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British Journal
of
Nutrition
(1988),
59,
389-396
389
The effect
of
three types
of
saponin on iron and zinc absorption
from a single meal in the rat
BY
SUSAN SOUTHON, A.
J.
A. WRIGHT,
K.
R. PRICE,
AFRC Institute
of
Food Research, Norwich Laboratory, Colney Lane, Norwich NR4 7UA
S.
J.
FAIRWEATHER-TAIT
AND
G.
R.
FENWICK
(Received
20
August 1987
-
Accepted
8
Decernher 1987)
1.
Iron and zinc retentions in young male rats, given
.3
g starch-sucrose paste containing
120
pg Fe as
FeSO, or 139 pg Zn as ZnC1, (extrinsically labelled with
"1-e
or
"Zn) and increasing amounts of
Gypsophila
saponins, were measured by whole-body counting. The results were compared with whole-body Fe and Zn
retention from a meal containing crude or purified saponin fractions. In
a
separate experiment Fe retention from
a meal containing
Gypsophila
saponins, soyasaponin
I,
or
saponins extracted from lucerne
(Medicago sativa)
plant
tops, was measured in older rats.
2.
Results indicated that Fe absorption decreased with increasing concentration of
Gypsophila
saponins. This
was significant at a saponin
:
Fe molar value of approximately
1,
with maximum effect occurring at molar ratios
of
4
and above, when Fe absorption was reduced by approximately 17
%.
Gypsophila
saponins had no effect on
Zn absorption from a test meat.
3.
Fe absorption was similar in groups given purified
or
crude
Gypsophila
saponins at the same saponin: mineral
molar value of
8,
demonstrating that the 'non-saponin' fraction of the commercial preparation does not affect
the absorption of this mineral.
4.
Saponins extracted from lucerne plant tops, fed at a saponin
:
Fe value of approximately
8,
also reduced Fe
absorption from a single meal. Fe absorption from a meal containing a similar amount
of
soyasaponin
I
was not
significantly different from controls.
5.
These results indicate that some dietary saponins may reduce Fe absorption and hence have an adverse effect
on Fe status in man and simple-stomached animals.
It has been demonstrated recently that the consumption of a semi-synthetic diet containing
12 g
Gypsophila
saponins/kg for a
3
week period, had a detrimental effect on iron status
in growing rats (Southon
et al.
1988). In view
of
this finding, and the suggestion that the
consumption of saponins by man should be encouraged as a means of reducing
hypercholesterolaemia (Malinow
et al.
1980), the present study was undertaken to
investigate this effect in greater detail.
The haemolytic properties of saponins are well-known but the normal environment of
the erythrocytes counteracts much
of
their haemolytic activity and it is generally agreed
that they are not readily absorbed (Bondi
et
al.
1973; Cheeke, 1980). It
is
more likely,
therefore, that the lower Fe status observed in rats consuming saponins was a consequence
of reduced Fe absorption, rather than metabolic disturbances caused by saponins entering
the bloodstream.
In the present study the effect of saponins on Fe absorption was investigated by
measuring whole-body 59Fe retention in rats from a single test meal containing extrinsically-
labelled ferrous sulphate, together with varying amounts of saponins. The effect of
saponins on zinc absorption from zinc chloride, extrinsically labelled with 65Zn, was also
investigated using a similar technique. The three saponins used in the present study were
a commercially available
'
non-food
'
saponin extracted from the roots of
GypsuphiZa
sp.,
soyasaponin
I
which is the dominant saponin found in most legumes (Price
et al.
1986) and
therefore consumed in significant quantities by man, and saponin extracted from lucerne
(Medicago sativa
;
commonly called alfalfa) plant tops which are more usually consumed by
animals.
390
SUSAN
SOUTHON
AND
OTHERS
MATERIALS
AND
METHODS
Three experiments were performed, the first to determine whether there was any indication
that
Gypsophila
saponins influenced Fe absorption from a meal at low saponin
:
Fe molar
values, and the second at higher saponin: Fe molar values. Test meals given to rats in Expts
1
and 2 contained saponin: Fe molar values of 05,
1,2
and 4, and
8,
16
and 32 respectively.
The first experiment also included an investigation of Zn absorption from meals containing
saponin. Test meals contained either untreated crude saponin
(‘
saponin white
’)
obtained
from Sigma Chemical Co. Ltd, Poole, or crude saponin which had been purified by means
of reversed-phase flash chromatography as previously described (Price
et
al.
1987
;
Southon
et
al.
1988). Quantitative analysis of the commercially supplied material demonstrated that
it contained 604 g saponin/kg dry weight.
In the third experiment Fe absorption from meals containing different saponins was
measured.
Gypsophila
saponins, soyasaponin
T
or saponins extracted from lucerne plant
tops, given at a saponin
:
Fe molar value of approximately
8,
were studied. Soyasaponin
I
was isolated from lucerne seeds according to the method patented by Kitagawa (1984). The
isolated saponin was shown to be homogenous by thin-layer chromatography (TLC) using
two solvent systems. The saponins present in lucerne plant tops were extracted from the
tissue according to the method of Gestetner (1971). The saponin fraction was shown to be
homogenous by TLC. Detailed information
on
the occurrence and probable structures of
these saponins has been presented in a recent review (Price
et
al.
1987).
Expt
1
Male Wistar rats (120), weighing approximately
80
g, were given a control (saponin-free)
semi-synthetic diet for 9 d, and trained to meal-feed in the final 2 d. The diet was of similar
composition to that described previously (Fairweather-Tait
&
Wright, 1984) with the
addition of
2.5
g methionine/kg diet. Rats were allocated to twelve groups of ten, with
similar mean body-weights, fasted overnight and each animal given a test meal of 3 g
cooked starch-sucrose (1
:
1,
w/w) paste containing either 120 pg Fe as ferrous sulphate in
0.1
M-hydrochloric acid, labelled with 18.5 kBq 59Fe (FeCl,, 11&740 MBq/mg Fe,
Amersham International, Amersham, Bucks) or 139 pg Zn as zinc chloride in 0.1 M-HC~,
labelled with 37 kBq 65Zn (ZnCl,, 3.7-92.5 MBq/mg Zn, Amersham International).
Fe absorption was measured in the first six groups, the saponin content of the meals
being as follows: group
1,
no
added saponin; groups 2,
3,
4 and
5,
purified
Gypsophila
saponins in saponin:Fe molar ratios of
0.5,
1,
2 and 4 respectively; group
6,
crude
Gypsophila
saponins in a saponin: Fe molar ratio of
0.5
(assuming a molecular weight of
1500 for the saponins and a purity of 604 g/kg for the crude saponin extract).
Zn absorption was measured in the remaining six groups. Test meals were prepared to
give saponin:Zn molar ratios similar to the saponin:Fe molar ratios used in the Fe
absorption study.
In all three experiments, the control semi-synthetic diet was provided
ad
lib.
4 h after the
test meal until the end of the experiment.
Expt
2
Fifty male Wistar rats, weighing approximately 60 g, were given a semi-synthetic diet for
10
d. Rats were trained to meal-feed during the final
3
d
of this period and then allocated
to five groups of ten such that the mean body-weights of the groups were similar. After an
overnight fast, each animal was given a 3 g starch-sucrose test meal containing
120
pg
Fe
labelled with 59Fe and an appropriate amount
of
Gypsophila
saponins. Group
1
received a
meal with no added saponin; groups
2,
3 and
4
were given purified saponins
in
amounts
Saponins and mineral absorption
39
1
calculated to give saponin:Fe molar ratios of approximately 8, 16 and 32 respectively;
group
5
was given crude
Gypsophila
saponins at a saponin: Fe molar value of 8.
Expt
.3
Sixty male Wistar rats, weighing approximately 200 g, were given a semi-synthetic diet for
14 d and trained to meal feed in the final 2 d of this period. Rats were allocated to four
groups of fifteen, with similar mean body-weights and, after an overnight fast, given a
59Fe-labelled test meal as described in Expt 2. The type of saponin added to each meal was
as follows: group
1,
no added saponin; group 2, soyasaponin
I;
group 3, lucerne-plant-top
saponins; group
4,
Gypsophila
saponins. Meals given to rats in groups 2,
3
and 4 each
contained 16 mg saponin, giving a saponin: Fe molar ratio of approximately 8, assuming
a molecular weight of 1000 for each of the saponins.
Whole-body counting
Whole-body radioactivity was measured using a NE 81 12 small animal whole-body counter
(Nuclear Enterprises, Edinburgh) as described by Fairweather-Tait
&
Wright (1984).
Whole-body Fe retention was calculated by measuring the 59Fe content of the whole
body immediately post-dosing and again
7
d later when all the unabsorbed Fe from the test
meal had been excreted. The amount of 59Fe remaining at this time was taken to be an
estimate of Fe absorption from the meal (Fairweather-Tait
&
Wright,
1984).
Zn absorption was estimated by counting the animals immediately after consuming the
meal, and then 5,7,9 12 and 13 d after the dosing; it was assumed that all the unabsorbed
65Zn would be excreted within
5
d of dosing. The counts obtained at each time point (after
correcting for counting efficiency, background and decay) were expressed as a percentage of
the counts present in the animal immediately after consuming the test meal to give whole-
body retention (apparent absorption). The logarithm of the percentage whole-body
retention at each time point was plotted against time, and from regression analysis an
estimate of true ‘j5Zn absorption from the meal was obtained. This technique makes
allowances for endogenous losses of 65Zn over the experimental period (Heth
&
Hoekstra,
1965).
Statistical analysis
Both regression and one-way analysis of variance were performed using the statistical
package GENSTAT (Alvey
et al.
1977). Where analysis of variance showed a treatment
effect, ,approximate
t
tests between two means
(x,
and
x2)
having
n
replicates
(n,
and
n,)
were performed using the standard error of the difference
of
the means
(SED)
calculated
from the residual mean square (RMS)
(SED
=
v’RMS.((l/n,)+(l/n,));
t
=
(x,-x,)/sED).
The residual degrees of freedom used to estimate the level of significance
oft
are shown in
the appropriate tables of results.
RESULTS
Expt
1
Mean values for Fe absorption
(%
of dose), in rats weighing 139
(SEM
1)
g, from 120pg
Fe in a test meal of
3
g cooked starch-sucrose paste, containing saponin: Fe molar values
of approximately
0.5,
1,2 and 4 are shown in Table
1.
Animals that ate less than two-thirds
of the meal were excluded from the experiment
to
minimize the possibility of a dose-related
response (Bannerman
et al.
1962). Results indicated that Fe absorption decreased with
increasing concentration of
Gypsophila
saponins, the effect becoming significant at a
saponin: Fe value of 1.
No
significant reduction in 59Fe absorption was observed at a
saponin: Fe value of
0.5,
for either the purified or crude extracts.
392
SUSAN
SOUTHON
AND
OTHERS
Table
1.
Expt
1.
59Fe and "'Zn absorption in rats from a 3g starch-sucrose
(l:l,
w/w)
?est meal containing
120pg
Fe or 139pgZn and variable amounts
of
Gypsophila
sapon ins
(Values are means with their standard
errors
for the no. of observations indicated)
-.
59Fe absorption
(%
of dose) 65Zn absorption
(%
of dose)
molar value
n
Mean
SE
n
Mean
SE
Saponin
:
Fe
or
Zn
0
8
69.9 1.4
10
44.8
1.9
0.5
10
67.1 1.9
10
44.0
2.3
0.5t
10
69.4 2.0
10
44.1
3.6
1
10
63.8*
1
-6
10
51.3
2.2
2
10
62.Y 1.3
9 45.5
3.3
4 10
58.2***
3.0
9 41.7
4.4
Residual mean square
37.95
Residual df
52
NA
NA
NA, not applicable as variance ratio
(F)
for one-way analysis of variance was not significant at
P
-=
005.
Values within a vertical column were significantly different from the control group (saponin:
Fe
molar value
0):
t
Crude saponin extract; purified saponins were given in all other test meals.
*P
<
0.05,
***P
<
0.001
(for details of statistical treatment,
see
p.
391).
Table
2.
Expt
2.
59Fe absorption in rats from a 3 g starch-sucrose
(I
:
I,
w/w)
test meal
containing
120
pg
Fe and variable amounts
of
Gypsophila
saponin
(Values are means with their standard errors for the no. of observations indicated)
__
"Fe absorption
(X
of dose)
Saponin
:
Fe
molar value
n
Mean
SE
0
10
62.2
1.7
8 8
50%*
4.3
8t
7
48.6** 2.2
16
5
51.6*
3.8
Residual mean square 69.65
Residual df 26
~
..
~~
..
~-
Values within a vertical column were significantly different from the control group (saponin: Fe molar value
0):
f
Crude saponin extract; purified saponins were given in all other test meals.
*P
<
0.05,
**P
<
0.01 (for details of statistical treatment, see
p.
391).
Gypsophila
saponins had no effect on Zn absorption from the test meal, as shown in
Table 1.
Expt
2
Values for Fe absorption
('YO
of
dose), in rats weighing
113
(SEM
1)
g,
from test meals
containing saponin
:
Fe molar values
of
8
and
16
are shown in Table
2.
At higher levels
of
Gypsophila
saponins the test meals were obviously increasingly unpalatable and all animals
from
group
4
(saponin
:
Fe
32)
had to be excluded, as less than two-thirds
of
the test meal
had been consumed by each rat. However, there were sufficient numbers in the other groups
Saponins and mineral absorption
393
Saponin: Fe molar value..
.
0
1
2
4 8
Saponin
(mg) . .
.
(0)
(3) (6)
(12)
(24)
16
(48)
Fig.
1.
Percentage '"Fe absorption, relative to the mean absorption of the appropriate saponin-free
control group, from
3
g
starch-sucrose (l:l, w/w) paste containing
120,ug
Fe, 18.5
kBq
'"Fe
and
varying amounts of purified
Gypsophila
saponin. Results are plotted
Y.
saponin:
Fe
molar values and
absolute amounts of saponin. Points are mean values with their standard
errors
represented by vertical
bars.
Table
3.
Expt
3.
59Fe absorption
in
rats from a
3g
starch-sucrose
(l:l,
w/w)
test meal
containing
120
pg
Fe
and
16
mg soyasaponin
I,
lucerne
(Medicago sativa)-plant-top
saponins or
Gypsophila
saponins
(saponin
:
Fe molar value aproximately
8)
(Values are means with their standard errors for the no. of observations indicated)
____
~
~-
"Fe absorption
(%
of dose)
Type
of
saponin
in test meal
n
Mean
SE
Control (no added saponin) 15 64.0
1.5
Soyasaponin
I
14
59.1
2.5
Lucerne 11 53.4;;; 1.7
Gypsophila
12 49.5*** 2.3
.
~-
Residual mean square
54.9
1
Residual df 48
Values within
a
vertical column were significantly different from the control group (saponin
:
Fe
molar value
0):
***P
<
0001 (for details of statistical treatment,
see
p.
391).
to demonstrate that rats fed on meals containing saponin absorbed significantly less 5vFe
than those given the
'
saponin-free
'
meal, and that the crude and purified saponin extracts,
fed at a saponin
:
Fe molar value
of
8,
produced a similar reduction in 59Fe absorption. Data
from Expts
1
and
2
were expressed as
YO
Fe absorption relative to the appropriate control
value, and combined to give an indication
of
the relation between the amount of saponin
consumed and whole-body "Fe retention. The resulting curve indicates that the effect
is
saturable and that it reaches a maximum at
a
saponin
:
Fe value
of
approximately
4,
when
Fe absorption was reduced by
17
YO
(Fig.
1).
394
SUSAN
SOUTHON
AND
OTHERS
Expt
3
59Fe absorption from a meal containing
16
mg purified
Gypsophila
saponins (saponin
:
Fe
molar value of approximately
8)
was once again shown to be significantly lower than that
of controls (Table
3).
The reduction in Fe absorption in the mature rats, weighing
319
(SEM
3)
g, was similar to that observed in younger animals given meals containing a similar
amount of saponins (Table
2).
Lucerne-plant-top saponins
(16
mg/meal) also reduced 59Fe absorption from a test meal,
but the same quantity of soyasaponin
I
had no significant effect when compared with the
control group (Table
3).
However, the mean value for 59Fe absorption for the soyasaponin
I
group was not significantly different from that for rats given lucerne saponins, and
statistical comparison of percentage Fe absorption in the soyasaponin
I
v.
the
‘
saponin-
free’ control group gave a
P
value of
<
0.1,
indicating a marginal effect.
DISCUSSION
Saponins comprise a structurally diverse class of chemicals widely found in the plant
kingdom (Price
et al.
1987).
They are characterized by the presence of a lipophobic
aglycone (which may be neutral, acidic or, in the case of the potato glycoalkaloids, basic)
and a lipophilic carbohydrate moiety or moieties. Whilst these contrasting lipo-
philic-lipophobic characteristics give saponins their surface-active properties (Gohtani
et al.
1985),
other properties (biological and chemical) are now seen
to
be very much
dependent on individual structural requirements. Only relatively recently have chemical
techniques enabled the nature and amounts of individual saponins in plant foods and
feedstuffs to be determined reliably (Price
et al.
1986;
Ireland
&
Dziedzic,
1987).
Analyses
have now clearly shown that saponins vary qualitatively and quantitatively with the plant
species and its age, the part examined, and environmental and agronomic conditions during
growth (Ota
et al.
1986), which supports the view that many studies
of
the biological effects
of saponins are difficult to interpret because of the use of ill- or non-defined extracts and
crude isolates. It was for this reason that the present study has been conducted using
defined
Gypsophila,
lucerne and soya-bean saponins. Chemically the former is the most
complex and is similar to saponins found in ‘health’ supplements being widely advocated
in the orient (Sonnenborn,
1987);
the lucerne saponin (containing medicogenic acid as
aglycone) is typical of those found in lucerne leaves and tops (Nonaka,
1986),
whereas the
seeds contain neutral soyasaponins structurally similar
to
those found in beans such as
haricot
(Phaseolus vulgaris),
kidney
(Phaseolus vulgaris)
and soya
(Glycine max)
(Kitagawa,
1984).
In a previous study it was found that the addition of a crude preparation of
Gypsophila
saponins to a semi-synthetic diet had a detrimental effect on Fe status in growing rats
(Southon
et al.
1988),
but Zn status as judged by femur Zn was unaffected. The present
study demonstrated that the amount of Fe absorbed from a test meal could be reduced in
the presence
of
these saponins, which indicates that the effect on Fe status was probably
due to reduced Fe absorption. Zn absorption was not affected by the saponins, which is in
agreement with our earlier finding of no change in femur Zn. The fact that crude and
purified
Gypsophila
saponins, given at a saponin
:
Fe molar value of approximately
8
(24
mg saponin/meal) were found to give similar values for the proportion of Fe absorbed
from a meal, indicates that the changes in Fe status observed after long-term consumption
of the crude extract were due to the saponin fraction of the material.
Lucerne-plant-top saponins, fed at a saponin: Fe value of approximately
8
(16
mg/meal),
also significantly reduced Fe absorption, and there were indications that a similar amount
Saponins
and
mineral
absorption
395
of soyasaponin
I
had a marginal effect, which might be important in rapidly growing
animals consuming the saponin over a long period. Comparison of the ability of these
saponins to increase intestinal permeability in vitro shows a similar pattern of response.
Gypsophila
saponins produced the greatest rate of decline in transmural potential difference
(PD),
lucerne-plant-top saponins had a slightly less potent effect, and soyasaponin
I
decreased the PD only marginally (Johnson
et
al.
1986; lucerne values, I. T. Johnson,
unpublished result).
It was suggested in our previous work (Southon
et
al.
1988) that saponins may interfere
with Fe metabolism, either by forming complexes with the dietary Fe thereby rendering it
unavailable for absorption, or by producing changes in mucosal function with long-term
consumption, thus reducing the efficiency
of
nutrient absorption. At first glance, the results
from the present study, where saponins were consumed in a single meal, appear to support
the first of these two hypotheses. However, the dose-response curve obtained by combining
the results from Expts
1
and
2,
indicates a more complex mechanism. Combining data this
way is not ideal but the resulting curve provides some useful insight into the relation
between dietary saponin concentration and the degree
of
reduction in Fe absorption. It
appears that the effect
of
Gypsophila
saponins plateaux at a saponin:Fe value of
approximately 4
(12
mg saponins/meal), and the observed reduction in Fe absorption
cannot be exaggerated by the addition of more saponin (up to 48 mg/meal). This suggests
that the reduction in absorption is due to an effect on Fe transport into or across the
mucosal cell, rather than a chemical binding in the intestinal lumen, since the latter would
be expected to exhibit
a
more marked concentration dependence (Morck
et
al.
1983) than
was observed in the present study. In vitro-binding studies are necessary to clarify this
point. It is possible that even short-term exposure of the mucosal cells to saponin, such as
from a single meal, can cause impairment in
a
sensitive component of the Fe transport
mechanism.
As
mentioned earlier in the discussion, other studies have clearly shown that
some saponins can readily make intestinal absorptive cells permeable in vitro and destroy
their capacity for the accumulation of sugars (Johnson
et
al.
1986); the accumulation and
transport of certain other nutrients may therefore be just as readily affected. The absence
of any reduction in Zn absorption from a meal containing saponins demonstrates, however,
that any change in intestinal function does not affect the absorption of all mineral
nutrients.
The saponin
:
Fe molar values used in the present study are likely to be higher than would
be generally found in the human diet, but the present study gives no indication as to the
relative importance of the absolute amounts
of'
saponin and Fe
v.
the saponin
:
Fe molar
value in determining the proportion of the mineral available for absorption. Recent
calculations indicate a mean daily intake
of
approximately
20
mg saponins per person
(primarily soyasaponins) for the
UK,
with vegetarians, and people who regularly consume
such products as soya-bean-based foods (370
mg
saponins/kg) and baked beans (240 mg
saponins/kg), having considerably higher intakes, in excess of
100
mg/d
(C.
L.
Curl,
K.
R. Price,
I.
T.
Johnson and
G.
R.
Fenwick, unpublished results). The intake of lucerne
saponins by man is generally very low but regular consumption of lucerne sprouts, tablets
and juice would lead to the consumption of significant amounts of these saponins. It is
possible, therefore, that the consumption of
foods
rich in saponin (which may also contain
other inhibitors of mineral absorption such as phytate and tannins) could result in dietary
saponin
:
Fe values demonstrated to influence Fe absorption adversely, particularly when
Fe intakes are marginal.
The identification of the hypocholesterolaemic effects of dietary saponins has led to the
suggestion that saponin intakes be increased to regulate plasma cholesterol (Sidhu
et
al.
1987). However, findings presented in the present study and other studies (Johnson
et
al.
396
SUSAN
SOUTHON
AND
OTHERS
1986;
Southon
et
al.
1988),
suggest that further work is needed to evaluate more fully the
implications
of
such a dietary change, particularly in relation to intestinal function, and the
absorption and utilization of essential minerals.
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