J. Sci. Food Agric.

198I, 32, 273-278

Saponin Content of Soya Beans and

Some Commercial Soya Bean Products
Dorothy E. Fenwick and David Oakenfull
CSIRO Division of Food Research, PO Box 52, North Ryde, New South Wales 2113, Australia
(Manuscript received 19 May 1980)

Quantitative thin layer chromatography has been used to estimate the saponin
content of whole soya beans (Glycine max L. Merrill, cv. Williams) and a number of
commercial soya bean products such as protein isolates and lecithin. Saponins were
present in all these materials at concentrations ranging from 56 g kg-1 (on a dry
weight basis) for whole soya beans to 3 g kg-1 for the protein isolate ‘Promine-D’.
Previous estimates indicate a saponin content of whole soya beans of only about
5 g kg-l. It is suggested that this is an underestimate resulting from loss of material
during the extraction procedures used in earlier methods of analysis.

1. Introduction
Saponins are steroid or triterpenoid glycosides which occur in a wide variety of These
compounds seem to be of considerable nutritional significance since dietary saponins lower plasma
cholesterol concentrations in experimental animals4j5 and possibly have the same effect in man.6
Soya beans contain at least ten triterpenoid sap on in^^,^^* and the presence of these may account
for the cholesterol-lowering effect of various soya bean products when present in human and animal
diets.5,6.9-11
Therefore, the saponin content of whole soya beans (Glycine max L. Merrill, cv. ‘Williams’)
and a number of commercial soya bean products have been estimated: protein isolate, tofu (bean
curd), defatted flour, and lecithin. Essentially, the method of KartnigI2 was followed-using thin
layer chromatography (t.1.c.) to separate the saponins from a crude extract of the plant material
and a densitometer to estimate the quantity of saponins on the chromatogram. All the test materials
contained significant amounts of saponins.

2. Experimental
2.1. Extraction of saponins
The material was dried for 16 h at 110°C. A weighed sample (about 20 g) was exhaustively extracted
with acetone in a Soxhlet extractor to remove lipids, pigments, etc. The solvent was then changed
to methanol and the extraction continued for at least 36 h. The methanolic extract was allowed to
cool and made up to 200 ml with methanol. This solution contained the saponins, but also other
compounds such as flavonoids.

2.2. Thin layer chromatography

Standard samples (5-50 pl) of the methanolic extract were spotted on to pre-coated silica gel t.1.c.
plates (Merck, Kieselgel 60 F-254). The plates were developed with n-butanol-ethanol-concen-
trated ammonia (3.5: 1 :2.5). To visualise the spots the plates were sprayed with a 100 ml litre-1
solution of sulphuric acid in ethanol and heated at 110°C for 30 min. A typical chromatogram is
shown in Figure 1 in which an extract prepared from whole soya beans is compared with a purified
commercial saponin (saponin white, Ajax Chemicals, Sydney).
0022-5~42/81/0300-0273$02.00 @ 1981 Society of Chemical Industry
213
274 D. E. Fenwick and D. Oakenfull

2A

0 Q
0
ot 0 Q
at 0 Q
2s+ 0
0

0 0

0 %
Q
Q 0
5x 5X 5A 5A 5OA 5h
Soyo Saponin Soya Saponin SOYO Soponin
saponins white soponins white saponins white

Figure 1. Chromatogram of a methanolic extract of whole soya beans compared with saponin white. Saponin
spots are marked with an arrow and those saponins found to he relatively depleted in protein isolates are marked
with an asterisk.
Figure 2. Chromatograms of a methanolic extract of whole soya beans. Various loadings are indicated: (A)
sprayed with anisaldehyde acetic acid, (B) treated with silver nitrate, (C) treated with a suspension of chicken
erythrocytes in isotonic buffer and gelatin. The shading indicates the relative intensities of the spots.

Since the methanolic extract contains other compounds it is necessary to identify those spots on
the chromatogram which are due to saponins. Three different procedures were used : (a) A chroma-
togram was sprayed with a mixture of anisaldehyde (1 rnl), sulphuric acid (2 ml) and acetic acid
(98 ml) and heated for 10 min at 100°C [see Figure 2(A)]. This reagent gives characteristic purple-
mauve colours with steroids or triterpenesL3 (but also with several other classes of compound.
(b) A chromatogram was dipped for 1 min into a solution containing 2 . 7 aqueous~ silver nitrate
(2 ml) in acetone (400 ml). It was then dried and sprayed with 0 . 5 sodium
~ hydroxide in ethanol.
Reducing sugars give a dark brown coloration on a light background.14 This method was not as
sensitive as the anisaldehyde spray. A heavier loading of methanolic extract was required resulting
in poorer resolution [Figure 2(B)]. (c) A chromatogram was coated with a suspension of washed
erythrocytes (from a domestic hen) in an isotonic phosphate buffer solution (pH 7.4) containing
45 g litre-l of gelatin.I5 The coated plate was then held at 100 % relative humidity at room tempera-
ture (28°C) for 16 h. Most saponins are strongly haemolytic and their presence on the plate is
revealed as a white spot against a red background. Soya bean saponins are not as strongly
haemolytic as saponin whitel6 [see Figure 2(C)]. A heavy loading of methanolic extract was again
required, resulting in poor resolution.
None of these tests individually can be considered to give a specific and unequivocal indication
of the presence of saponins on a thin layer chromatography plate. However, collectively, they give a
reliable indication of which of the spots on the original chromatogram (in Figure 1) are due to
saponins-these are marked in Figure 1 by arrows.

2.3. Quantitative thin layer chromatography

For this purpose, chromatograms on glass-backed plates were sprayed with a 10% solution of
sulphuric acid in ethanol and heated at 110°C for 30 min (von Biedermann, C. H., private com-
munication). The soya bean saponin extracts were always spotted alongside a standard solution
( 5 g litre-l) of saponin white as a reference. The intensity of the saponin spots was obtained by
using a densitometer (Schoefell Rapid-Scan SD 2000) and measuring the appropriate peak areas
Saponin content of soya beans 275

Figure 3. Plot of the relative peak area (see text)

against loading for purified soya bean saponins.

Loading ( ~ 1 )

on the recorder chart with a planimeter. The result was expressed as the ratio of the combined
saponin peak areas of the unknolvn sample to those of the adjacent saponin white standard. A
calibration curve was constructed by measuring chromatograms of known different loadings
of a purified soya bean saponin mixture prepared by the method described by Kitagawa e i al.7-
this is shown in Figure 3. The quantity of saponins in the unknown samples was calculated from
this curve.
It has been estimated that the precision of this method is only 5 20 %. Although, in principle,
better than 2 % is obtainable by quantitative thin layer chromatography,l7 the situation described
here is far from ideal. It is necessary to measure a composite series of peaks and the base line is not
always clearly defined.
2.4. An alternative method of estimation based on that of Gestetner et aZ.l*
The soya sapogenins were quantitatively extracted from defatted soya bean flour. Dried flour
(1.84 g) was refluxed for 8 h with a mixture of 0 . 6 7 aqueous
~ sulphuric acid (150 ml) and dioxane
(50 ml). The product was filtered and the solution extracted with ether (100 ml then 3 x 50 ml).
The combined ether extracts were washed with water until free from acid and then evaporated.
The solid residue was dissolved in toluene (30 ml) and the solution poured on to an activated
alumina column (Woelm, neutral, activity grade 1) which had been treated with a trace of acetic
acid and washed with toluene to remove impurities. The column was eluted with a 30 ml litre-1
solution of methanol in toluene (3 x 500 ml). The combined eluant solutions were then evaporated
to dryness and the solid residue weighed. The yield of sapogenins was 0.0227 g.
Gestetner et ~ 1 . 1 8showed that the sugar: sapogenin ratio was constant at 1 :1 in saponins pre-
pared from different varieties of soya beans so that the saponin content could be calculated from
the yield of sapogenins.

3. Results
The moisture and saponin contents of the soya bean products that were investigated are given in
Table 1. The manufactured products all contained some saponins-but less than whole soya beans.
19
216 D. E. Fenwick and D. Oakenfull

In the protein isolates, the proportions of the different saponins, as indicated by the relative inten-
sity of the spots on the chromatogram, were different from those of whole beans or defatted flour.
The protein isolates were depleted of the saponins of lower RF (indicated in Figure I ) . These
saponins could be more readily washed from protein during the manufacturing process. It is also
possible that some hydrolysis of the saponins could occur during the preparation of protein iso-
lates. The resulting sapogenins would then be lost during the acetone extraction.

Table 1. Moisture and saponin content of soya beans and some soya bean
products

Saponins
Moisture (g kg-l on a dry
Material (g kg-l) weight basis)

Whole soya beans 97 56b

Defatted soya flour 82 122
125’
Soya hulls 78 20
Protein isolate: ‘Promine-D’b 92 3
‘GL-750’C 91 8
‘Maxten c”$ 84 19
‘Maxten E’d 74 25
Lecithin: ‘Crown’’ 55 53
‘Vitaplex’f 52 29
Tofu9 850 21

Estimated as described in section 2.4.

Central Soya Co. Inc., Illinois.
Griffith Laboratories Pty Ltd, Victoria.
Miles Laboratories (Australia) Pty Ltd.
Crown Vitamins Pty Ltd, Kogarah, New South Wales.
f Vitaplex Pty Ltd, Chatswood, New South Wales.

9 The Soy Bean Factory, Surrey Hills, New South Wales.

4. Discussion
Soya beans have previously been reported to contain only 5 g kg-l s a p ~ n i n s ~ J ~ , ~ g - ttimes
e n less
than reported here. It is possible that the cultivar which was examined had an abnormally high
saponin content but this seems unlikely.
Previous analyses have been used on either (a) measuring the yield of purified saponins from a
given weight of plant material;22 or (b) preparing and isolating the sapogenins as described in
section 2.4 and estimating them colorimetrically.18It is possible that substantial amounts of material
are lost during the various extractions. It was found, for example, in following the method of
Birk et u Z . ~ ~ @ that a large proportion of the saponins were lost in a step in which the saponins were
precipitated from aqueous solution by addition of calcium oxide. When the sapogenin method of
Gestetner et u1.18 was used, but the product weighed instead of estimating it colorimetrically,
a yield of purified sapogenins was obtained which indicated a saponin content for defatted soya
flour of 25 g kg-l-in excellent agreement with that obtained by quantitative t.1.c. (see Table 1).
The results reported here appear to be consistent with the saponin contents of other legumes
reported by Applebaum et d . 2 1 (see Table 2). These data were also obtained by measuring the yield
of saponins extracted from a given weight of plant material but here the authors recognised that
not ail of the saponins were precipitated by calcium oxide (precipitated and non-precipitated sapo-
nins are combined in Table 2). The results are also consistent with an estimate of the saponin
content of defatted soya flour recently reported by Hudson and EI-Difrawi.22They used a method
based on that of Birk et al. to isolate the saponins and obtained a yield of 19 g kg-l.
Saponin content of soya beans 277

Table 2. Saponin content of some legumes as reported

by Applebaum et al.zl

Seed

Chickpeas (Cicer arietinum) 60

Garden peas (Pisum safivum) 62
Lentils (Lens culinaris) 66
Broad beans (Vicia faba) 63
Haricot bean (Phaseolus vulgaris) 32
Peanuts (Arachis hypogaea) 16

Calculated on the basis of lipid-free meal as 1 kg.

Soya bean protein isolate has generally been assumed to be free from sap on in^.^,^^ It has recently
been shown though that in protein fractions prepared from lucerne (alfalfa) leaves saponins are
concentrated at a higher level than in the original lucerne.23 Since saponins are strongly surface
active and bind to proteins it is reasonable to suppose that they would be carried with the protein
during the various isolation procedures.
The high levels of saponins found in soya bean flour and some soya protein isolates appear to
explain the cholesterol-lowering effect of these materials when fed to man6 or animal^.^^^ The low
values reported previously are too low to be consistent with this effect.

Acknowledgement
The authors are greatly indebted to Miss C. H. von Biedermann for much practical help and advice.

References
I . Kofler, L. Die Saponine Verlag von Julius Springer, Vienna, 1927.
2. Bau, N. ; Rastogi, R. P. Triterpenoid saponins and sapogenins. Phytochemistry 1967, 6, 1249-1268.
3. Bondi, A.; Birk, Y.; Gestetner, B. Chemistry and Biochemistry of Herbage (Butler, G. W.; Bailey, R. W., Eds),
Academic Press, New York, 1973, pp. 51 1-528.
4. Oakenfull, D. Saponins in food. Food Chem. (in press).
5 . Cheeke, P. R. Nutritional and physiological properties of saponins. Nufr. Rep. Int. 1976, 13, 315-324.
6. Potter, J. D.; Topping, D. L.; Oakenfull, D. Soy products, saponins and plasmacholesterol. Lancet 1979, i,
223.
7. Kitagawa, I.; Yoshikawa, M.; Yosioka, I. Saponin and sapogenol. XIIi. Structures of three soybean saponins;
soysaponin I, soysaponin I1 and soysaponin 111. Chem. Pharm. Bull. 1976,24, 121-129.
8. Wolf, W. J.; Thomas, B. W. Thin layer and anion exchange chromatography of soybean saponins. J. Am.
Oil Chem. Soc. 1970,47, 86-90.
9. Carroll, K. K. The role of dietary protein in hypercholesterolemia and atherosclerosis. Lipids 1977, 13,
360-365.
10. Sirtori, C. R.; Agradi, E.; Conti, G.; Montero, 0.; Gatti, E. Soybean-protein diet in the treatment of type I1
hypercholesterolaemia. Lancet 1977, i, 275-277.
I I. Simons, L. A. The effects of oral lecithin and clofibrate o n cholesterol metabolism. Artery 1978, 4, 167-182.
12. Kartnig, von Th. ; Ri, C. Y.Diinnschichtchromatographische Untersuchungen an den Saponin aus Cortex
quillajae. Planta Med. 1973, 23, 269-277.
13. Lisboa, B. P. I n Lipid Chromatographic Analysis Vol. 2 (Marinetti, G. V., Ed.), Marcel Dekker, New York,
1976.
14. Trevelyan, W. E.; Procter, D. P ; Harrison, J. S. Detection of sugars o n paper chromatograms. Nature (London)
1950, 166, 444-445.
i5. Smoczkiewica, M. A. ; Nitschke, D. ; Stawinski, T. M. Mikrobestimmung von spuren von Saponinglykosiden
in ‘nicht-saponin fuhren dem’ pflanzen Material. Mikrochim. Acra 1977, 11, 597-605.
16. Birk, Y.; Bondi, A.; Gestetner, B.; Ishaaya, I. A thermostable haemolytic factor in soyabeans.
Nature (London) 1963, 197, 1089-1090.
17. Touchstone, J. C.; Dobbins, M. F. Practice of Thin Layer Chromatography John Wiley and Sons, New York,
1978, p. 235.
18. Gestetner, B.; Birk, Y.; Bondi, A.; Tencer, Y. Soya bean saponins. VII. A method of the determination of
sapogenin and saponin contents in soya beans. Phytochemistry 1966,5, 803-806.
278 D. E. Fenwick and D. Oakenfull

19. Smith, A. K.; Circle, S. J. Soybeans: Chemistry and Terhnology Avi Publishing C o . , Westport, 1972.
20. Gestetner, B.; Ishaaya, 1.; Birk, Y . ; Bondi, A. Soybean saponins 111. Fractionation and characterisation.
Israel J. Chem. 1963, 1, 461-469.
21. Applebaum, S. W.; Marco, S . ; Birk, Y.Saponins as possible factors of resistance of legume seeds to the attack
of insects. J. Agric. Food Chem. 1969, 17, 618-622.
22. Hudson, B. J. F.; El-Difrawi, E. A. The sapogenins of the seeds of four lupin species. J . Pkmt Foods 1979, 3,
181-186.
23. Livingston, A. L . ; Knuckles, B. E.; Edwards, R. H.; de Fremery, D.; Miller, R. E.; Kohler, G . 0. Distribution
of saponin in alfalfa protein recovery systems. J. Agric. Food Chem. 1979, 27, 362-365.