Sterols from Crude Soybean Oil H. R. KRAYBILL, M. H. THORNTOX,
AND
K. E. ELDRIDGE
Purdue University Agricultural Experiment Station and the U. S. Regional Soybean Industrial Products Laboratory’, Lafayette, Ind.
The approximate sterol curt-. ECAUSE of its chemical A combined adsorption and extraction t e n t of t h e o i l s w a s d e t e r - . structure, stigmasterol is method for the production of a phosphamined by saponifying a 100an excellent material For tide-free sterol concentrate from soybean gram sample, extracting the the synthesis of the hormone oil, having fifteen to twenty times the sterol sterols with petroleum ether, progesterone. For that reason content of crude oil, is described. From recrystallizing onc3 from ethathe increased clinical use in nol] drying, and weighing. recent years of sex hormones this concentrate without saponification a By this method the crude oil. has stimulated interest in the large portion of the mixed sterols crystalwas found to contain approxi.. commercial production of stiplized readily and may be recovered by filtramately 0.2 per cent of sterols masterol. Stigmasterol occurs tion. The remaining sterols, some of while the nonbreak oil obtained as a component of the sterol which are combined as esters, are readily by filtration of the crude oil mixture in many different t h r o u g h t h e a d s o r b e n t conplant?, but the only source of recovered by the saponification of a relatained approximately 0.1 per commercial importance is the tively small volume of oil. cent of sterols. These results mixed sterols of the soybean This method results in the production of indicated that about half of in which it occurs to the e-+ c‘nonbreak” soybean oil and also permits t h e s t e r o l s were a d s o r b e d tent of 20 to 26 per cent of the the recovery of the greater part of the sterol along with the phosphatides. total sterols. Stigmasterol and T h e adsorbed sterols were the hormones synthesized from glucosides present in the crude oil. s u b s e q u e n t 1y completely rei t have b e e n m a d e c o m m e r The method was applied to the producmoved from the oil-saturated cially in Germany for several tion of soybean sterols on a large laboratory a d s o r h e n t w h e n it was ex-years, but production in this scale. In one experiment 234 grams of tracted with acetone. Acetone c o u n t r y h a s b e e n extremely sterol glucosides and 1778 grams of mixed cxtraction of the adsorbent alsa limited. removed a large part of t h e sterols were obtained. From 20 to 23 per The plant sterols or phytosterol glucosides which had sterols are always obtained as cent of stigmasterol was separated from been adsorbed from the oil, mixtures of several different different portions of these mixed sterols. but practically none of the adsterols, some of which are exsorbed p h o s n h a t i d e s . After tremely difficult to separate in evaporation of the acetone from the-extract, a dark colored the pure state. Ronstedt (1) separated soybean sterols into oil was obtained which contained a flocculent insoluble solid, stigmasterol and a mixture of sitosterols by bromination. consisting of sterol glucosides. The sterol glucosides were From the sitosterol mixture he further separated dihydroseparated from the dark colored oil by filtration. The oil; sitosterol and various other fractions representing isomeric remaining after removal of the insoluble glucosides contained sitosterols. These fractions could not be purified satisfac0.6 to 1.0 per cent of sterols and was practically free from: torily. From the work of Rosenheim and Webster (3) and phosphatides. others it also seems probable that soybean sterols contain a Further treatment with the adsorbent of the oil obtained1 small amount of ergosterol. by acetone extraction from the above operation does not re-. Plant sterols usually are obtained by extraction of the unsult in much greater concentration of the sterols since t h e , saponifiable residue after saponification of the fat, followed by sterols themselves are not appreciably adscrbed from the oil i fractional crystallization of the unsaponifiable fraction. except in the presence of the phosphatides. Steiger and Reichstein ( 6 ) used this procedure to obtain approximately 1500 grams of sterols from 1000 kg. of soybean oil, This method requires the use of large quantities of solConcentration of Sterols by Solvent ETwaction vents and is laborious and costly. According to the patent obtained by Schwieger (d), The adsorption method described resulted in a considerable sterols are recovered from materials such as the acetone concentration of the sterols, but further concentratLon was washings of crude phosphatides by treating them with desired to reduce to a minimum the amount of oil t9 be saponialcohol, allowing them to crystallize in the cold, recoverfied. This was accomplished by re-extraction of the sterol ing them by filtration, and extracting the sterols after saponiconcentrate derived from the adsorption process,by a solvent fication. in which the sterols were soluble but which ma$ substantially immiscible with the oil itself. Both methanol and ethanol can
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Concentration of Sterols by Adsorption from Crude Soybean Oil Filtration of crude soybean oil through a column of aluminum silicate adsorbent ( 2 ) resulted in the removal of the phosphatides and partial removal of the sterols from the oil.
1 A cooperative organieatlon particlpated In by the Bureaus Of 4gricultural , Chemistry a n d Englneerlng and of Plant Industry of the Upited Stqtes Department of Agriculture, and the Agricultural Experiment S$??tluna of the North Central States of Illinois, Indlana, Iowa, Kansas, Mlchigan, himnesota, Xfissourl, Sebraska, North Dakota, Ohlo, Squtj Dakota, and 1 Wisconsin
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1138
AUGUST, 1940
INDUSTRIAL AND ENGINEERING CHEMISTRY
be used, but methanol was better adapted to this purpose because of its lower solvent power for the oil. The oil recovered from the adsorbent by acetone extraction was therefore refluxed with methanol. After cooling, the methanol solution was separated from the oil. The methanol extraction was repeated three times. After distillation of the solvent from the methanol solutions, the residues of oil were combined and saponified. The sterols were then extracted from the unsaponifiable matter. I n this way 0.44 gram of sterols was recovered from 100 grams of the oil obtained by acetone extraction of the adsorbent. Saponification of an equal quantity of the same material and recovery of the sterols from the unsaponifiable matter yielded 0.5 gram. Thus, practically complete recovery of the sterols was effected by the procedure described. Since refluxing with a solvent was slow and inefficient, in subsequent experiments the phosphatide-free sterol concentrate obtained by acetone extraction of the adsorbent was extracted with methanol in a continuous extractor of the liquidliquid type. The results of a typical experiment are as follows : Fifteen hundred grams of the oily concentrate, obtained by acetone extraction of the material adsorbed from approximately 80 pounds (36.3 kg.) of crude soybean oil, were extracted for several days with methanol; then the methanol v a s removed from the extract by evaporation. The fraction obtained by methanol extraction amounted to 318 grams or about 21 per cent of the material which was subjected to extraction with methanol. This residue contained 6.52 grams o r 2.05 per cent of sterols. The amount of extract and its sterol content varied considerably with the length of time and the thoroughness of hhe extraction. I n some cases the sterol content of the extract was as high as 4 to 5 per cent. Thus, the combined adsorption and extraction processes are capable of effecting a twenty fold concentration of the sterols. Moreover, when a phosphatide-free oil with a sufficiently high sterol concentration is obtained, the sterols crystallize directly from i t a t room temperature and may be recovered by simple filtration. When phosphatides are present in the material to be extracted, they are completely removed by the alcohol and are concentrated in the extract to such an extent as to prevent crystallization of the sterols. The crude mixed sterols obtained by crystallization from the oily concentrate are fairly free from color and are easy to purify. Since some of the sterols are present in the oil in combined form (esters, glycosides), all of the sterols do not crystallize. If total recovery is desired, the residue remaining after filtering off the crystalline sterols may be saponified and treated in the conventional manner for recovery of sterols.
Large-Scale Production of Soybean Sterols The adsorption method for concentrating sterols was applied on a somewhat larger scale in a laboratory-type filter press. The adsorbent was mixed mith the oil and the mixture pumped through the press until i t was completely filled -with adsorbent. The oil n-as then recirculated through the press one or more times. I n this experiment 8 pounds (3.6 kg.) of adsorbent and 106 pounds (48 kg.) of crude expeller soybean .oil mere used. After passage through the press, much of the oil was nonbreak and bleached to a light color on heating. The adsorbent was removed from the press and extracted with acetone. Four and three tenths grams of sterol glucosides were recovered from this extract. After complete removal of the solvent, the acetone extract weighed 11 pounds (5 kg.) and contained approximately 0.6 per cent of sterols. These sterols were further concentrated by extraction with methanol, and were finally recovered by th- crystallization and saponifica-
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tion procedure already outlined. The yield was approximately 27 grams. A second experiment was carried out in a commercial soybean expeller plant. Approximately 10,000pounds (4535 kg.) of crude expeller soybean oil were pumped through a filter press filled with the adsorbent. The phosphatides were completely removed from the oil passed through the press during the early part of the filtration, but after the adsorbent became more saturated the phosphatides were not removed conipletely from the oil. The adsorbent was removed from the filter press and extracted with acetone, and the acetone was removed by evaporation. There were obtained 710 pounds (322 kg.) of oil having a sterol content of about 0.6 per cent. After filtration of this oil, 234 grams of sterol glucoside were obtained. The filtered oil was then extracted with methanol in a continuous extractor. I n order to extract the 710 pounds of oil, a metal extractor which had a capacity of about 30 liters was built. From 3 t o 5 days were required for the complete extraction of the sterols from a batch of oil in this extractor. After removal of the methanol by distillation, the extract amounted to 130 pounds (59 kg.) of oil or 18.3 per cent of the oily concentrate recovered from the adsorbent. This oil contained about 3 per cent of sterols, and sterols crystallized from i t on standing. These mixed sterols were filtered off and purified by recrystallization from alcohol. A total of 768 grams of purified mixed sterols was recovered in this way. After the crystalline sterols were removed by filtration, the remaining oil was saponified and the soap solution extracted with petroleum ether in the extractor mentioned above. A total of 1010 grams of purified mixed sterols was recovered from this operation. Thus, 1778 grams of purified mixed sterols were obtained from the 130 pounds of sterol concentrate. This amount is equivalent to the total sterol content of about 2000 pounds (910 kg.) of crude oil.
Properties of Soybean Sterols The mixed sterols which crystallized from the concentrate were freed from oil by recrystallization from cold petroleum ether and were recrystallized from ethanol, and the last traces of color were removed by treatment with vegetable charcoal. Those obtained by saponification were purified by crystallization from ethanol after treatment with vegetable charcoal. The purified mixed sterols, melting point 134-136' C., [~]'.0 (CHCL) - 37.20' (c = 1.2, 1 = 2), gave an acetate, melting point 133-134' C., [a]': (CHC1,) - 42.58' (c = 3.2, 1 = 2). These sterols were obtained as white shining plates and were found t o give a positive Liebermann-Burchard reaction. Sterols similar to these were also obtained by hydrolysis of the sterol glucoside. Numerous experiments in the separation of stigmasterol from the mixed sterols by the bromination method of Windaus and Hauth (6) indicated that they contain approximately 20 to 25 per cent of stigmasterol.
Literature Cited (1) B o n s t e d t , K., 2. physiol. Chem., 176, 269 (1928). (2) K r a y b i l l , H. R . , B r e w e r , P. H., a n d T h o r n t o n , M. H., U. S. P a t e n t 2,174,177 ( S e p t . 26, 1 9 3 9 ) . (3) R o s e n h e i m . O., and W e b s t e r . T. A., Biochem. J . , 21, 389 (1927). (4) Schwieger, A.. C . S. Patent 2,046, 345 ( J u l y 7 , 1936). ( 5 ) Steiger, M., and R e i c h s t e i n , T., Helv. Chim. Acta, 20, 1040 (1937). (6) W i n d a u s , A., a n d H a u t h , A.. Ber., 39, 4378 (1906).
PRESENTED before the Division of -4gricultural and Food Chemistry a t the 98th Meeting of the American Chemical Society, Boston, .\lass. A portion of the data is from a thesis presented by 112. H. Thornton in partial fulfillment of the requirements for the degree of doctor of philosophy.
