloroacetic acid (TCA) in a ratio
of 1:2 and incubated at 90° C for 15 minutes (Olson and
Hamlin, 1969) . After protein precipitation, samples were
cooled and centrifuged at 1500 x g for 15 min. Iron standards
were also prepared and treated with TCA in the same way- as
samples and then analyzed for iron by AAES.

In Experiment II, plasma was analyzed for copper and zinc
only. Plasma samples were diluted 1:1 and 1:7 with deionized
water and analyzed for copper and zinc, respectively by AAS
(Perkin-dlmer Model 2380, Norwalk, CT). Standard solutions
were made by diluting stock standards with 50 and 12.5%
glycerol solutions for copper and zinc, respectively. The
glycerol solution was used to compensate for the viscosity of
the plasma.

Tissues: Livers, kidneys and tibias were freeze-dried
(Vacudyne, Chicago, IL) to a constant weight. Liver samples
were crushed manually. Duplicate 500 mg liver samples , one
kidney (approximate 0.28 g) and one tibia (approximate 0.43 g)
were soaked overnight in 10 ml concentrated HN03 to allow
tissues to dissolve in the acid. Samples were further heated

1 to 2 days until dry; then 5 to 10 ml of 3202 was added to

48

complete ashing. Ashed samples were diluted with 0.1 N HCl:
5 ml for copper, 25 ml for liver and.kidney zinc and iron, 50
ml for tibia zinc and iron. If a white ash was not obtained
or particles were present in the flask when diluted with 0.1
N HCl, repeat ashing was necessary.

NIST Standard: Standard reference materials of bovine
liver and wheat flour were digested and analyzed using the
same procedures as for tissues samples to check the validity

of the methodology.

W

Ceruloplasmin activity of plasma samples was determined
by a colorimetric procedure described by Schosinsky et a1.
(1974). This assay relies on the diamine oxidase activity of
ceruloplasmin which reacts with the diamine groups of 0-

dianisidine dihydrochloride.

Win

The concentration of hemoglobin in blood samples was
determined using the cyanomethemoglobin method described by
Crosby et al. (1954). Hemoglobin concentration was expressed

as g/dl.

49

$§Q§i§§i§§l_5n§l¥§£§

EXperiment I: Data were subjected to a one-way analysis
of variance at the 95% confidence level to determine the
significance of differences between means. Significant
differences among the treatments were determined by Fischer's
least significant differences (LSD) test when appropriate.

Emperiment II: Student's t-test was applied to compare
all data. In order to see the overall effect of the hulls
diet in both experiments, data were analyzed using a 2 x:2

randomized ANOVA.

RESULTS

PRELIMINARY ANALYSES

The tannin concentration of the ten different kinds of
dry beans varied widely, ranging from non-detectable to 1139
mg catechin equivalent (CE) [1009 whole bean with an extraction
time of 20 min (TABLE 6). With an extraction time of 24 h
values ranged from non-detectable to 232 mg CE/100 g whole
bean. Tannins were not detectable in the white seeded beans
such as small white and lupin beans. .Among the colored seeds,
pinto beans contained more tannins than did the kidney beans.
Light kidney beans had more tannins than did dark kidney
beans, whereas dark.colored.back eye pea.had.the lowest tannin
concentration of these beans. Tannin concentration in all ten
dry beans after 24 h extraction time, the recommended
extraction time (Burns, 1971), had actually decreased.

Data on relative distribution of tannin in four brands of
pinto beans are shown in TABLE 7. Different brands of pinto
beans had similar tannin concentrations in the whole beans.
Brown's Best had the highest tannin value both in the whole
beans and in hulls. The lowest whole bean tannin value was
seen in the Morrice brand. Cotyledons from all the brands

consistently contained non-detectable amounts of tannins.

50

51

 

TABLE 6

Tannins concentration of ten different dry beans1

 

mg Catechin Equivalent / 100 g dry wt of beans

Dry Beans

20 min extraction 24 h extraction

 

Black eye peas 250:15 ND
Domino black 320:28 158:10
Faba 166115 ND
Lentil 391127 ND
Lupin ND ND
Kidney dark 460i 7 461 4
Kidney light 575:18 156:12
Pinks 233:16 ND
Pinto 1139:17 232:22
Small white ND ND

 

1 Mean t SEN (n=3); ND

not detectable.

52

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53

TABLE 7 also summarizes the relative distribution of
mineral concentrations in the four brands of pinto beans.
Cotyledons contained somewhat lower concentration of iron than
whole beans, while hulls were the most abundant source of iron
in these beans, irrespective of the brand. The iron
concentration both in hulls and cotyledons was quite similar
irrespective of brand or the length of soaking time. For
zinc, both cotyledons and hulls contained amounts similar to
those in whole beans. A wide range (4.8 to 11.6 ppm) of
copper concentrations was found in hulls in the various
brands; however similar concentrations of copper were observed

in cotyledons.

ANIMAL STUDIES

W

In Experiment I, food intake and body weight gain of rats
fed diet diets containing 1.6% catechin or pinto bean hulls
with an equivalent tannin concentration were not significantly
different from those of controls (TABLE 8). Weight gain
tended to be somewhat lower in rats fed the pinto hulls diet
and resulted in a significantly decreased efficiency of food
utilization compared to rats fed the control diet. Although
food intake was similar among the three groups, total copper,
zinc and iron intakes were different because the analyzed

concentrations of minerals in the diets were different (TABLES

54

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55
5 and '9) . Rats fed the pinto hull diet had higher total
intakes of copper and zinc but lower intake of iron than rats
fed control and catechin diets.

Compared to controls, rats fed 25% pinto bean hulls in
Experiment II showed significantly higher food intake but not
body weight gain or food efficiency (TABLE 8). Although diets
had similar concentrations of minerals, total copper, zinc and
iron intakes were higher in the hull-fed group because of
their greater intake (TABLE 9).

mm

Results in both experiments showed rats fed the pinto
bean hulls diet had smaller relative liver weights (TABLE 10).
In Experiment I, liver weight and kidney dry weight were
significantly lower in hull-fed rats (TABLE 11). Tibia dry
weight was higher for rats fed the catechin diet. In
Experiment II, the weights of liver, kidney and tibia were
similar in control and hull fed rats.

992931:

In both experiments rats fed bean hulls showed
significantly elevated plasma copper (TABLE 12) . In
Experiment II, hull-fed rats had a significantly higher plasma
ceruloplasmin activity and a tendency toward higher values was
also seen in Experiment I (TABLE 12). No dietary effect on
plasma copper or plasma ceruloplasmin activity, however, was

found in the catechin-fed group compared to the control group.

56

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60
The total copper in tissues was altered by the.hulls diet
treatment in both studies (TABLE 13). Hull-fed rats had a
significantly lower total amount of copper in the liver
compared to those of the control rats. Similar results were
found in kidney and tibia in Experiment I. Liver
concentration of copper was similar in the three groups in
Experiment I, but lower values were found in rats fed the
hulls diet compared to control rats in Experiment II. Results
showed no effect of the catechin diet on either concentration
or total content of copper in tissues.
line
No differences in plasma zinc concentrations were found
in either experiment (TABLE 12). In both studies, rats fed
the hulls diet had a significantly higher liver zinc
concentration compared to control rats (TABLE 14). A similar
increase in liver zinc was found in.rats fed the catechin.diet
in Experiment I. Conflicting results were found in the two
experiments for the effect of the hulls diet on tibia zinc
concentration. Tibia zinc concentration was lower in the
hull-fed rats in Experiment I compared to controls but was
higher in Experiment II. Total tibia zinc was significantly
higher in the catechin-fed rats compared to controls and hull-

fed rats in Experiment I.
11:92

The dietary treatments had no effect on plasma iron,

hematocrits or hemoglobin values (TABLE 12).

61

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DISCUSSION

PRELIMINARY ANALYSES

It is difficult to compare the tannin concentration of
dry beans as reported by different investigators, primarily *
because of the differences in methods and beans sources used
(Price et a1., 1978; Desphande and Cheryan, 1985). The
tannin concentration for pinto beans in the present
investigation was 1139 mg catechin equivalent (CE)/100 mg
whole bean. Using a similar method, Desphande and Cheryan
(1985) reported that pinto beans had a tannin concentration of
306-562 mg catechin equivalent/100 g whole bean. The factors
that may influence the tannin content include storage time,
soil and growing season (Salunkhe et a1., 1990).

Results showing low or no condensed tannins in white
colored beans, compared to colored beans confirms the results
of Cabrera and Martin (1986) and Deshpande et al. (1982). A
wide variation in the tannin concentrations in pigmented beans
was observed ranging from 250 to 1139 mg catechin equivalent/
lOOg dry wt in present study. Studies on the inheritance of
tannin content have shown a high broad-sense heritability
(Salunkhe et a1., 1990). Although black-seeded beans
contained higher amounts of tannin, few plants with nonblack

but colored seed had high tannin concentrations. Tannins were

63

64

detected only in the colored beans; however, no direct
relationship was observed between the intensity of color and
tannin content in the present study which was in agreement
with the observation of Desphande et a1. (1982) . Light kidney
beans had more tannins than did dark kidney beans, whereas
dark colored black eyed peas had the lowest tannin content of
these beans.

The extraction time is an important factor influencing
tannin analysis. In the literature, extraction times of 20
min to 24 hr are used. Many phenols tend to oxidize during
sample preparation and extraction (Salunkhe et a1., 1990) .
Hence, the longer the extraction time, the lower the tannin
content was. Price et a1. (1978) reported that when the
extraction time was extended to 24 hr, there was a decrease in
absorbance of 10 to 15% in assayable tannins. Extraction for
24 hr gave overall 40 to 70% lower estimates of tannin for all
the sorghum varieties studied by Price et a1. (1978) as
compared to a 1 hr extraction. Desphande and Cheryan (1985)
reported that with prolonged extraction periods (> 48 hr) only
10-20% of the total tannins were detected. This decrease
related, to’ extraction ‘times Ihas been. attributed to ‘the
destruction of tannins by HCl, since, even after the extracts
were separated from the grain, the absorbance continued to
decrease with time (Salunkhe et a1., 1990). Deshpande and
Cheryan (1987) observed that the tannin extraction pattern of

dry beans (Phaseolus vulgaris L.) is markedly affected by the

65
solvent used. With absolute methanol, maximum absorbance at
500 nm using the 0.5% vanillin assay of Price et a1. (1978)
was obtained within 10 to 20 min of extraction, and the values
slowly decreased with longer extraction times, presumably due
to tannin oxidation.

Results showing that tannins are mainly located in the
hulls, with negligible amounts in the cotyledons confirms
reports in the literature (Rao and Prabhavathi, 1982).
Desphande and Salunkhe (1982) found that removal of seed coats
lowered the assayable tannin concentration of beans by 68 to
95% and dehulled. pinto beans had. a 94.6% reduction. of
assayable tannin concentration. By determining the tannin
concentration in seed coats, Laurena et a1. (1984) also
detected 2 to 3 times greater concentration of assayable
tannins in eight varieties of cowpeas. Desphande and Cheryan
(1985) showed that when expressed on a total seed weight
basis, seed coats had a 1.1 to 2.5 times greater concentration
of assayable tannins compared to that of dry bean flours.

Different commercial brands of pinto beans had a similar
tannin concentration in whole beans. A wide variation was
observed in the tannin concentration of hulls, 3.5 to 11.8 g
CE/100 g; The length of soaking time for removal of the hulls
may have been the major reason for the difference between the
Morrice and Brown's Best beans. Soaking winged beans and
cowpeas for 24 hr reduced tannin concentration.by 50% and.over

50% as reported by Sathe and Salunkhe (1981) and Laurena et

66

al. (1986), respectivelyu Desphande and Cheryan. (1983)
observed that pinto beans with the highest tannin
concentration showed the greatest reduction in tannin
concentration after soaking, and the leaching of bean tannins
on soaking in distilled water increased with the time of
soaking. They reported tannin losses averaging 17%, 41%, and
49% when beans were soaked in water for 6, 12, and 18 hr,
respectively. Since tannins are present in the outermost
layers of the seed, during soaking they may diffuse into the
seed and bind with proteins (Salunkhe et al., 1990).
Desphande Tand. Cheryan (1985) further suggested. that ‘the
apparent loss of tannin was caused by the formation of
insoluble tannin-protein complexes which were not assayable by
the method employed to determine the tannin content.

Deshpande and Cheryan (1985) reported 6.85 g CE/ 100g
hulls for pinto beans without soaking. The data in the
present study showed 3.5 g CE/100 g hulls with 17 hr soaking
and 11.8 g CE/100 g hulls with 4 hr soaking. The conflicting
results in the two studies could be related to differences in
source of beans, preparation or time of storage. Deshpande
and Cheryan (1985) stated the storage time was an important
factor affecting the assayable tannin concentration of beans
especially after milling. They reported a steady decline in
tannin concentration of pinto beans with time after milling.
In addition, the beans used in their study had been harvested

in 1980 and stored for 5 years before analyzing.

67

Little attention has been paid to the distribution of
mineral content in various bean fractions. In present study,
hulls were the most abundant source of iron in these beans,
irrespective of the brand. Cotyledons had somewhat lower
amounts of iron than whole beans. The iron concentration of
pinto bean hulls ranged from 140 to 161 ppm. Srisuma (1989)
investigated four cultivars of navy beans and reported a wide
range (60 to 172 ppm) for the iron.concentration.in.the hulls.
For zinc and copper, both cotyledon and hulls contained levels
similar to those in whole beans. In the present study, the
zinc value in hulls was from 28.2 to 33.7 ppm; Srisuma (1989)
showed a similar range from 16.7 to 35.1 ppm. Values for the
copper concentration of whole pinto beans, 4.8 to 11.6 ppm,
were similar to those reported in the literature (Srisuma,

1989).

ANIMAL STUDIES

Results for the effect of diets containing 25% pinto bean
hulls on food intake and efficiency were different in the two
experiments. In Experiment I rats fed 25% pinto bean hulls
had a reduction in food efficiency and no change in food
intake, but in Experiment II hull-fed rats had a higher food
intake and no change in food efficiency. When results from
the two experiments were combined statistically using 2x2

ANOVA, no significant influence on food intake and body weight

68
was shown. Similarly, Garcia-Lopez et a1. (1990) reported no
effect on total food intake and food utilization in anemic
rats fed 2.4% red kidney bean hulls for 3 weeks. Edwards et
al. (1973) found no evidence of growth depression after
feeding sheep 62% faba bean hulls for 28 days. Levrat et a1.
(1993) stated that in rats food intake and weight gain were
not affected by addition of 1% condensed tannins extracted
from quebrancho to the diet. Chang et a1. (1994) reported
that feeding rats 0.057% condensed tannin isolated from tea or
cowpeas for 28 days did not significantly change the food
intake or body weight. Jansman et a1. (1994) observed that
food intake and weight gain of rats consuming a diet of 1.41%
tannins extracted from faba hulls did not differ significantly ‘
from those of control rats. However Jansman et a1. (1994) , in
the same study found significantly decreased food intake and
lower weight gain in rats fed 60% of faba bean hulls which
were estimated to contain 1.99% condensed tannin. Jansman et
a1. (1993) showed that in piglets incorporation of 20% faba
bean hulls estimated to contain 3.3% condensed tannins had a
tendency to decrease food consumption and resulted in a
significant reduction in weight gain. Moreover, Marquardt et
a1. ( 1977) previously reported that chicks fed a diet
containing 3.9% of condensed tannins purified from faba bean
had markedly reduced food intake compared to those of control.
In Experiment 1, rats fed the 1.6% catechin diet ate normally

but tended to have a somewhat lower food efficiency. This

69

finding is consistent with the observation of Greger and Lyle
(1988) using paired-fed rats. Conversely, Joslyn and Glick
(1970) reported that diets containing 2% and 4% catechin
caused a depression effect of 17% and 45%, respectively, on
growth in rats compared to controls. These conflicting
results for the effect of tannins on food intake and food
efficiency might be explained by both the level and the source
of the tannins as well as the animal species involved. Some
authors found evidence for differences in the sensitivity
among animal species for tannins in sorghum (Elkin et a1.,
1990; Mehansho et a1., 1987) and faba bean (Ford and Hewitt,
1979) . These data also suggested a threshold effect of
condensed tannins on food intake in rats at a level of about
2% of the diet.

Tannic acid when fed to different animal species has been
shown to affect different internal organs. Chang and Fuller
(1964) reported fatty livers in chicks fed diets containing
tannic acid. Karim et a1. (1978) observed necrosis of the
liver and kidney of chicks fed diets containing 1-3% tannic
acid. Jansman (1994) concluded that the effects on the liver
and kidney indicate that either tannic acid itself or
degradation products of tannic acid (e.g. gallic acid) are
absorbed from the small intestine and cause toxic effects.
However, it is not completely clear from the literature
whether the condensed tannins affect internal organs,

especially liver and kidney. This is related to limited

70

knowledge about whether or not condensed tannins are absorbed
from the digestive tract. In the present study, rats fed the
hulls diet had significantly diminished liver relative weight
and tended to have decreased wet and dry liver weights
compared to control-fed rats. Similarly, Marquardt et a1.
(1977) reported that incorporation of 3.9% condensed tannins
extracted from faba bean decreased liver weight in chicks. In
contrast, Chang et al. (1994) found that diets containing
0.057% tannins isolated from cowpea had no effect on either
liver weight or relative liver weight in rats. Moreover,
Jansman et a1. (1993) observed no effect on liver weights in
piglets fed 3.3% of condensed tannins isolated from.faba beans
hulls. These conflicting results might be explained by specie
differences in sensitivity to the condensed tannins. However,
results in the present study showing relative liver weights in
rats fed 1.6% catechin similar to those of controls suggest
that factors other than condensed tannin in hulls may have
contributed to the decrease in relative liver weight.

Components of pinto bean hulls appeared to alter copper
bioavailability. Ingestion of 25% pinto bean hulls elevated
plasma copper and ceruloplasmin activity but.diminished total
liver copper significantly and consistently in the two
studies. The reduction of total liver copper may have been
the consequence of a reduction of liver weight in Experiment
I or decrease of liver copper concentration in Experiment II.

These findings of decreased liver copper in hull-fed rats

71
occurred in spite of higher total dietary intakes of copper.
If the total liver content of copper, however, is expressed as
a percentage of the total dietary copper intake, copper
deposit in hull-fed rats was much lower (5.1%) instead of
higher compared to those of catechin-fed (7.8%) or control-fed
(8.5%) rats. Since the levels of dietary copper (s 2ppm) were
considered to be marginally deficient, the efficiency of
copper absorption and the percentage of copper deposition into
the liver would be expected to be high in all groups. ‘Various
factors may have contributed to the decrease in total liver
copper observed in the hull-fed rats compared to controls.
Stabel et a1. (1993) reported that the liver is particularly
sensitive to changes in copper status. Suttle et a1. (1975)
demonstrated that administration of copper after a period of
copper depletion, priority was given to repletion of copper in
plasma followed.by copper pools such.as the liver» Therefore,
release of liver copper stores with consequent decrease in
total liver copper content may explain the increased levels of
serum copper and ceruloplasmin seen in hull-fed rats.
DiSilvestro and Harris (1983) found that injection of catechin
elevated lysyl oxidase activity in the aortas of chicks.
Since lysyl oxidase is a copper containing-enzyme and responds
rapidly in copper deficient animals, they suggested that
catechin might enhance copper absorption. Greger and Emery
(1987) observed that rats fed coffee and decaffeinated coffee

that contained 5% polyphenols had elevated liver copper. In

 

72
contrast to the present results, Greger and Lyle (1988)
reported that ingestion of black tea, estimated to contain
1.17% condensed polyphenols, enhanced copper absorption
leading to elevated levels of copper in plasma and in liver by
anemic rats. However, feeding rats the same level of catechin
showed. no change in. plasma copper or in tissue copper
concentration (Greger and Lyle, 1988). They concluded that
catechin in. black. tea. was unlikely' responsible for the
alteration in copper bioavailability. In the present study,
thereby, it is likely that pinto bean hulls but not condensed
tannins (or at least catechin) in hulls were responsible for
the change in plasma copper and total liver copper content
because similar effects were not seen in catechin-fed rats.
Aoyagi et a1. (1993) estimated copper bioavailability from
feed ingredients derived from plants (peanut hulls and soy
mill run) and animals (livers from chicken, beef, sheep and
turkey). They concluded that the copper in feed ingredients
from plants was less bioavailable than that from animals.
They suggested these results were likely due to phytate
binding of copper in the plant material. However, it would
appear that some other unidentified factor other than phytate
in pinto bean hulls was responsible for the alteration in
copper bioavailability in the present study because the
majority (up to 95%) of phytate is present in the cotyledon of

pinto beans (Salunkhe et a1., 1990).

 

73

Dietary treatments affected liver zinc concentration.
Significantly higher liver zinc concentrations were found in
rats fed the hulls and catechin diets. However, the hulls
diets overall had no effect on tibia zinc, because opposite
effects were shown in the two studies. The reason for the
conflicting results is not clear. Tibias in rats fed the
catechin diet tended to have a higher zinc concentration and
significantly elevated.total zinc content. Similarly, Greger
and Emery (1987) observed that rats fed coffee and
decaffeinated coffee that contained 5% polyphenols had
elevated tibia zinc. Moreover, Greger and Lyle (1988)
reported a significant elevation of tibia zinc concentration
in rats fed catechin or tea; however, the mechanism(s)
responsible for these changes are unclear. Pinto bean hulls
and catechin had no effect on plasma zinc in the two studies.

No effect was observed in iron bioavailability since the
hemoglobin and.hematocrit did.not change in different dietary
treatments. These results confirm the observations of Brune
et a1. (1989), Garcia-Lopez et a1. (1990) and House and Van
Campen (1994) that condensed tannins per se were not

responsible for the low iron bioavailability of legumes.

CONCLUSIONS

Ingestion of diets containing 25% pinto bean hulls led to
a decrease in relative liver weight in both experiments. The
significance of this change is unclear. The overall results
indicated that feeding rats pinto bean hulls tended to alter
copper bioavailability as shown by the decreased total liver
copper content and increased plasma copper. However, these
effects were not seen in the rats fed the catechin diet.
Since both the hulls and catechin diets contained a similar
concentration of assayable condensed tannins, we suggest that
pinto bean hulls but not condensed tannins are responsible for
the alterations which were observed. There could be some
other factor(s) present in bean hulls that caused the copper
release from liver stores into the plasma.

The catechin and pinto bean hulls diets had no effect on
serum zinc but elevated the liver zinc concentration. In the
hull-fed rats, this increase might be related to the higher
total zinc intake but this can not explain the results in the
catechin-fed rats. Condensed tannins, therefore, could be the
potential factor responsible for this change. However, the
mechanism involved is unclear. Interestingly, we found that
the dietary treatment enlarged the size of the tibia in rats
fed the catechin diet, but not in rats fed the hulls diet. If

this increase in tibia size was related to condensed tannins,

74

75
i.e. , catechin, it is suggested that there is some other
unidentified factors in hulls which overcomes the effect of

condensed tannins.

RECOMMENDATIONS

Because increased consumption of beans is being
recommended in order to increase dietary intake of complex
carbohydrates and fiber, it is important to have a better
understanding of other components in beans such as tannins
which traditionally have been considered as antinutritional
factors. Ideally such research would be done using tannins
purified from different varieties of beans in order to fully
understand their nutritional effects. The commercially
available catechin is extracted from tree bark, and hence it
is questionable to use it as a standard reference in bean
tannins studies. Obviously bean hulls contain numerous other‘
chemical compounds as well as a variety of different types of
tannins. The most common method used to isolate and purify
condensed tannins was described by Hagerman and Butler (1980) . ‘

Additional research also should focus on investigating if
the condensed tannins in the pinto bean hulls were the factor
responsible for the alterations in copper and zinc
bioavailabilty in rats that were observed in the present
experiments. Experimental approaches might include feeding
rats diets containing: (1) PVP (polyvinylpyrrolidone), an
agent known to bind condensed tannins (2) purified tannins

isolated from the pinto bean hulls.

76

77
Because of recent evidence suggesting a possible
beneficial effect of tannins, other future research in this
area should be aimed at investigating the possible role of
bean tannins in cancer prevention or their pharmacological

properties as an antifungal agent.

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