United States Patent (19) 11) Patent Number: 4,602,003 Malinow (45) Date of Patent: Jul

United States Patent (19) 11) Patent Number: 4,602,003 Malinow (45) Date of Patent: Jul. 22, 1986

54 SYNTHETIC COMPOUNDS TO INHIBIT Effect on Birds, Mammals and Cold-Blooded Organ INTESTNAL ABSORPTION OF isms', J. Sci Fal. Agric. 20:433-436 (1969). CHOLESTEROL IN THE TREATMENT OF Malinow et al., "Effect of Alfalfa Saponins on Intestinal HYPERCHOLESTEROLEMIA Cholesterol Absorption in Rats', Am. J. Clin. Nutr. (75) Inventor: M. René Malinow, Portland, Oreg. 30:2061-2067 (1977). Malinow et al., "Cholesterol and Bile Acid Balance in 73) Assignee: Medical Research Foundation of Macaca fascicularis', J. Clin. Invest, 67:156-162 (1981). Oregon, Portland, Oreg. Morgan et al., "The Interactions Between Dietary Sap (21) Appl. No.: 379,098 onin, Cholesterol and Related Sterols in the Chick', (22) Filed: May 17, 1982 Poultry Sci. 51:677-682 (1972). Newman et al., “Dietary Saponin, A Factor Which 51 Int. Cl...... A01N 31/00; A61K 31/705; May Reduce Liver and Serum Cholesterol Levels', A61K 31/58; A61K 31/56 Poultry Sci. 37:42-46 (1958). 52 U.S. Cl...... 514/26; 536/5; Oakenfull, "Effects of Saponins on Bile Acids and 260/239.55A; 260/397.2; 514/172; 514/169 Plasma Lipids in the Rat', Br. J. Nutr, 42:209-216 58) Field of Search ...... 260/239.55 A, 397.2; (1979). 563/6, 6.1; 424/238, 180 Reshefet al., "Effect of Alfalfa Saponins on the Growth (56) References Cited and Some Aspects of Lipid Metabolism of Mice and Quails', J. Sci Fal. Agric. 27:63-72 (1976). U.S. PATENT DOCUMENTS Segal et al., "Hemolytic Properties of Synthetic Glyco 3,160,626 12/1964 Oxley ...... 260/239.55 A 3,303,187 2/1967 Rubin ...... 260/239.55 A sides”, Biol. Abs, 67:57966 (1979). 3,992,315 11/1976. Dibb et al...... 536/5 Sofowora et al., "Synthesis of 3-6-Glycosides of Di 4,188,379 2/1980 Pegel ...... 424/182 osgenin, Yanogenin and Gitogenin', Biol. Abs. 63:4467 4,198,401 4/1980 Pegel ...... 424/195 (1977). 4,242,502 12/1980 Malinow et al...... 424/182 Topping et al., "Effects of Dietary Saponins on Fecal 4,254,111 3/1981 Pegel et al...... 424/182

Bile Acids and Neutral Sterols, Plasma Lipids, and 4,260,603 4/1981 Pegel et al. . ... 424/182 Lipoprotein Turnover in the Pig', Am. J. Clin. Nutr. 4,265,886 5/1981 Pegel ...... 424/182 33:783-786 (1980). FOREIGN PATENT DOCUMENTS Topping et al., "Prevention of Dietary Hypercholester 2425859 12/1977 France . olemia in the Rat by Soy Flour High and Low in Sapo nins', Nutr. R. Intl. 22:513-519 (1980). OTHER PUBLICATIONS Wilcox et al., "Serum and Liver Cholesterol, Total Kintya et al., "Search for Hypochotesferemic Agents Lipids and Lipid Phosphorus Levels of Rats Under Among a Group of Steroid Glycosides', Kim. Farm. Various Dietary Regimens', Am. J. Clin. Nutr. Zh (1981) 15:9, pp. 56–60. 9:236-243 (1961). M. R. Malinow, et al., “Prevention of Hypercholester Merck Index (1976), pp. 439 and 1217. olemia in Monkeys (Macaca fascicularis) by Digitonin', “Steroids' by Fieser et al (1959), p. 28. Am. J. Clin. Nut. 31:814-818 (1978). R. J. Morris, et al., "Isolation, Purification, and Struc Primary Examiner-Elbert L. Roberts tural Identity of an Alfalfa Root Saponin'", J. Org. Attorney, Agent, or Firm-Klarquist, Sparkman, Chem, 26:1241-1243 (1961). Campbell, Leigh & Whinston Anderson, "Effect of Alfalfa Saponin on the Perfor 57 ABSTRACT mance of Chicks and Laying Hens', Chem. Abs. 52:2194f (1958). Synthetic sapogenin and sterol compounds, adminis Bite et al., “Synthesis of Steroid Glycosides', Chen. tered orally to warm-blooded animals, inhibit the ab Abs. 66:38204v. (1967). sorption of cholesterol and are useful in the treatment of Cayen et al., “Effect of Diosgenin on Lipid Metabolism hypercholesterolemia. Particular compounds suitable in Rats', J. Lipid Research 20:162-173 (1979). for such purposes include glycosides with spirostane, Griminger et al., "Dietary Saponin and Plasma Choles spirostene, or cholesterol aglycones, and esters of terol in the Chicken', Proc. Soc. Exp. Biol. Med., spirostanes, spirostenes and cholesterol. 99:424-426 (1958). Ishaaya et al., "Soybean Saponins, IX., Studies of Their 15 Claims, No Drawings 4,602,003 1. 2 It was previously reported that toxicity of plant sapo SYNTHETIC COMPOUNDS TO INHIBIT nins is decreased in rats, mice, and birds by cholesterol INTESTINAL ABSORPTION OF CHOLESTEROL in the diet (J. O. Anderson, “Effect of Alfalfa Saponin IN THE TREATMENT OF on the Performance of Chicks and Laying Hens." Poult. HYPERCHOLESTEROLEMIA Sci. 36:873-876, 1957; I. Ishaaya, et al., "Soyabean Sap onins. IX. Studies of Their Effects on Birds, Mammals This invention was made with government support and Cold-blooded Organisms." J. Sci Food Agric, under Grant No. 5 P51 RR00163 "Support for Regional 20:433-436, 1969; G. Reshef, et al., “Effect of Alfalfa Primate Research Center” awarded by the Department Saponins on the Growth and Some Aspects of Lipid of Health and Human Services, Division of Research O Metabolism of Mice and Quails.” J. Sci Food Agric. Resources. The government has certain rights in this 27:63-72, 1976; E. B. Wilcox, et al., "Serum and Liver invention. Cholesterol, Total Lipids and Lipid Phosphorus Levels BRIEF SUMMARY OF THE INVENTION of Rats Under Various Dietary Regimes.” Am. J. Clin. Nutr., 9:236-243, 1961). The invention relates to synthetic compounds which, 15 when administered orally to warm-blooded animals, Despite these encouraging results, it has remained a inhibit the absorption of cholesterol. problem that plant extracts, which are of variable com Certain water/alcohol soluble extracts from plant position, contain a volume of nonuseful chemical sub sources have been found to reduce cholesterolemia in stances. It is difficult, due to the variations in composi chicks, pigs and rats (P. Griminger, et al., "Dietary 20 tion, to set a standard dosage or predict the impurities Saponin and Plasma Cholesterol in the Chick.” Proc. present. Thus, such extracts are not well suited for use Soc. Exp. Biol. Med. 99:424-426, 1958; H. A. I. New by humans. Furthermore, purification of plant extract man, et al. "Dietary Saponins, a Factor Which May substances and synthesis of saponins suspected to exist Reduce Liver and Serum Cholesterol Levels.' Poultry in plants are likely to be very costly due to the antici Sci. 37:42-46, 1958; B. Morgan, et al., "The Interactions 25 pated complexity of the required procedures. Between Dietary Saponin, Cholesterol and Related It has now been discovered that certain synthetically Sterols in the Chick." Poultry Sci. 51:677-682, 1972; D. produced, pure "sapogenin-derived' compounds, e.g., L. Topping, et al, “Effects of Dietary Saponins in Fecal substances compounded from spirostane, spirostene, or Bile Acids and Neutral Sterols, Plasma Lipids, and sterol-derived' compounds are nontoxic. Such com Lipoprotein Turnover in the Pig.' Am. J. Clin. Nutr. 30 pounds depress cholesterol absorption more effectively 33:783-786, 1980; D. G. Oakenfull, et al., “Effects of than alfalfa extracts on a weight basis and thus can be Saponins on Bile-acids and Plasma Lipids in the Rat.' administered in reasonably sized doses. Because the Br. J. Nutr 42:209-216, 1979; and D. L. Topping, et al., chemical compositions of these substances are known "Prevention of Dietary Hypercholesterolemia in the and because they can be synthesized at a high degree of Rat by Soy Flour High and Low in Saponins.' Nutr. 35 purity, they are suitable for use by any warm-blooded Rep. Int, 22:513-519, 1980.) animal, including humans. Precursor substances for use More specifically, extracts from alfalfa hay are in the synthesis are available as by-products of present known to be active in reducing the absorption of dietary industrial processes. For example, one spirostane com cholesterol. Although such alfalfa extracts are of un pound (tigogenin) is currently a wasted by-product of known composition, they are found to contain saponins 40 digitalis manufacture. identifiable by thin-layer chromatography. The alfalfa Unless administered in massive amounts, pure sapo extracts contain, in addition to saponins, unspecified genins do not significantly bind cholesterol or inhibit its amounts of carbohydrates, amino acids, peptides, pig absorption. It is only when compounded with another ments, and free aglycones removed from alfalfa hay by moiety that sapogenins have the desired effect. Exam the water:alcohol solvent used during their preparation. 45 ples of such sapogenin compounds are compounds of Such crude extracts are sometimes referred to herein as tigogenin and diosgenin, particularly glycosides having "alfalfa saponins' as an operational definition. These tigogenin or diosgenin as an aglycone. Although it is alfalfa extracts reduce the intestinal absorption of cho not established that the size of the nonsapogenin moiety lesterol in rats and monkeys (M. R. Malinow, et al., has any effect on a compound's ability to inhibit choles "Cholesterol and Bile Acid Balance in Macaca fascicula 50 terol absorption, at least one glycoside with a relatively ris. Effects of Alfalfa Saponins." J. Clin. Invest. longer sugar moiety has an increased biological activity 67:156-162, 1981). The capacity of such alfalfa extracts in regard to cholesterol absorption. Specifically, cel to interfere with cholesterol absorption is enhanced by lobiose-sapogenins are more active than glucose partial acid hydrolysis as reported in M. R. Malinow, et sapogenins having the same aglycone. Of such sub al., "Effect of Alfalfa Saponins on Intestinal Cholesterol 55 stances, cellobiose-tigogenin is particularly active to Absorption in Rats.” Am. J. Clin. Nutr 30:2061-2067, inhibit cholesterol absorption. Somewhat less active is 1977; and U.S. Pat. No. 4,242,502 (Malinow, et al.)). cellobiose-diosgenin, perhaps due to the presence of a Digitonin binds cholesterol in vitro, inhibits the intes double bond in Cs of the spirostene aglycone of diosge tinal absorption of cholesterol, and prevents the hyper 1. cholesterolemia expected in monkeys ingesting high-fat, 60 highcholesterol foods (M. R. Malinow, et al, "Preven DETAILED DESCRIPTION tion of Hypercholesterolemia in Monkeys (Macaca fas Certain synthetic compounds, when administered cicularis) by Digitonin, Am. J. Clin. Nutri, 31:814-818, orally, inhibit the absorption of cholesterol by warm 1978). But, digitonin is costly. Diosgenin has also been blooded animals, and consequently may lower plasma reported to inhibit the absorption of cholesterol in rats 65 cholesterol levels and induce regression of atheroscler when given in a massive dose (1000 mg/kg) (M. N. osis. These include compounds of sterols and of "spiros Cayen, et al., "Effect of Diosgenin on Lipid Metabolism tane substances' such as diosgenin, tigogenin, simila in Rats.”, J. Lipid Res. 20: 162-174, 1979). genin and the like. One group of such compounds in 4,602,003 3 4 cludes compounds of diosgenin (5,20a,22d,25d-spiros tan-3,6-ol) as represented by the following formula: O O HO-C-C-O-R wherein R is a sapogenin such as diosgenin or tigogenin or a sterol such as cholesterol. Tests were performed on laboratory animals accord ing to the procedure outlined in M. R. Malinow, et al., O "Effect of Alfalfa Saponins on Intestinal Cholesterol Absorption in Rats.” Am. J. Clin. Nutr., 30, December 1977, pps. 2061-2067. The tests were performed in groups of six animals each (mean-ESE). The result of the tests are summarized in Table I: HO 15 TABLE I Another group according to the invention include Effect of Sapogenin Esters on intestinal Absorption of compounds of tigogenin (5a,20o,22d,25D-spirostan-3,6- Cholesterol in Rats. Tests performed in groups of ol) represented by formula: six animals each (mean E SE) 2O Intestinal absorption of cholesterol (% of I.D.) Substance mg/rat Controls Experimental P tigogenin maleate 15 80.5 - 0.6 62.9 - 10 000 diosgenin maleate 15 79.6 t 1.4 56.0 - 2.2 0.00 tigogenin 15 79.1 - 5 81.2 - 11 N.S. diosgenin 15 73.5 2.0 76.4 2.6 N.S. maleic acid 5 79.1 - 1.5 78.1 0.8 N.S. maleic acid/tigogenin 5/10 77.0 2. 78.1 - 0.8 N.S. Maleic acid/diosgenin 5/10 77.0 2.1 71.2 - 2.8 N.S.

In vitro tests for micellar cholesterol binding were performed according to the method of M. R. Malinow, et al., "Prevention of Hypercholesterolemia in Monkeys (Macaca fascicularis) by Digitonin.” Am. J. Clin. Nutr. A. Esters 35 31:814-818, 1978. These tests confirmed that choles Esters of the above formulas can be prepared by terol was bound by the sapogenin maleates. The results reacting an anhydride with the "spirostane substance'. of these in vitro tests appear in Table II: Any of numerous organic anhydride substances could TABLE II be used in such molecules, although not all such mole 40 In Vitro Micellar Cholesterol Binding cules would be effective or be sufficiently nontoxic for Bound cholestero general use. Substance (mg/mg substance) Esters have been formed from the following anhy Tigogenin maleate 0.20 drides which react with the hydroxyl group of the sapo diosgenin maleate 0.24 genin or sterol: 45 digitonin 0.33 alfalfa saponins 0.24 tigogenin O Anhydrides Used in the Synthesis of Sapogenin and Sterol Esters diosgenin O phtalyl-DL-glutamic cis-1,2-cyclobutane dicarboxylic 50 1-octenyl-succinic citraronic When tested in primates, however, the sapongenin glutaric 3-nitrophtalic maleates induced vomiting when administered orally. nonenylsuccinic methylsuccinic trans-1,2-cyclohexane 3-methylglutaric Thus, the maleates may be unacceptable for administra dicarboxylic tion to humans, or even be toxic. cix-1,2-cyclohexanedicarboxylic 2,3-dimethyl maleic 3,3-dimethyl glutaric 1,2,3,4-cyclobutane 55 B. Glycosides tetracarboxylic trans-1,2-cyclohexanedi- diphenyl Sapogenin and sterol compounds according to the carboxylic invention, specifically glycosides of tigogenin, diosge 2-dodecen-1-ylsuccinic maleic nin and cholesterol successfully bind cholesterol and dichloromaleic 60 inhibit its absorption by the digestive system of warm blooded animals. Thus far, no toxic effect has been Of the esters produced, only maleic esters proved to associated with such substances. And, due to their be effective in the inhibition of intestinal absorption of structure, it is likely that they are substantially nontoxic. cholesterol in laboratory animals. The glucosidic compounds were formed by reacting Such maleic esters were synthesized by combining 65 a carbohydrate-containing molecule with the hydroxyl maleic anydride and the desired sapogenin or sterol group of the aglycone substance. The carbohydrate substance in chloroform at 50 for a period of four days. containing molecule can, for example, be a-D-(-)- The result was a maleic ester of the formula: glucose of the formula: 4,602,003 5 6 The glycosidic bonds between the carbohydrate-con taining molecule and the aglycone could be either an a glycosidic bond:

5 -O H O 4. H OH O or 6-D-(+)-glucose of the formula: 9 or a f3 glycosidic bond:

6CH3OHCH2 H O

15 4.

H OH For compounds which are to bind cholesterol, it is anticipated that a g glycosidic bond will be preferred in or longer carbohydrate molecules such as (+)-cellobi- 20 most instances since compounds having a g glycosidic ose(3-anomer) otherwise known as 4-O-(S-D- bond are less likely to be hydrolyzed in the intestine of glucopyranosyl-D-glucopyranose): the subject animal. Thus, although an a-cellobiose-tigogenin of the fol lowing formula might be suitable,

H OH g-glucose a-glucose tigogenin cellobiose (a-anomer) a-cellobiose-tigogenin

it is anticipated that a 6-cellobiose-tigogenin, such as one of the following formula, would be more effective:

7 4,602,003 -continued f3-glucose {3-glucose tigogenin cellobiose (g-anomer) Nmm1f3-cellobiose-tigogenin Glucosides having tigogenin and diosgenin aglycones were synthesized and tested according to the following fractions with the above solvent and after 150 ml, use procedures. 10 methanol to elute the saponins. EXAMPLE 1 k. Perform thin-layer chromatography (TLC) on a small amount (around 10 g) with chloroform:me Synthesis thanol:water (65:38: 10) as the solvent. Use a Cu ace A series of saponins were synthesized by glycosyla tate solution for charring. Saponins typically have tion of the hydroxyl group of the two sapogenins, tigog 15 retardation factor (Rf) values around 0.6 to 0.8 and enin (5a,20a,22d,25D-spirostan-3p3-ol) and diosgenin give a bluish or brownish color. (5a,20a,22a,25D-spirosten-3,6-ol). Synthesis of the sap This procedure probably synthesized saponins with onins was accomplished with a modified version of the fg-glycosidic bonds, or, in the case of cellobiose, a mix method described by Rosevear at al., "Alkyl Glycoside ture of 6- and a-glycosides. It was possible to synthe Detergents' A Simpler Synthesis and Their Effects on 20 size the a-isomer, by using glucose pentaacetate (a- Kinetic and Physical Properties of Cytochrome COxi anomer) and ZnCl2 instead of AgCO3 as catalyst. dase.' Biochemistry, 19:4108-4115, 1980. Glucosides having tigogenin and diosgenin aglycones a. Weigh 1 mmol (678 mg) of cellobiose octoacetate were tested for in vitro binding of cholesterol according (3-anomer) or 1 mmol (390 mg) of glucose pentaace to the following procedure: tate (g-anomer) into a foil-covered reaction vessel. 25 While stirring magnetically, add 5 ml of glacial acetic EXAMPLE 2 acid and stir for a few minutes. Quickly add 5 ml of In Vitro Binding of Cholesterol 31% HBr in glacial acetic acid. Immediately shut in a. Prepare a micellar suspension of cholesterol by plac ... the fumes with a stopper and stir vigorously for 45 to ing in a flask caprylic acid (1.2x 10-3M), glyceryl 55 min (cellobiose) or 30 to 40 min (glucose). 30 monoleate (0.6X 10-3M), and 4-14C) cholesterol b. Add 10 ml of choloform for cellobiose or dichloro (0.3X 103M; specific activity ~25,000 dpm/mg), methane for glucose. Pour into a separating funnel and agitating these for 1 h at 38 C. with sodium containing 30 ml of ice and water. Shake for 2 min taurocholate (10x10-3M) in 0.1M phosphate buffer, and extract the bottom nonaqueous layer into 30 ml pH 6.2 (20). The suspension contains approximately of a cold saturated sodium bicarbonate solution previ 35 58 ug of cholesterol/ml. Centrifuge the suspension at ously saturated with chloroform or dichlormethane. 10,000 rpm for 30 min. before use. Shake for 2 min. Repeat NaHCO3 extraction twice or b. Place 100 ug of the synthetic saponin dissolved in until the pH of the aqueous layer is >7.0. Wash the methanol in a 50-ml screw-cap tube and evaporate the nonaqueous phase three times with cold solvent solvent under N2 in a warm-water bath. saturated distilled water. 40 c. Dry the extracted solvent layer over 500 mg. of mag c. Add 4 ml of the micellar suspension and shake it for nesium sulfate while stirring for 30 min. Centrifuge 2 hat room temperature. and wash the precipitate with dry solvent. - d. Transfer the medium to centrifuge tubes. Centrifuge d. Pour the dried filtrate into a foil-covered, 50-ml at 3,000 rpm for 20 min. screw-cap tube and evaporate to about 10 ml with N2 45 e. Determine the radioactivity in 0.5 ml of the upper at low heat. solvent phase with 10 ml of toluene-based scintilla e. Keeping the reaction vessel protected from light and tion fluid. air moisture as much as possible, add: 1 mmol of f. Treat blanks as above without adding the synthetic tigogenin ordiosgenin; 200 mg of dry silver carbon saponin. ate; one small iodine crystal; and 1 g of 4. A molecular 50 Results of these tests showed that binding of choles sieves. Stir in the dark for 12 to 24 h. terol occurs with the synthetic saponins. Representative f. Centrifuge at a low speed for 10 min. and transfer the data from the test appears in Table III: supernatant to a foil-covered, 50-ml screw-cap tube. TABLE III Wash the precipitate twice with 5 ml of solvent and In Vitro Binding of Cholesterol by Synthetic Saponins" combine the solvents. 55 Mass/flask Cholesterol bound g. Evaporate the combined solvent with N2 to a cloudy Saponin (ug) (pig) syrup of about 1 to 2 ml. Add a stir bar and 10 ml of None O O Cellobiose-tigogenin 250 21 a solution of triethylamine:methanol:water (1:2:1). Cellobiose-tigogenin 500 37 Stir for 30 min. and let stand overnight. Cellobiose-tigogenin 750 46 h. Transfer the solution quantitatively into dialyzing 60 Cellobiose-tigogenin 000 60 tubing with 40 ml of water. Dialyze it against tap Cellobiose-diosgenin 1000 26 water for 48 h. Glucose-diosgenin 1000 49 i. Transfer the solution to a container for freeze-drying. Glucose-tigogenin 1000 40 Results are average of three determinations. Evaporate the water overnight. Mixture of a- and B-glycosides. j. Dissolve the resulting white powder in dichlorome- 65 Mainly 8-glycoside. thane-methanol (10:1, vol/vol) and transfer to a chro matography column filled with silica gel type 60 Further experimentation was conducted to determine (230-400 mesh, E. Merck Reagents). Separate 7 ml the effect of the synthesized saponins on living animals. 4,602,003 9 10 effects. It is an advantage that such synthetic com EXAMPLE 3. pounds are pure substances with known or determin Effects of Synthetic Glycosides in Vivo able chemical structures and may be synthesized in Effects of the synthetic glycosides on the intestinal sufficient purity so as to be used to treat human beings. absorption of cholesterol in rats were tested according As indicated by their ability to prevent the absorption of to the procedure of M. R. Malinow, et al., "Effect of cholesterol, synthetic sapogenin and sterol compounds Alfalfa Saponins on Intestinal Cholesterol Absorption may reduce deaths due to atherosclerotic disease, a in Rats.' Am. J. Clin. Nutr. 30:2061-2067, 1977. The most significant cause of death in the adult population animals were fed semipurified cholesterol-free food of the Western world. from 8 a.m. to 10 a.m. for ten days. On the day of the 10 Having given examples of preferred embodiments of experiment, the rats were anesthetized at about 10:30 my invention, it will be apparent to those skilled in the a.m. and the test substance, as well as a pulse dose of art that changes and modifications may be made with radioactive cholesterol, were given per gastric tube. out departing from my invention in its broader aspects. The excretion of 14C-neutral steroids was determined in I therefore intend the appended claims to cover all such feces collected for 72 h after intragastric administration 1. 5 changes and modifications as fall within the true spirit of the test substances and 2 mg of 4-1C) cholesterol and scope of my invention. (specific activity ~0.25 uCi/mg). I claim: As shown in Table IV, these rate experiments demon 1. A method of treating a human or other warm strated that the synthetic glycosides inhibit the intestinal blooded animal subject to reduce digestive absorption absorption of cholesterol. No significant inhibition was 0 of cholesterol, the method comprising administering to observed with sapogenins. Better results were obtained the subject an effective amount of a nontoxic, substan with a longer sugar moiety (cellobiose) than with a tially pure, synthetic, glycosidic compound selected shorter moiety (glucose). from the group consisting of glycosides having tigoge TABLE IV Effects of Sapogenins and Synthetic Glycosides on Cholesterol Absorption in Rats' Intestinal Student's Number Absorption of t test, Substance of Weight Dose cholesterol P versus Relative Series administered rats (g) (mg/rat) (I.D.) controls Absorption I lone 6 242 - 3 0 74.6 2.3 100 glucose- 6 246 - 7 i4 46.2 1.8 <0.001 62 tigogenin glucose- 6 245 5 14 526 - 3.7 <0.001 71 diosgenin alfalfa 6 2499 14 60.2 - 3.7 <0.01 81 extract I One 6 290 - 7 0 74.8 - 1.6 100 cellobiose- 6 291 - 2 14 39.6 t 1.8 <0.001 53 tigogenin cellobiose- 6 287 - 4 14 53.7 - 13 <0.001 72 diosgenin alfalfa 6 275 it 8 14 56.3 1.1 <0.01 75 eXtract III none 6 268 - 5 O 78.0 2.1 100. tigogenin 6 259 - 5 15 80.4 - 1.4 N.S. 103 IV none 6 328 - 4 O 73.5 - 2.0 100 diosgenin 6 330 - 4 15 76.4 2.6 N.S. 04 V none 6 276 - 7 O 77.7 2.2 100 cellobiose- 6 282 - 3 15 69.5 - 1.3 0.01 89 cholesterol glucose- 6 273 - 8 15 72.5 + 1.0 N.S. 93 cholesterol alfalfa 6 277 5 15 68.4 - 2.3 0.02 88 extract Values are mean SE. Abbreviations: I.D., injected dose; N.S., not significant. The excretion of 'C-neutral steroids was determined in feces collected for 72 h after intragastric administration of glycosides or sapogenins and 2 mg of E4'C-cholesterol. The mechanism whereby cholesterol absorption is inhibited by sapogenin and sterol compounds is un- 55 nin aglycones. known. However, it is possible that the compounds 2. The method of claim 1 wherein the glycoside is a form an insoluble complex with cholesterol in the intes glucoside. tinal lumen and thereby prevent the absorption of di 3. The method of claim 2 wherein the glycoside is a etary cholesterol. Additionally, the compounds may glucose-tigogenin. bind biliary cholesterol and cholesterol from desqua 4. The method of claim 2 wherein the glycoside is a mated intestinal cells and, thus, may induce negative cellobiose-tigogenin. cholesterol balance through the increased excretion of 5. The method of claim 4 wherein the glycoside is a endogenous cholesterol. As-cellobiose-tigogenin. In the above described experiments, synthetic sapo 6. The method of claim 1 wherein the glycoside is genin and sterol compounds inhibited the absorption of 65 administered orally. exogenous cholesterol in laboratory animals. It is thus 7. A method of treating a human or other warm anticipated that they will also be effective in preventing blooded animal subject to reduce digestive absorption atherosclerosis or inducing its regression without toxic of cholesterol, the method comprising administering an 4,602,003 11 12 12. The method of claim 1 wherein the glycoside effective amount of a nontoxic cholesterol glycoside to consists of a carbohydrate bonded to the aglycone by a the subject. fe-oriented glycosidic bond. 8. A method of treating a human or other warm 13. A method of treating a human or other warm blooded animal subject to reduce digestive absorption blooded animal subject to reduce digestive absorption of cholesterol, the method comprising administering to of cholesterol, the method comprising administering to the subject an effective amount of an ester selected from the subject an effective amount of a glucose-cholesterol. the group consisting of diosgenin maleate, tigogenin 9. The method of claim 8 wherein the glucose-choles maleate, and mixtures thereof. O 14. The method of claim 8 wherein the glucose terol is a cellobiose-cholesterol. cholesterol is administered orally. 10. The method of claim 9 wherein the glucose 15. A method of treating a human or other warm cholesterol is a g-cellobiose-cholesterol. blooded animal subject to reduce digestive absorption of cholesterol, the method comprising administering to 11. The method of claim 7 wherein the cholesterol 15 the subject an effective amount of cholesterol maleate. glycoside is administered orally. k s k ck k