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Interaction of lucerne saponins with sterols
181
BIOCHIMICAET BIOPHYSICA ACTA BBA 56045
INTERACTION
B.
OF LUCERNE
SAPONINS
WITH
STEROLS*
GESTETNER, Y. ASSA, Y. HENIS, Y. TENCER, M. ROTMAN, Y. BIRK and A. BOND1 of Agriculture, The Hebrew University of Jerusalem, Rehovot (Israel)
Faculty
(Received
December
6th, 1971)
SUMMARY
The structural features of sterols, which enable them to interact with lucerne saponins, were studied. It was found that an intact steroid ring structure having the conformation of cholestanol, to which a side chain characteristic to cholesterol or phytosterols is attached, is essential for the formation of a sterol-saponin addition product. Unlike the complex formed between digitonin and cholesterol, these addition products are extremely unstable and their formation is not dependent on the presence of a 3+hydroxyl group. In addition products formed with cholesterol or 7-dehydrocholesterol an approximately I :5 molar ratio exists between saponin and sterol, whereas in the case of other sterols this ratio is approximately I : 8. Although lucerne saponins readily form addition products with a variety of sterols, a preferential affinity to cholesterol and 7-dehydrocholesterol was found when the addition product was formed while cholesterol and one of the other sterols were present simultaneously in the reaction mixture. These preferential affinities were demonstrated also by bioassays conducted with the fungus Sclerotiwn rolfsii Sacc.
INTRODUCTION
The ability of various sterols to form water-insoluble complexes with steroid saponins, i.e. digitonin, is known for a long time and the prerequisites of forming such complexes are well establishedl. This feature is utilized also in isolation procedures of lucerne saponinsap3*4. Very little is, however, known about the nature of interaction between sterols and saponins from the triterpene series, to which lucerne saponins belong. The hemolytic activity of saponins is generally attributed to their interaction with cholesterol in the red cell membrane .516. In the case of lucerne saponins this activity could be abolished by the presence of extraneous cholesterol or 5-stigmastene3t%01 on the reaction mixture’. Larval growth inhibition caused by luceme saponins can be prevented by the presence of various sterols in the diets, but with regard to counteracting fungal growth inhibition, only cholesterol was effective8. These findings * Part VI in a series of “Lucerne
Saponins”.
Part V, Phytochemistry, Biochim.
Biophys.
IO (1971)
Acta,
2221.
270 (1972)
181-187
182
B. GESTETNER
t?t ai
seem to imply that the biological activities exerted by lucerne saponins involve sterol in the organism. In order to elucidate the nature of the biological activities of lucern saponins on different organisms, it has been of interest to study their interrelationship with various sterols. MATERIALS
AND METHODS
Preparation of saponins Lucerne saponin extract was prepared from roots of lucerne, Hairy Peruviar variety, by the method of Shany et aLlo. A lucerne saponin (0$-D-glucopyranosyl-(I + 6)-,%D-glucopyranosyl-(r --f 3) /3-n-glucopyranosylmedicagenic acid)ll was isolated according to Gestetner et aLs. Interaction of sterols with Lucerne saponins The following sterols were used for interaction with lucerne saponins: cholester 01, 7-dehydrocholesterol, ergosterol, cholecalciferol, ergocalciferol and $?-cholestane 3/?-01 (Sigma Chemicals Co.); cholesterol acetate, 5-androstene-3~,r7,9-diol and it! diacetate, 3/3-hydroxy-5-cholen-24-oic acid (Ikapharm, Israel) and 5-stigmastene. 3,901 (@-sitosterol) (Fluka A.G.) composed of approx. 60% 5-stigmastene-3,%ol approx. 20% 24-methyl-5-cholestene-3b-ol and approx. 20% 5,22_stigmastadiene. 3B-01. Pure samples of the two latter substances were donated by Dr M. Katz Cholesterol and 5-stigmastene-38-01 were crystallized twice from abs. ethanol befon use. The sterols were determined to be chromatographically pure by gas-liquid chromatography in a Packard No. 7300/7400 gas chromatograph equipped with z flame ionization detector, on 2 m x 0.32 cm columns of 5% OV-IOI on GCQ at ar operating temperature of 260 “C12. Lucerne saponins were allowed to interact with sterols by refluxing, for 3c min, a mixture of IOO mg saponin extract and I g of either of the sterols in IOO ml water. After removal of unbound sterols, by leaching with water and benzene, respectively, the sterol-saponin addition product was dissociated by boiling in a mixture benzene-ethanol. The participating sterols and saponins were identified by thin-layer chromatography. Detailed procedures of preparation of sterol-saponin addition products and their analyses were described previously8. The sterols were determined quantitatively by the method of Abel1 et al. 13. The saponins were subjected to acid hydrolysis and their aglycone and sugar contents were determined8+. The comparative affinity of various sterols for lucerne saponinswith respect tc cholesterol, was examined by preparing the sterol-saponin addition product in the simultaneous presence of cholesterol and one of the other sterols. After removal of unbound material from the reaction mixture, bound sterol was liberated as described above and identified by gas-liquid chromatography. Retention times are quoted relative to cholesterol. Effect of lucerne saponins on fungal growth in the presence of sterok Bioassays with Sclerotium rolfsii Sacc. were carried out according to Gestetner et al.s. The culture medium contained 0.005% lucerne saponins, with or without various amounts of the sterols.
Biochim. Biophys. Acta, 270 (1972)
181-187
INTERACTION OF LUCERNE SAPONINS WITH STEROLS
183
RESULTS
The structural requirements of sterols to form an addition product with lucerne saponins were investigated with a variety of sterols, which differ from cholesterol, in Steroln
albe
to
precipitate
Sterols
HO5-Stigmastene-
unalble
H
to
precipitate
5&Cholestane-
38-01
‘3
-+‘I
&J
I3
“<2 C"3
HO 543-
Cholestaneone
-Hydroxy-B-cholen-24-oic Ergosterol
acid =",
CHS
HO
Ergocalciferol
HO
Cholecalciferol
HO 24-
Methyl-
5 -
="3
- cholestene-
-38
-01
CH’ “3
5,22- Stigmastadiene -3 /I -01
Fig. I. Structural formulas of sterols, which do or do not interact with lucerne saponins. Biochim.
Bio$hys.
Ada,
270 (1972) 181-187
184
B. GESTETNER
etd.
one or more features of their structure (Fig. I). The results of these experiments indicate that the ability of the various sterols to interact with lucerne saponins is not affected by the following structural changes in the molecule: (I) presence of additional double bonds in the ~y~lo~e~tanoper~y~ophenanthrene ring or in the isoctyl side chain; (2) presence of additional methyl or ethyl groups in the side chain; (3) saturation of the double bond in ring B; and (4) oxidation of blocking the 3B-hydroxyl group. On the other hand, compounds which differ from cholesterol in the following structural features, are unable to combine with lucerne saponins: (I) the spatial relationships of rings A and B is cis to each other; (2) opening of B ring, and subsequent stereochemical change (i.e. calciferols) ; (3) elimination of the side chain; and (4) shortening or lengthening the side chain and introducing various functional groups into it. Thus it seems that the presence of an intact steroid ring structure, having the conformation of cholestanol, to which a side chain characteristic to cholesterol or phytosterols is attached, are essential for interaction of sterols with lucerne saponins. Although lucerne saponins could be precipitated by interaction with different sterols, the attempts to isolate a sterol-saponin addition product were not successful, since it dissociated under the con~t~ons employed. Examination of the saponins, which were isolated by interaction with the different sterols showed that they were ~hromato~aphi~~ly indistin~ishable from each other as well as from saponins precipitated by cholesterol and j+sitosteroP. The chemical composition of the reacting saponins was also identical, i.e. medicagenic acid being the sole aglycone attached to a variety of sugars@. The various sterols can be divided into two distinct groups, one consisting of cholesterol and 7-dehydrocholesterol which form addition products at an approx. 1:5 molecular ratio, unlike the other sterols, in the addition products of which an approx. x :8 molecular ratio exists (Table I). These molecular ratios are based on an average molecular weight of lucerne saponins, of 1000 to 1200~7~.The homogeneous lucerne saponin gave with cholesterol I : 4.82 and with 5-stigmastene-3#I-ol I : 8,zo molecular ratios. These ratios are in good agreement with those obtained with the mixture of saponins present in the whole extract. The results given in Table I can also be interpreted in terms of affinity for TABLE1 FORMATION
~
OF STEROL-LUCERNESAPONIK
ADDITION
PRODUCTS
_~~
I.91 16.5 Cholesterol 7-De~ydro~oiesterol 16.0 2.04 5-Stigmastene-3j!-ol** 20.0 3.26 Ergosterol 17.5 3.52 5x-Cholestan-3-one 15.0 3.42 Pm * Based on an average molecular weight of lucerne saponins of xooo ** This commercial sample contains approx, 60% 5-stigmastene-3/?-01, 5-cholestene-3/?-ol and approx. 20% 5,zz-stigmastadiene-3B-01. B&whim. Biophys.
Acta, 270 (1972) 181-187
I/4.9
115.3 X17.9 I/Q
1/8.8 to
1200.
approx. 20%
q-methyl-
INTERACTION OF LUCERNE SAPONINS WITH STEROLS
I&
saponins, which is much more pronounced by cholesterol and 7-dehydrocholesterol, than by the other sterols. This statement was further substantiated by forming addition products with saponins in the presence of more than one sterol in the reaction mixture. By using cholesterol as reference compound, it was found (Fig. 2) that when sterol-saponin addition product is formed in the presence of cholesterol and one of the other sterols, the bulk of the addition product was formed with cholesterol, whereas the other sterols, except for 7-dehydrocholesterol, interacted with lucerne saponins only to a negligible extent. The preferential affinity of cholesterol and 7-dehydrocholesterol for lucerne saponins could also be demonstrated by bioassays with the fungus S. rolfsii. This fungus contains cholesterol and its growth is strongly inhibited by lucerne saponins. This growth inhibition can, however, be prevented by the additon of cholesterol, but these bioassays, it could be not /I-sitosterol to the culture medium s. By extending shown (Fig. 3) that similarly to cholesterol, 7-dehydrocholesterol can also prevent the growth inhibition caused by lucerne saponins, whereas the other sterols are ineffective in this respect even at much higher concentrations.
7
I Time
3
m&n
2. Gas chromatographic identification of sterols which form addition products with lucerne saponins. (a) Control, consisting of a mixture of sterols. I, cholesterol; z, 7-dehydrocholesterol; 3, gee-cholestan-s-one; 4, ergosterol; 5, 24-methyl-S-cholestene-3/?-01; 6, 5,22-stigmastadiene-@ol; 7. 5-stigmastene-3/l-01. (b-g) Sterols from the saponin addition product formed with: (b) cholesterol and 7-dehydrocholesterol, (c) cholesterol and ga-cholestane-s-one, (d) cholesterol and ergosterol, (e) cholesterol and nq-methyl-g-cholestene-$01, (f) cholesterol and 5,22-stigmastadiene-$?-ol, (g) cholesterol and 5-stigmastene-$ol.
Biochim. Bio$hys. Ada, 270 (1972) 181-187
B. GESTETNER
-I
@==%-
I
~.005
-f
AAc!-------
0
I
0025
Concentration
I
0.050 of sterols
et cd.
I
0075 (%)
1
Fig. 3. Effect of different concentrations of various sterols on the growth impairment of S. rolfsii Sacc. caused by 0.005% sterol precipitable lucerne saponins. 0, cholesterol; A, g-stigmastene38-01; 0, 7-dehydrocholesterol; A, ergosterol; 0, ga-cholestan-3-one. DISCUSSION
The structural features of sterols, which enable them to interact with lucerne saponins were studied. Similarly to the complexes formed between digitonin and various sterols, only sterols belonging to the cholestanol series are able to interact with lucerne saponins But unlike to complexes formed with digitonin, the sterol-lucerne saponin addition products are not dependent on the presence of an equatorial 3-phydroxyl group, in the absence of which digitonin does not form complexes with sterolsl. In the case of lucerne saponins the nature of the steroid side chain seems to be more important for the formation of addition products, than the modifications introduced into the steroid ring structure. This becomes evident, when one considers the preferential affinities displayed by cholesterol and 7-dehydrocholesterol. The ability of cholesterol and 7-dehydrocholesterol to prevent fungal growthinhibition, caused by lucerne saponins, can also be attributed to the preferential affinities of these sterols for lucerne saponins. Other sterols, when added to the culture medium, form also addition products with the saponins. However, when these unstable products undergo dissociation, the liberated saponins are able to interact with the membraneal cholesterol and consequently fungal growth is arrested. If, on the other hand, cholesterol or 7-dehydrocholesterol are added to the culture medium, they are able to compete with the native fungal sterols, and thus prevent growth inhibition. The extreme instability of the sterol-lucerne saponin addition product prevented its isolation. It should be, however, pointed out that also complexes of digitonin formed with sterols other than cholesterol, display similar instability’. The finding that the 3b-OH group is not involved in the interaction of sterols with lucerne saponins, points towards the improbability of covalent binding between them. On the other hand, one of the factors which is undoubtedly essential for the Biochim. Biophys.
Acta, 270 (1972) 181-187
INTERACTION OF LUCERNE SAPONINS WITH STEROLS
187
formation of the addition product, is the presence of unsubstituted carboxyl groups, at C, and C,, in medicagenic acid. By blocking these groups the interacting ability of lucerne saponins with sterols was abolished (Y. Assa, unpublished). It was also found that soybean saponins, which contain sapogenins devoid of carboxyl groups, are not able to form such addition product9. Some of the biological activities exhibited by saponins, such as hemolysis,6~6~15 are generally attributed to the interacor inhibition of growth of microorganisms 1ev17 tion of saponins with membraneal cholesterol. Since the structural requirements of cholesterol to interact with lucerne saponins differ from those existing between cholesterol and digitonin and since soybean saponins, which do not form addition products with sterols are also strongly hemolytic18, neither the extent, nor the mode of action of various saponins on biomembranes should be expected to be similar. Therefore, the examination of the action of different saponins may prove to be a useful tool in the elucidation of the function and significance of cholesterol in cell membranes, in particular in those of microorganisms. ACKNOWLEDGEMENT
This work was supported by the U.S. Department of Agriculture, Grant No. FG-1~~46. REFERENCES I P. Bladon, in R. P. Cock, Cholesterol, Academic Press, New York, 1958, p. 84. 2 E. D. Walter, G. R. van Atta, C. R. Thompson and W. D. Maclay, J. Am. Chem. Sot., 76 (1954) 2271. 3 C. R. Thompson, G. R. van Atta, E. M. Bickoff, E. D. Walter, A. L. Livingston and J. Guggolz, Tech. Bull. U.S. Agric., 1161 (1957) 53. 4 C. B. Coulson, J. Sci. Food Agric., g (1958) 281. 5 R. R. Dourmashkin, R. M. Dougherty and R. J. C. Harris, Nature, 194 (1962) 1116. 6 A. M. Glauert, J. T. Dingle and J. A. Lucy, Nature, rg6 (1962) 953. 7 S. Shany, Alfalfa Saponins-Isolation, Characterization and Biological Significance, Ph.D. Thesis, The Hebrew University of Jerusalem, 1971, p. 42. 8 S. Shany, B. Gestetner, Y. Birk and A. Bondi, J. Sci. Food Agric., 21 (1970) 508. g B. Gestetner, Y. Assa, Y. Henis, Y. Birk and A. Bondi, J. Sci. Food Agric., 22 (1971) 168. ro S. Shany, Y. Birk, B. Gestetner and A. Bondi, J. Sci. Food Agric., 21 (1970) 131. II B. Gestetner, Phytochemistvy, IO (1971) 2221. 12 C. Grunwald. Anal. Biochem.. 14 (1970) 16. 13 L. L. Abell, B. B. Levy, B. B. B&die and I?. E. Kendall, J. Biol. Chem., 195 (1952) 357. 14 G. Noelting and P. Bernfeld, Helv. Chim. Acta, 31 (1948) 286. 15 E. Ponder, Haemolysis and Related Phenomena, Grnne and Stratton, New York, 1948. 16 M. M. Weber and S. C. Kinsky, J. Bacterial., 89 (1965) 350. 17 B. Wolters, PZanta Med., 16 (1966) 77. 18 Y. Birk, A. Bondi, B. Gestetner and I. Ishaaya, Nature, rg7 (1963) 1089. Biochim.
Biophys.
Acta,
270 (1972) 181-187
BIOCHIMICAET BIOPHYSICA ACTA BBA 56045
INTERACTION
B.
OF LUCERNE
SAPONINS
WITH
STEROLS*
GESTETNER, Y. ASSA, Y. HENIS, Y. TENCER, M. ROTMAN, Y. BIRK and A. BOND1 of Agriculture, The Hebrew University of Jerusalem, Rehovot (Israel)
Faculty
(Received
December
6th, 1971)
SUMMARY
The structural features of sterols, which enable them to interact with lucerne saponins, were studied. It was found that an intact steroid ring structure having the conformation of cholestanol, to which a side chain characteristic to cholesterol or phytosterols is attached, is essential for the formation of a sterol-saponin addition product. Unlike the complex formed between digitonin and cholesterol, these addition products are extremely unstable and their formation is not dependent on the presence of a 3+hydroxyl group. In addition products formed with cholesterol or 7-dehydrocholesterol an approximately I :5 molar ratio exists between saponin and sterol, whereas in the case of other sterols this ratio is approximately I : 8. Although lucerne saponins readily form addition products with a variety of sterols, a preferential affinity to cholesterol and 7-dehydrocholesterol was found when the addition product was formed while cholesterol and one of the other sterols were present simultaneously in the reaction mixture. These preferential affinities were demonstrated also by bioassays conducted with the fungus Sclerotiwn rolfsii Sacc.
INTRODUCTION
The ability of various sterols to form water-insoluble complexes with steroid saponins, i.e. digitonin, is known for a long time and the prerequisites of forming such complexes are well establishedl. This feature is utilized also in isolation procedures of lucerne saponinsap3*4. Very little is, however, known about the nature of interaction between sterols and saponins from the triterpene series, to which lucerne saponins belong. The hemolytic activity of saponins is generally attributed to their interaction with cholesterol in the red cell membrane .516. In the case of lucerne saponins this activity could be abolished by the presence of extraneous cholesterol or 5-stigmastene3t%01 on the reaction mixture’. Larval growth inhibition caused by luceme saponins can be prevented by the presence of various sterols in the diets, but with regard to counteracting fungal growth inhibition, only cholesterol was effective8. These findings * Part VI in a series of “Lucerne
Saponins”.
Part V, Phytochemistry, Biochim.
Biophys.
IO (1971)
Acta,
2221.
270 (1972)
181-187
182
B. GESTETNER
t?t ai
seem to imply that the biological activities exerted by lucerne saponins involve sterol in the organism. In order to elucidate the nature of the biological activities of lucern saponins on different organisms, it has been of interest to study their interrelationship with various sterols. MATERIALS
AND METHODS
Preparation of saponins Lucerne saponin extract was prepared from roots of lucerne, Hairy Peruviar variety, by the method of Shany et aLlo. A lucerne saponin (0$-D-glucopyranosyl-(I + 6)-,%D-glucopyranosyl-(r --f 3) /3-n-glucopyranosylmedicagenic acid)ll was isolated according to Gestetner et aLs. Interaction of sterols with Lucerne saponins The following sterols were used for interaction with lucerne saponins: cholester 01, 7-dehydrocholesterol, ergosterol, cholecalciferol, ergocalciferol and $?-cholestane 3/?-01 (Sigma Chemicals Co.); cholesterol acetate, 5-androstene-3~,r7,9-diol and it! diacetate, 3/3-hydroxy-5-cholen-24-oic acid (Ikapharm, Israel) and 5-stigmastene. 3,901 (@-sitosterol) (Fluka A.G.) composed of approx. 60% 5-stigmastene-3,%ol approx. 20% 24-methyl-5-cholestene-3b-ol and approx. 20% 5,22_stigmastadiene. 3B-01. Pure samples of the two latter substances were donated by Dr M. Katz Cholesterol and 5-stigmastene-38-01 were crystallized twice from abs. ethanol befon use. The sterols were determined to be chromatographically pure by gas-liquid chromatography in a Packard No. 7300/7400 gas chromatograph equipped with z flame ionization detector, on 2 m x 0.32 cm columns of 5% OV-IOI on GCQ at ar operating temperature of 260 “C12. Lucerne saponins were allowed to interact with sterols by refluxing, for 3c min, a mixture of IOO mg saponin extract and I g of either of the sterols in IOO ml water. After removal of unbound sterols, by leaching with water and benzene, respectively, the sterol-saponin addition product was dissociated by boiling in a mixture benzene-ethanol. The participating sterols and saponins were identified by thin-layer chromatography. Detailed procedures of preparation of sterol-saponin addition products and their analyses were described previously8. The sterols were determined quantitatively by the method of Abel1 et al. 13. The saponins were subjected to acid hydrolysis and their aglycone and sugar contents were determined8+. The comparative affinity of various sterols for lucerne saponinswith respect tc cholesterol, was examined by preparing the sterol-saponin addition product in the simultaneous presence of cholesterol and one of the other sterols. After removal of unbound material from the reaction mixture, bound sterol was liberated as described above and identified by gas-liquid chromatography. Retention times are quoted relative to cholesterol. Effect of lucerne saponins on fungal growth in the presence of sterok Bioassays with Sclerotium rolfsii Sacc. were carried out according to Gestetner et al.s. The culture medium contained 0.005% lucerne saponins, with or without various amounts of the sterols.
Biochim. Biophys. Acta, 270 (1972)
181-187
INTERACTION OF LUCERNE SAPONINS WITH STEROLS
183
RESULTS
The structural requirements of sterols to form an addition product with lucerne saponins were investigated with a variety of sterols, which differ from cholesterol, in Steroln
albe
to
precipitate
Sterols
HO5-Stigmastene-
unalble
H
to
precipitate
5&Cholestane-
38-01
‘3
-+‘I
&J
I3
“<2 C"3
HO 543-
Cholestaneone
-Hydroxy-B-cholen-24-oic Ergosterol
acid =",
CHS
HO
Ergocalciferol
HO
Cholecalciferol
HO 24-
Methyl-
5 -
="3
- cholestene-
-38
-01
CH’ “3
5,22- Stigmastadiene -3 /I -01
Fig. I. Structural formulas of sterols, which do or do not interact with lucerne saponins. Biochim.
Bio$hys.
Ada,
270 (1972) 181-187
184
B. GESTETNER
etd.
one or more features of their structure (Fig. I). The results of these experiments indicate that the ability of the various sterols to interact with lucerne saponins is not affected by the following structural changes in the molecule: (I) presence of additional double bonds in the ~y~lo~e~tanoper~y~ophenanthrene ring or in the isoctyl side chain; (2) presence of additional methyl or ethyl groups in the side chain; (3) saturation of the double bond in ring B; and (4) oxidation of blocking the 3B-hydroxyl group. On the other hand, compounds which differ from cholesterol in the following structural features, are unable to combine with lucerne saponins: (I) the spatial relationships of rings A and B is cis to each other; (2) opening of B ring, and subsequent stereochemical change (i.e. calciferols) ; (3) elimination of the side chain; and (4) shortening or lengthening the side chain and introducing various functional groups into it. Thus it seems that the presence of an intact steroid ring structure, having the conformation of cholestanol, to which a side chain characteristic to cholesterol or phytosterols is attached, are essential for interaction of sterols with lucerne saponins. Although lucerne saponins could be precipitated by interaction with different sterols, the attempts to isolate a sterol-saponin addition product were not successful, since it dissociated under the con~t~ons employed. Examination of the saponins, which were isolated by interaction with the different sterols showed that they were ~hromato~aphi~~ly indistin~ishable from each other as well as from saponins precipitated by cholesterol and j+sitosteroP. The chemical composition of the reacting saponins was also identical, i.e. medicagenic acid being the sole aglycone attached to a variety of sugars@. The various sterols can be divided into two distinct groups, one consisting of cholesterol and 7-dehydrocholesterol which form addition products at an approx. 1:5 molecular ratio, unlike the other sterols, in the addition products of which an approx. x :8 molecular ratio exists (Table I). These molecular ratios are based on an average molecular weight of lucerne saponins, of 1000 to 1200~7~.The homogeneous lucerne saponin gave with cholesterol I : 4.82 and with 5-stigmastene-3#I-ol I : 8,zo molecular ratios. These ratios are in good agreement with those obtained with the mixture of saponins present in the whole extract. The results given in Table I can also be interpreted in terms of affinity for TABLE1 FORMATION
~
OF STEROL-LUCERNESAPONIK
ADDITION
PRODUCTS
_~~
I.91 16.5 Cholesterol 7-De~ydro~oiesterol 16.0 2.04 5-Stigmastene-3j!-ol** 20.0 3.26 Ergosterol 17.5 3.52 5x-Cholestan-3-one 15.0 3.42 Pm * Based on an average molecular weight of lucerne saponins of xooo ** This commercial sample contains approx, 60% 5-stigmastene-3/?-01, 5-cholestene-3/?-ol and approx. 20% 5,zz-stigmastadiene-3B-01. B&whim. Biophys.
Acta, 270 (1972) 181-187
I/4.9
115.3 X17.9 I/Q
1/8.8 to
1200.
approx. 20%
q-methyl-
INTERACTION OF LUCERNE SAPONINS WITH STEROLS
I&
saponins, which is much more pronounced by cholesterol and 7-dehydrocholesterol, than by the other sterols. This statement was further substantiated by forming addition products with saponins in the presence of more than one sterol in the reaction mixture. By using cholesterol as reference compound, it was found (Fig. 2) that when sterol-saponin addition product is formed in the presence of cholesterol and one of the other sterols, the bulk of the addition product was formed with cholesterol, whereas the other sterols, except for 7-dehydrocholesterol, interacted with lucerne saponins only to a negligible extent. The preferential affinity of cholesterol and 7-dehydrocholesterol for lucerne saponins could also be demonstrated by bioassays with the fungus S. rolfsii. This fungus contains cholesterol and its growth is strongly inhibited by lucerne saponins. This growth inhibition can, however, be prevented by the additon of cholesterol, but these bioassays, it could be not /I-sitosterol to the culture medium s. By extending shown (Fig. 3) that similarly to cholesterol, 7-dehydrocholesterol can also prevent the growth inhibition caused by lucerne saponins, whereas the other sterols are ineffective in this respect even at much higher concentrations.
7
I Time
3
m&n
2. Gas chromatographic identification of sterols which form addition products with lucerne saponins. (a) Control, consisting of a mixture of sterols. I, cholesterol; z, 7-dehydrocholesterol; 3, gee-cholestan-s-one; 4, ergosterol; 5, 24-methyl-S-cholestene-3/?-01; 6, 5,22-stigmastadiene-@ol; 7. 5-stigmastene-3/l-01. (b-g) Sterols from the saponin addition product formed with: (b) cholesterol and 7-dehydrocholesterol, (c) cholesterol and ga-cholestane-s-one, (d) cholesterol and ergosterol, (e) cholesterol and nq-methyl-g-cholestene-$01, (f) cholesterol and 5,22-stigmastadiene-$?-ol, (g) cholesterol and 5-stigmastene-$ol.
Biochim. Bio$hys. Ada, 270 (1972) 181-187
B. GESTETNER
-I
@==%-
I
~.005
-f
AAc!-------
0
I
0025
Concentration
I
0.050 of sterols
et cd.
I
0075 (%)
1
Fig. 3. Effect of different concentrations of various sterols on the growth impairment of S. rolfsii Sacc. caused by 0.005% sterol precipitable lucerne saponins. 0, cholesterol; A, g-stigmastene38-01; 0, 7-dehydrocholesterol; A, ergosterol; 0, ga-cholestan-3-one. DISCUSSION
The structural features of sterols, which enable them to interact with lucerne saponins were studied. Similarly to the complexes formed between digitonin and various sterols, only sterols belonging to the cholestanol series are able to interact with lucerne saponins But unlike to complexes formed with digitonin, the sterol-lucerne saponin addition products are not dependent on the presence of an equatorial 3-phydroxyl group, in the absence of which digitonin does not form complexes with sterolsl. In the case of lucerne saponins the nature of the steroid side chain seems to be more important for the formation of addition products, than the modifications introduced into the steroid ring structure. This becomes evident, when one considers the preferential affinities displayed by cholesterol and 7-dehydrocholesterol. The ability of cholesterol and 7-dehydrocholesterol to prevent fungal growthinhibition, caused by lucerne saponins, can also be attributed to the preferential affinities of these sterols for lucerne saponins. Other sterols, when added to the culture medium, form also addition products with the saponins. However, when these unstable products undergo dissociation, the liberated saponins are able to interact with the membraneal cholesterol and consequently fungal growth is arrested. If, on the other hand, cholesterol or 7-dehydrocholesterol are added to the culture medium, they are able to compete with the native fungal sterols, and thus prevent growth inhibition. The extreme instability of the sterol-lucerne saponin addition product prevented its isolation. It should be, however, pointed out that also complexes of digitonin formed with sterols other than cholesterol, display similar instability’. The finding that the 3b-OH group is not involved in the interaction of sterols with lucerne saponins, points towards the improbability of covalent binding between them. On the other hand, one of the factors which is undoubtedly essential for the Biochim. Biophys.
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formation of the addition product, is the presence of unsubstituted carboxyl groups, at C, and C,, in medicagenic acid. By blocking these groups the interacting ability of lucerne saponins with sterols was abolished (Y. Assa, unpublished). It was also found that soybean saponins, which contain sapogenins devoid of carboxyl groups, are not able to form such addition product9. Some of the biological activities exhibited by saponins, such as hemolysis,6~6~15 are generally attributed to the interacor inhibition of growth of microorganisms 1ev17 tion of saponins with membraneal cholesterol. Since the structural requirements of cholesterol to interact with lucerne saponins differ from those existing between cholesterol and digitonin and since soybean saponins, which do not form addition products with sterols are also strongly hemolytic18, neither the extent, nor the mode of action of various saponins on biomembranes should be expected to be similar. Therefore, the examination of the action of different saponins may prove to be a useful tool in the elucidation of the function and significance of cholesterol in cell membranes, in particular in those of microorganisms. ACKNOWLEDGEMENT
This work was supported by the U.S. Department of Agriculture, Grant No. FG-1~~46. REFERENCES I P. Bladon, in R. P. Cock, Cholesterol, Academic Press, New York, 1958, p. 84. 2 E. D. Walter, G. R. van Atta, C. R. Thompson and W. D. Maclay, J. Am. Chem. Sot., 76 (1954) 2271. 3 C. R. Thompson, G. R. van Atta, E. M. Bickoff, E. D. Walter, A. L. Livingston and J. Guggolz, Tech. Bull. U.S. Agric., 1161 (1957) 53. 4 C. B. Coulson, J. Sci. Food Agric., g (1958) 281. 5 R. R. Dourmashkin, R. M. Dougherty and R. J. C. Harris, Nature, 194 (1962) 1116. 6 A. M. Glauert, J. T. Dingle and J. A. Lucy, Nature, rg6 (1962) 953. 7 S. Shany, Alfalfa Saponins-Isolation, Characterization and Biological Significance, Ph.D. Thesis, The Hebrew University of Jerusalem, 1971, p. 42. 8 S. Shany, B. Gestetner, Y. Birk and A. Bondi, J. Sci. Food Agric., 21 (1970) 508. g B. Gestetner, Y. Assa, Y. Henis, Y. Birk and A. Bondi, J. Sci. Food Agric., 22 (1971) 168. ro S. Shany, Y. Birk, B. Gestetner and A. Bondi, J. Sci. Food Agric., 21 (1970) 131. II B. Gestetner, Phytochemistvy, IO (1971) 2221. 12 C. Grunwald. Anal. Biochem.. 14 (1970) 16. 13 L. L. Abell, B. B. Levy, B. B. B&die and I?. E. Kendall, J. Biol. Chem., 195 (1952) 357. 14 G. Noelting and P. Bernfeld, Helv. Chim. Acta, 31 (1948) 286. 15 E. Ponder, Haemolysis and Related Phenomena, Grnne and Stratton, New York, 1948. 16 M. M. Weber and S. C. Kinsky, J. Bacterial., 89 (1965) 350. 17 B. Wolters, PZanta Med., 16 (1966) 77. 18 Y. Birk, A. Bondi, B. Gestetner and I. Ishaaya, Nature, rg7 (1963) 1089. Biochim.
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