Abstract--The effects of different tannins were compared in feeding trials utilizing rata fed diets that were equal in their total phenolics content. The drug propranolol was used to suppress praline-rich salivary protein production in some animals in order to examine the defensive potential of these proteins against dietary tannins. Differences were evident in the way different tannins affected digestion and the post- digestive assimilation of nutrients into new body mass. Different tannins also interacted differently with propranolol and were recovered in fecal material in different quantities. While the two condensed tannin- containing diets and one hydrolysable tannin-containing diet each had similar effects on overall weight gain, they appeared to cause their effects in different ways. These results are discussed in the context of the frequently made assumption that all tannins act as feeding deterrent allelochemicals in a uniform manner.
Introduction Tannins and related plant polyphenolics have been regarded as generalized anti- herbivore allelochemicals for some time. As originally viewed by Feeny (1976) and Rhoades and Cates (1976) this group of allelochemicals was supposed to act by reducing the digestibility of nutrients following their ingestion by herbivores. Recent evidence for both insects and mammals (Bernays et al., 1989; Mole et al., 1990a) now suggests that the primary anti-nutritional activity of tannins is not via digestibility reduction, but by the inhibition of metabolic events occurring after the digestion and absorption of nutrients, i.e. systemic effects.
In spite of these recent advances, tannins are still regarded as a rather homogenous group with respect to their biological and ecological effects. In this way our under- standing of the biochemical ecology of tannins lags behind recent advances in understanding the diversity of their chemical structures (Hemingway and Karchesy, 1989). The present report provides evidence that chemically different tannins have different effects with regard to manner in which they reduce animal growth. We re-evaluate the evidence concerning the in vivo effects that tannins produce in mammalian herbivores and we question the homogeneity of tannins as a group defined in terms of their biological activity.
In this paper we use the term "tannin" in the operational sense (Mole and Waterman, 1987a) referring to plant extracts that contain phenolics and have the ability to precipitate proteins from aqueous solutions. The chemically more specific terms "condensed tannin" or "hydrolysable tannin" are used when appropriate for specific substances such as tannic acid or for plant extracts known to contain tannins of one predominant type.
Tannins and the use of iso-phenolic diets In these experiments, animals were fed diets that contained tannins derived from
different sources. Despite chemical differences in the types of tannin fed, a key feature ~:Present address: School of Biological Sciences, University of Nebraska at Lincoln, NE 68588, U.S.A.
(Received 11 February 1992)
667
668 S. MOLE ETAL.
of the present work is that all our experimental diets were identical in their total phenolic contents. We have termed these "isophenolic" diets because they contained the same quantities of phenolic material as determined by a total phenolics assay (Price and Butler, 1977).
The rationale for working with isophenolic diets stems from the fact that total phenolics assays have been routinely used in ecological work to quantify phenolic allelochemicals. For this reason, it is of importance to know the variation in biological activity between diets which are identical in their total phenolics content. We are unaware of previous feeding trial comparisons employing such diets.
We chose to feed commercially available tannins which are routinely used as standards in chemical assays of total phenolics and condensed tannins. These were tannic acid (Sigma Chemical Co.) and quebracho tannin (Trask Chemical Co.), the former being a relatively pure hydrolysable tannin and the latter being a crude extract of the heart wood of Schinopsis quebracho-colorado (Anacardiaceae). Both of these have been used in constructing diets for feeding trials (Mole and Waterman, 1987b; Bernays et al., 1989) which adds to the utility of the side by side investigation as presented here.
Liquid/liquid partition between water and ethyl acetate was used to divide the quebracho tannin into polar and non-polar fractions which were re-dried by lyophilization and rotary evaporation at room temperature respectively. Approximately two-thirds (w/w) of the commercially supplied quebracho enters the polar fraction and the remainder enters the non-polar fraction. Separate investigations of the two fractions were made to examine the potential heterogeneity to be found within a single tannin, as such crude tannins are often used in feeding studies.
Proline-rich salivary proteins and tannins A second goal of this work was to examine the effects of suppressed salivary
proline-rich protein (PRP) production on animals fed different tannins. The present experiments extend and complement our previous report (Mole et al., 1990a) in which a more extended review of the function of these proline-rich proteins is given. In essence, animals which produce salivary PRPs are thought to gain some protection against the activity of dietary tannins in the gastrointestinal tract, because the tannins are specifically bound to salivary PRPs and are thus removed from the digestive process (Mole et al., 1990b).
Application of Waldbauer methodology We again used the growth analyses due to Waldbauer (Mole et al., 1990a;
Waldbauer, 1968) which describe growth (GR) as a function of feed consumption (CR) and the efficiency with which animals convert ingested food into new body substance (ECI). ECI is further analyzed as a function of the approximate digestibility of the food (AD) and the efficiency with which digested and absorbed food is then converted to new body mass (ECD). Equations which define these terms are given in Table 1.
According to this analysis, if the only effect of tannins is to reduce digestibility, then AD will be diminished while the ECD term should not be affected because it is a measure of the post-absorptive fate of the nutrients. There is a possibility that a depression of AD could significantly reduce ECD if a large proportion of nutrients were diverted from growth to maintenance processes such as basal metabolism. In practice, we are interested in the relative effects of tannins on AD and ECD. If tannins depress ECD without any substantial effect on AD, then an explanation of their effects in terms of processes other than reduced digestion is indicated.
Determination of AD and ECD in a feeding trial requires all the component measure- ments to be made in the same units, otherwise the relationship ECI = ADXECD will not
GROWTH REDUCTION BY TANNINS 669
TABLE 1. EQUATIONS DEFINING TERMS USED TO ANALYZE THE CONSUMPTION AND UTILIZATION OF FOOD, ADAPTED FROM WALDBAUER (1968)
1. Growth Rate (GR)=Animal Weight Gain During Experiment
2. Consumption Rate (CR) = Feed Ingested During Experiment.
3. Efficiency of conversion of ingested food to body matter (ECI) is calculated as:
ECI = (Animal Weight Gain/Feed Ingested) x 100%
Where all weights are made on a dry wt. basis GR can be expressed in terms of CR and ECI, i.e, GR = CR × ECL
4. Approximate Digestibility (AD) is calculated as:
Weight of Feed Ingested-Weight of Feces AD = -× 100%
Weight of Feed Ingested
5. Efficiency of conversion of digested food to body matter (ECD) is calculated as:
Animal Weight Gain ECD = x 100%
Weight of Feed Ingested--Weight of Feces
Where all weights are made on a dry wt. basis ECI can be expressed in terms of AD and ECD, i.e. ECI = AD x ECD and thus GR =CR XADX ECD.
Equations defining terms used to analyze the consumption and utilization of nitrogen are similar, thus...
Nitrogen Ingested--Nitrogen in Feces 6. AD(N) = X100%
Nitrogen Ingested
hold. We report measurements on both a dry weight basis and a Kjeldahl nitrogen basis.
In order to test the hypothesis that PRPs diminish the effects of tannins on rats, we replicated the experiments using animals which were and were not able to produce PRPs. The drug propranolol, a I]-antagonist, was used as this is known to suppress PRP production (Mehansho et al., 1987). In the absence of propranolol, PRPs are induced during the first 3 days that animals are fed high tannin diets (Mole et al., 1990a). When present, propranolol suppresses this induced effect, l[3-antagonists, as a class of drugs, are known to produce other physiological effects and the potential for tannins to interact with these is discussed below as a potential limitation to this technique.
Materials and Methods Animals and formulation of diets. We used weanling rats of the Sprague-Dawley strain (Harlan Laboratories, Indianapolis, IN) fed diets containing ground sorghum grain. The diets were of ground sorghum (cultivar IS0469) supplemented with vitamins, minerals and lipids to satisfy National Research Council (1977) require- ments. The sorghum grain was from the 1984 crop grown at the Purdue University Agronomy Farm. Table 2 summarizes the construction of Diets 1-8.
Feeding and sample collection. Fifty-two male rats were fed Rodent Block (Wayne Feeds) for 1 day after delivery. Forty animals were then weighed and allocated to eight treatment groups of five animals based on their body weight, so that the mean weights of each group at the start of the experiment were approximately equal. The remaining 12 animals also had a similar group mean weight. The 20 experimental animals to be fed diets without propranolol were individually housed in metabolism cages and fed Diet 1 for 1 week and then placed on their experimental diets for a 2-week period. The other 20 animals allocated to experimental diets containing propranolol were also individually housed but were fed Diet 5 for 1 week, after which they were placed on the four propranolol-containing diets for 2 weeks. Of the 12 animals not allocated to the experimental diets, six were fed Diet I for 1 week and six were fed Diet 5 for 1 week. At the end of this week, these animals were killed with CO 2 gas and deep frozen for future use. This was done for the remaining 40 animals 2 weeks later at the end of the feeding trial.
During the 2-week period of the experiment in which the experimental diets were fed to the eight groups of animals, the animals were fed and watered adlibitum and the weight of feed consumed was recorded. Fecal pellets were collected every 2 days from a wire retaining grid under the cages. Collections of fecal material were stored frozen.
Laboratory procedures. After the fresh wt. of the 40 rats from the main experiment were recorded, the frozen bodies were rapidly crushed into small pieces and lyophilized (Mole et aL, 1990a). The initial live wt. of the
*Numbers 1-8 are used for reference in the text. tThe basal diet contained the following ingredients as a percentage of the diet: Sorghum (cultivar IS0469), 81.34; corn starch,
8.25; mineral mix (AIN76), 3.5; vitamin mix (AIN76), 1.0; lysine.HCI, 0.6; choline chloride 0.25; BHT, 0.01 ~Where present, tannins were added to give the same level of total phenolics by the Prussian Blue assay in all tannin
containing diets. §Cellulose was added as an inert diluent for the other ingredients listed above.
20 animals which underwent the 2-week experiment were recorded directly, but their initial dry wt. had to be estimated from the dry wt. of the 12 animals killed at the beginning of the 2-week period during which the experimental diets were fed. The estimated values of initial dry wt. were calculated as the product of their initial live wt. and the mean fresh wt./dry wt. ratio of the six animals killed at the time the feeding trial began. Dry and fresh wt. gains were calculated as the differences between final and initial weights.
After weighing, the lyophilized material was ground to a fine powder in a Wiley mill. All the fecal material produced by each rat over the 2-week period was lyophylized, weighed, ground to a powder and bulked for each animal. Samples of the feeds were lyophylized to measure their fresh wt./dry wt. ratio and the weight of feed consumed by the animals during the experiment was converted to a dry wt. equivalent using these ratios.
Samples of feed, powdered rat and feces were analyzed for their total nitrogen content by the Kjeldahl method. The protein content in these materials was estimated as N%×6.25. The total quantities of protein in feed, fecal and animal body matter were determined from these and their dry wt. data. Body nitrogen gain was calculated as the difference between final and initial weights of protein in the body.
Samples (0.5 g) of feed and powdered feces were extracted with 80% (v/v) aqueous MeOH for 2 h and the filtered extracts were assayed for total phenolics by the Prussian blue method (Price and Butler, 1977) and for condensed tannins by the butanol/HCI method (Mole and Waterman, 1987a) in order to estimate the recoveries of these substances in the feces. Dry weighed samples of feed were also moistened with water to form a stiff paste and then incubated for 24 h at 37~C. This material was then dried and assayed for tannins and phenolics to measure the quantity of these substances rendered inextractable simply by wetting.
Results Tannins and other phenolics in the feeds The sorghum cult ivar IS0469 used to construct the basal diets was found to be tannin- free and to contain only trace levels of phenolics. Diets 1 and 5 were tannin-free controls and in these diets the various tannins in the other diets were replaced by cel lulose to keep the diets isocaloric. These other diets contained 1.05% tannic acid (Diets 4 and 8) or an equivalent quant i ty of total phenol ics (Diets 2, 3, 6 and 7). Direct proanthocyanid in assays of Diets 2, 3, 6 and 7 demonstrated the presence of condensed tannins. In these assays, Diets 2 and 6 (non-polar quebracho fraction) gave absorbances 1.7 t imes greater than those obtained for Diets 3 and 7 (polar fraction). So, in spite of their having the same total phenol ic contents, diets containing the non-polar fraction appeared to contain more condensed tannin than the diets
containing the polar fraction. The perils of over interpret ing ratios between condensed tannin and total phenol ic
assays have been documented elsewhere (Mole and Waterman, 1987a). However, these data do indicate that the aqueous (polar) quebracho extract (Diets 3 and 7) contains more phenol ics that are not chemical ly condensed tannins relative to the non-polar extract. The presence of non-tannin phenol ics in quebracho extract is wel l known (Gustavson, 1956) and so their presence comes as no surprise. Those condensed tannins that do part i t ion into the polar extract are also likely to be more highly polymer ized than those in Diets 2 and 6. This is because of the ethyl acetate/
GROWTH REDUCTION BY TANNINS 671
water partition and the relative insolubility of high molecular weight proanthocyanidins in ethyl acetate.
In choosing an ethyl acetate/water fractionation of crude quebracho, we initially assumed that condensed tannins would be insoluble in ethyl acetate; indeed, the material we obtained as our non-polar fraction was nearly insoluble in dry ethyl acetate. However, our fractionation was effectively between ethyl acetate saturated water and water saturated ethyl acetate. We believe this accounts for the presence of condensed tannins in what we have termed the non-polar fraction.
Weight gain The mean starting (live) weights for the groups of animals not fed propranolol (Diets
1-4) were 67.6, 68.4, 68.0 and 67.0 g respectively. These animals were all fed Diet 1 for a 1-week acclimatization period before the experiment. Animals fed propranolol during the experiment (Diets 5-8) were acclimatized on Diet 5 for 1 week, and had slightly lower mean starting (live) weights of 61.4, 67.8, 65.8 and 65.5 g, respectively. The particularly low group mean for the group fed Diet 5 was due to a single individual of low initial body weight.
Weight gain on the diets without tannin is far in excess of that on the tannin- containing diets (Table 3). Not only is there little weight gain seen with the groups of animals fed the tannin-containing diets (see Diets 2, 3 and 4 vs Diet 1), but also, in the presence of propranolol growth is much more strongly depressed. The most severe effect is seen with the polar fraction of quebracho where for Diet 7, GR is cut to less than 25% of that for Diet 3 which contains tannin but not propranolol. In contrast, the control diets for propranolol (Diet 1 compared to Diet 5) show much less of a reduction due to propranolol alone.
Use of Dunnett's procedure for comparing means with a control (Steel and Torrie, 1980) confirms that animals fed tannin had significantly (P<0.05) lower weight gains than animals fed the corresponding tannin-free diets (1 and 5). There were no significant differences in animal performance within groups of animals fed propranolol (Diets 6-8) but growth on Diet 2 was significantly (P<0.05) less than that on Diets 3 and 4.
Food consumption Table 3 also gives the amounts of feed consumed (CR) during the 2-week period.
Animals fed tannins and propranolol (Diets 6-8) consumed low quantities of feed. At the other extreme are the animals fed neither tannin nor propranolol (Diet 1).
Can the differences in growth rates seen above be explained by tannins acting to change CR, rather than ECI? For instance, did the animals fed Diet 1 grow well because they ate more than the animals fed any other diet? Superficially, the data for CR imply an affirmative answer, but this would fail to consider the size of the animals. The parameter relative consumption rate (RCR) is estimated here as RCR = CR/Median Dry Wt. (mean of the initial and final dry wts of animal during the experiment). Comparing the data for diets with and without tannins, RCR for tannin-containing diets is very similar for all diets irrespective of tannin content. This can be seen in Table 3 where this index of consumption can be compared to other factors impacting growth rate. While there is some significance between treatment variation in RCR, we do not see this the biologically important or major factor producing low growth on tannin-containing diets; there is substantially more variation in ECI.
Analysis of ECl ECI is much lower for the tannin-containing diets relative to the control diets without
tannin (see Table 3). Low ECI is the main component of low GR, where GR is estimated from the differences in animal dry wt. gain during the experiment. It is also evident
TA
BLE
3. W
EIG
HT
S, W
EIG
HT
GA
INS
AN
D N
UT
RIT
ION
AL
IND
ICE
S F
OR
TH
E 2
-WE
EK
FE
ED
ING
TR
IAL
(dry
wts
in
gram
s)
Die
t *
11"
2 3
4 5
6 7
8
Mea
sure
men
ts
Mea
n S
EM
M
ean
SE
M
Mea
n S
EM
M
ean
SE
M
Mea
n S
EM
M
ean
SE
M
Mea
n S
EM
M
ean
SE
M
Fin
al W
eigh
t 55
.6
3.4
29.6
1.
7 38
.0
2.4
35.9
1.
6 42
.3
2.0
24.0
2.
3 22
.7
1.3
23.4
1.
1
Wei
ght
Gai
n 35
.8
3.3
9.6
1.2
18.1
1.
9 16
.2
1.1
24.3
1.
4 4.
1 2.
0 3.
4 1.
2 4.
0 0.
9 F
eed
Inge
sted
(CR
) 27
8 13
18
6 5,
3 21
9 10
20
3 8
226
12
142
10
144
8 14
0 7
RC
R
18.2
0.
2 16
.8
0.5
17.6
0.
31
16.8
0.
6 17
.6
0.6
14.0
0.
5 14
.6
0.5
14.1
0.
7 E
CI
12.8
0.
7 5.
1 0.
5 8.
2 0.
5 8.
0 0.
3 10
.7
0.3
2.5
1.1
2.3
0.7
2.8
0.6
AD
87
.6
0.3
85.0
1.
0 83
.9
0.4
85.2
0.
4 88
.2
0,7
82.2
0.
7 82
.3
0.3
86.6
1.
18
EC
D
14.6
0.
9 6.
0 0.
7 9.
7 0.
6 9.
4 0.
4 12
.2
0.3
3.0
1.4
2.7
0.8
3.2
0.7
*Die
ts n
umbe
red
as in
Tab
le 2
, whi
ch g
ives
det
ails
of
the
ir c
onst
ruct
ion.
1M
eans
and
the
ir as
soci
ated
sta
ndar
d er
rors
are
giv
en f
or
the
grou
ps o
f fiv
e an
imal
s fe
d ea
ch d
iet.
GROWTH REDUCTION BY TANNINS 673
from Table 3 that this effect of tannin on ECI is much more severe when propranolol is also present in the diet. For example, there is a threefold difference when Diets 3 and 4 are compared with Diets 7 and 8. The effect of propranolol alone is comparatively minor.
Because low feed consumption does not appear to be the primary cause of low growth rate on tannin containing diets, then from the GR = CR×ECI model (see Table 1) an analysis of the components of ECI is called for. The values of both AD and ECD are lower for the tannin-containing diets. However, the depression of ECD is profound while AD is only very slightly lowered (see Table 3). Thus, digestibility reduction does not appear to be the main cause of reduced ECI. The data in Table 3 confirm our earlier observation for rats fed high and low tannin sorghum (Mole etaL, 1990a), that tannins do not act to reduce growth rate by digestibility reduction. On this account, the present data are even more explicit. Their particular interest lies in the uniformly small effect that the three tannin types have on AD.
Examination of the data for ECD shows large differences between data for the diets with and without tannin, a factor seen even more clearly in the presence of propranolol. The detail of interest is that differences between the tannins are manifested as differences in the degree to which they affect ECD and interact with propranolol. Specifically, the non-polar fraction of quebracho has severe effects and is less strongly potentiated by interaction with propranolol which diminished the ECD of all three tannin-containing diets to a uniformly low level.
Growth analysis on a nitrogen basis In these experiments animals on all diets consumed nitrogen from the same basal
diet, thus nitrogen consumption is directly and constantly proportional to feed consumption (Table 2). Unlike our previous experiments where weight losses were observed (Mole et aL, 1990a), animals in the present work gained weight and retained a relatively constant body composition so that nitrogen gain by body tissues is effectively estimated by dry wt. gain (Table 2). We begin the nitrogen-based growth analysis by examining ECI(N) and the data presented in Table 4.
As with ECI, ECI(N) is considerably lower for animals consuming tannins. Unlike the data for AD, AD(N) is rather more significantly depressed for animals consuming tannin. A particularly interesting effect is seen with propranolol. For the control diets (1 and 5) there is no reduction in AD(N) but for the two kinds of quebracho containing diets, propranolol substantially reduces digestion (compare Diets 2 and 3 with 6 and 7). Most interestingly, this interaction with propranolol does not occur with tannic acid. These results confirm our previous finding that propranolol has a deleterious effect on nitrogen digestion in the presence of condensed tannins. Given the frequently made assumption that tannins act within the gut lumen, then this remains consistent with a mechanism involving the suppression of proline rich protein production by this drug. The new and interesting result here is that where a hydrolysable tannin is present AD(N) remains high even in the presence of propranolol. By Dunnetts procedure, AD(N) for Diets 6 and 7 are significantly different (P<0.01) from the control Diet 5 while the value for Diet 8 is not significantly different (P<0.05). Diets 2-4 do have AD(N) values lower than Diet 1, and it is possible that with additional replication, a statistically significant depression in AD(N) would be seen for all diets relative to tannin-free controls. However, the striking effect is still the potentiation, by propranolol, of lowered AD(N) for the condensed tannin but not hydrolysable tannin-containing diets.
The result for condensed tannins is that AD(N) is more severely affected than AD, so that these tannins do appear to have a disproportionately larger effect on protein digestibility than on the digestibility of the diet as a whole. This is consistent with the view that tannins act primarily by reducing the availability of nitrogen to the body.
TA
BLE
4. G
RO
WT
H A
NA
LYS
IS O
N A
WE
IGH
T O
F N
ITR
OG
EN
BA
SIS
, FO
LLO
WIN
G C
ALC
ULA
TIO
NS
DE
TA
ILE
D IN
TA
BLE
1
Die
t*
It
2 3
4 5
6 7
8
Mea
sure
men
ts
Mea
n S
EM
M
ean
SE
M
Mea
n S
EM
M
ean
SE
M
Mea
n S
EM
M
ean
SE
M
Mea
n S
EM
M
ean
SE
M
EC
I (N
) 48
.2
2.7
19.6
2.
0 31
.0
1.8
30.2
1.
4 40
.5
9.1
10.1
4.
3 9.
1 2.
5 10
.9
2.0
AD
(N
) 81
.2
2.8
72.2
3.
1 71
.4
1.6
69.2
1.
0 82
.8
0.9
46.8
2.
9 57
.9
1.8
70.1
2.
7
EC
D (N
) 60
.0
5.1
27.4
3.
2 43
.5
2.5
43.8
2.
2 49
.0
1.1
19,8
7.
7 15
.7
4.3
15.5
3.
0
*Die
ts n
umbe
red
as in
Tab
le 2
, whi
ch g
ives
det
ails
of
thei
r co
nstr
uctio
n.
tMe
an
s an
d th
eir
asso
ciat
ed s
tand
ard
erro
rs a
re g
iven
fo
r th
e gr
oups
of
five
anim
als
fed
each
die
t.
TA
BLE
5. A
PP
AR
EN
T D
IGE
ST
IBIL
ITIE
S* (
%)
OF
CO
ND
EN
SE
D T
AN
NIN
S (C
T) A
ND
TO
TA
L P
HE
NO
LIC
S (T
P)
Die
tl-
Dig
estib
ility
25
3
4 6
7 8
mea
sure
men
ts
Mea
n S
EM
M
ean
SE
M
Mea
n S
EM
M
ean
SE
M
Mea
n S
EM
M
ean
SE
M
TP
(dr
y fe
ed)
78.2
2.
3 92
.3
1.0
97.1
1.
0 65
.2
5.8
82.0
1,
7 98
.0
0.6
TP
(w
ette
d fe
ed)
69.0
3.
3 82
.1
2.2
92.9
2.
6 50
.3
8.2
58.0
3.
9 95
.0
1.5
CT
(dry
fee
d)
39.6
4.
6 47
.4
1.8
25.6
4.
1 31
.9
2.6
CT
(wet
ted
feed
) 8.
4 1.
4 2.
1 3.
4 12
7 12
.5
-27
4.
9
*Dig
estib
ilitie
s w
ere
calc
ulat
ed o
n th
e ba
ses
of
feed
sam
ples
ana
lyse
d ei
ther
dry
or
afte
r th
ey
had
been
wet
ted
and
drie
d.
tDie
ts n
umbe
red
as in
Tab
le 2
, whi
ch g
ives
det
ails
of
thei
r co
nstr
uctio
n.
~:M
eans
and
the
ir as
soci
ated
sta
ndar
d er
rors
are
giv
en f
or
the
grou
ps o
f fiv
e an
imal
s fe
d ea
ch d
iet.
GROWTH REDUCTION BY TANNINS 675
Examination of the data for ECD(N) shows that animals on tannin-containing diets have disproportionately low ECD(N) values relative to tannin-free diets (1 and 5). Where diets contain propranolol and tannins, then ECD(N) is reduced even more than for the tannin alone. On a nitrogen basis as well as a dry wt. basis, the largest effect is on ECD rather than AD.
Fate o f dietary phenolics The major influence of dietary tannins and related phenolics on ECD and ECD(N)
suggests that the metabolic effects of these materials are not confined to the digestive tract. Dietary phenolics not eliminated in the feces may have been absorbed from the digestive tract, either without or after metabolic transformation by intestinal micro- organisms. A useful index of the extent to which tannins were absorbed into the body would appear to be their apparent digestibility. An apparent digestibility for tannins, AD(tannin), has been calculated according to the following formula:
The amount of tannin ingested is calculated as the product of the weight of food consumed and its assayed tannin content (% w/w) while the quantity of tannin egested is calculated from the weight of feces produced and the assayed tannin content (% w/w) of the feces. In the present study, as little as 2% of the total phenolics assayed in the freshly made, uneaten dry diet were recovered in the feces; i.e. their apparent digestibility is as high as 98% (see Table 5, "dry feed" data for Diet 8).
We do not interpret the apparent digestion of tannin to indicate its actual digestion, due to problems concerning the extractability of tannins from the feed and feces. Those phenolics which are present in the feces are difficult to detect and assay because in the moist environment of the intestine they form strong complexes with dietary components and with endogenous tannin-binding proteins (Mole et al., 1990a,b). This may prevent their extraction, particularly after oxidation during drying processes. As an attempt to control for this, total phenolics and tannins were assayed on diet samples which had first been wetted and then dried. For each diet the condensed tannin and total phenolics content of each diet was substantially lower relative to equivalent data obtained with dry unwetted food. Apparent digestibilities of tannins were then re-calculated using this data for the tannin content of the food and, as expected, lower digestibilities were obtained in every case (see Table 5). In extreme cases, negative data were obtained. These are readily understood if one considers that (i) wetting and drying the food renders tannins inextractable and that (ii) their subsequent passage through the gut may enhance their extractability because tannin complexing substances such as proteins may be digested and absorbed leaving tannins free for extraction.
Discussion Effect o f tannins on a mammalian system The new evidence we present concerns the particular means by which tannins reduce growth rate. The present study confirms our earlier observations that depressed ECI rather than depressed CR is the main contributing factor to growth reduction. Within the analysis of factors contributing to reduced ECI, lowered ECD is again seen to be the largest factor, rather than reduced AD (Mole et al., 1990a). The idea that the predominant effect of tannins is to reduce the consumption of digestibility of the diet can now be rejected for this mammalian system, for both hydrolysable and condensed tannins.
676 S, MOLE ETAL.
On a nitrogen basis, our results show a similar pattern to the data expressed on a dry wt. basis. The one important difference is that tannins can reduce nitrogen digestibility, particularly in the presence of propranolol. This both confirms our earlier observations and shows an agreement with numerous other studies that indicate this effect (Foley and Hume, 1987; Mole and Waterman, 1987b; Mole et al., 1990a). We believe the importance of our work lies in demonstrating a tannin-reduced ECD(N). We show that reduced nitrogen digestion and absorption is not the only or most important means by which tannins affect nitrogen nutrition. Overall, we show that tannins alter mammalian growth in ways similar to those seen for insects (Bernays et al., 1989). Comparable information using this type of analysis is unavailable for other mammals. We speculate that similar results would be obtained for other young monogastric mammals, since our work and other studies with insects have all been on growing stages of the life cycle.
In contrast to our earlier report (Mole et al., 1990a), these experiments used tannins added to other dietary ingredients rather than using a tannin-containing grain for the diet. The growth-reducing effects were similar but less severe in the present experiments (no animals lost weight). We attribute this to our having underestimated the levels of phenolics in the tannin containing sorghum by assuming that our extractions and thus assays were efficient. Such problems are rather intractable and preclude exact statements about the differences between artificially constructed versus naturally tannin containing feeds.
Tannins and the nutr i t ional signif icance o f PRPs The present results confirm our earlier report that the drug propranolol can act to
enhance the negative effects of tannins (Tables 4 and 5). Two way analyses of variance for ECI confirm the statistical significance of this interaction effect for tannic acid (P<0.01) and the polar fraction of quebracho (P<0.01). The interaction of propranolol with the nonpolar fraction is lower in magnitude. This fraction has such a growth depressing effect that the suppression of PRP production does not produce such great additional effects as with the polar fraction. We interpret the present evidence, together with that of our previous report (Mole et al., 1990a), to indicate that PRPs can alleviate some of the deleterious effects of dietary tannins. We are particularly encouraged in this by the repeated demonstration here that AD(N) is further lowered when both propranolol and condensed tannins are present in the diet. We feel this result to be encouraging because it is easy to envisage tannins in the gut lumen interacting with this drug via a PRP based mechanism. Results for the digestibility of phenolics in this study are also hard to interpret by any mechanism other than the PRP based model (see below). However, an imporLant caveat to our present and previous work (Mole et al., 1990a) is that 13-antagonists do have other physiological effects on the body and we cannot rule out the possibility of confounding and unforeseen interactions.
The exceptional result for AD(N) seen with tannic acid was that AD(N) was not lowered. This result may be the first evidence of differing biological activities for condensed and hydrolysable tannins with respect to nitrogen digestion. It is made all the more interesting that it is revealed via the interaction with propranolol. Irrespective of whether the mechanism of action involved PRPs or not, this serves to illustrate that important differences may exist in the ways tannins interact synergistically with other secondary metabolites, or drugs as in the present case.
Further evidence for between tannin differences and the involvement of PRPs is seen from the data on the apparent digestibility of phenolic materials in the feces. Where propranolol is absent and PRP production is not suppressed, the apparent digestibility of condensed tannins and total phenolics is relatively high (Diets 2-4). Where PRP production is suppressed with propranolol, these digestibilities are lower
GROWTH REDUCTION BY TANNINS 677
for condensed tannin-containing diets (6 and 7). In contrast, apparent digestibility for total phenolics remains high for the tannic acid-containing Diet 8.
It is hard to explain these data without assuming the involvement of PRPs, or at least some other endogenously produced tannin binding proteins. Our interpretation is that a large proportion of condensed tannins exit the gastrointestinal tract as insoluble complexes with proline-rich salivary proteins from which they cannot be extracted and assayed in the fecal material. In the absence of PRPs, condensed tannins exit the gastrointestinal tract in a more extractable and assayable form. For hydrolysable tannins, we believe the situation is different. The fact that suppressed PRP production does not further reduce AD(N) for tannic acid containing diets suggests that tannic acid does not normally exit the gastrointestinal tract bound to PRPs. While tannic acid may normally exit the tract bound to other material our results are also consistent with the idea that tannic acid is susceptible to hydrolysis during digestion. In this mammalian system, these issues are an interesting subtlety when set against the major ECD and ECD(N) reducing effects of both types of tannin. These effects point to the mediation of a tannin or tannin derived agent acting post absorptively in the body in the case of tannic acid. For the relatively crude quebracho tannin containing diets, tannin associated non-tannin phenolics may also be at play here.
Acknowledgements--We thank Rick Hauber for his assistance and Drs T. R. Cline, R. Elkin and three anonymous referees for their comments on the manuscript. This work was supported by USAID/INTSORMIL. Journal paper number 12907 from the Purdue University Agricultural Experiment Station.
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