SITOSTEROLS FROM CORN GLUTEN
Dec., 1950
100-ml. portions of ether. Remoyal of the ether left 4.5 g. of red solid which was crystallized tmce from water
t o yield O.g0g. of the hydrate of o-benzoylbenzoic acid, Its identity was confirmed by conversion m. p. 93-94 t o anthraquinone.
.
Summary
phenylacrylonitrile possesses a structure similar to that of FittigJs isatropic acids and is probably a substituted tetralin* 2. Several hydrolysis products of the dimer of a-phenylacrylonitrile are described.
1. It has been shown that the dimer of a- STAMFORD, CONN.
[CONTRIBUTION FROM
THE
MULTIPLEFELLOWSHIP OF THE CORN
PRODUCTS R E F I N I N G
RECEIVEDMARCH27, 1950
COMPANY, MELLONINSTITUTE]
Sitosterols from Corn Gluten BY DOROTHY M. RATHMANN AND LOUISE R. MORROW Anderson and co-workers’ found that the “sito- from which i t is apparent that the sitosterol fracsterols from corn” are a complex mixture con- tion of highest negative specific rotation yielded taining sitostanols and a small amount of stigmas- primarily i-y-sitosteryl methyl ether (I). Interol in addition to cy-, P- and y-sitosterols, and termediate fractions yielded a complex mixture of that relatively more sitostanol was obtained from i-sitosteryl methyl ethers (11) and sitosteryl the endosperm than from the germ. In the com- methyl ether (111). The sterol fraction of lowest mercial isolation of zein from corn gluten,2 one specific rotation yielded principally episitostanol stage consists in extraction of an alkaline isopro- (IVA) and sitostene (V) which may have been pyl alcohol solution with hexane to remove the derived from sitostanol in the course of preparing lipids. The resulting extract contains a t least the methyl ethers. 95Oj, of the oil originally present in the gluten. On the basis of this preliminary investigation, After washing the extract with water and evapo- i t is concluded that y- and @-sitosterolsare the rating off the hexane there is obtained a dark red- major components of the phytosterols from xanbrown oil, known as “xanthophyll oil,” about thophyll oil, that stigmasterol is absent, and that 11% of which consists of nonsaponifiables. The the content of sitostanol is much lower than was crude phytosterols are isolated in a yield of 8% found by Anderson in corn gluten. Many small (based on the oil), as a cream-colored solid having fractions have not been fully characterized. Speca sitosterol content of a t least 85%. A typical ulations regarding the possible occurrence of the batch of this sterol mixture constituted the start- a-sitosterols are consequently not warranted. ing material for the present investigation. Experimental Removal of the non-steroid contaminants and With the exception of the crude sitosterols, samples some degree of fractionation (Table I) was were dried over phosphorus pentoxide for several houts at achieved by recrystallization of the acetates which 60 and 2 mm. pressure before being analyzed. Specific rotations were determined at room temperature were then hydrolyzed. Further fractionation was obtained by the adsorption analysis of the i-sito- (25 * 2”) for a chloroform solution of the compound in a tube, using sodium light. The concentration steryl methyl ethers. Although adsorption has 2-dm. was 2.0 * 0.2 g. per 100 ml. of solution. Barton and Cox6 been employed3 as a means of freeing the i-steryl reported that, in the range used in the present work, the methyl ethers from unreacted sterols, there seems speciiic rotations of various sterol derivatives were indeto have been no investigation of the method as a pendent of temperature and concentration. Consequently these data are not listed for each compound. means of analyzing phytosterol mixtures. Carbon-hydrogen analyses were conducted by G. L. I n order to obtain data on the probable behavior Stragand, Microchemical Laboratory, University of of the ethers of the sitosterols during adsorption, Pittsburgh. The infrared spectra were obtained and ini-cholesteryl methyl ether and cholesteryl methyl terpreted by R. B. Hannan, Jr., Department of Researcb Chemical Physics, Mellon Institute. ether were prepared by the method of Stol14 and in Unless otherwise stated, the acetates were prepared by chromatographed. The results indicated that refluxing the sterol with 10 volumes of acetic anhydride the recovery of both types of ethers would be for thirty minutes. The benzoates were obtained from high and that the i-steryl methyl ether would the reaction of 1weight of sterol with 1 volume of benzoyl in 10 volumes of anhydrous pyridine for twentybe less strongly adsorbed than the corresponding chloride four hours at room temperature. After being isolated in steryl methyl ether. the usual fashion, these derivatives were recrystallized from Chromatography of the ethers derived froin the ethyl acetate. Properties of Sitosterols from Corn Qluten .-Crude sitosterol fractions is summarized in Table I1 O
(1) (a) Anderson and Moore, THISJOURNAL, 46, 1944 (1923); /b) Anderson, ibid., 46, 1450 (1924); ( c ) Anderson and Shriner, i b i d . , 48, 2976 (1926); (d) Anderson and Sbriner, J . Biol. Chcm., 72, 401 (1927); (e) Anderson, Nabenhouer m d Shiner, ibid., 71, 389
(19271. (2) Swallen, Ind. Eng. Chcm., 89, 394 (1941). JOURNAL. Sq 3346 (1940). (3) Fernholz and Ruigb. THIS (4) Stoll, 2. physiol. Chcm., 487, 147 (1982).
sitosterols isolated from xanthophyll oil consisted of slightly tan crystals containing 85 t o 90% sitosterols (digitonin-precipitable) and having total fatty acid conteflts as high as 674,depending on the method of iylation Melting points were in the range of 125 t o 135 RecrystsUi zation of a mixture of two such preparations from ethyl acetate gave: crop A, 74y0 yield, m. p. 134-136”, [ C V ] ~ D
.
(5) Barton and Cox, J . Chcm. Soc., 783 (1948).
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DOROTHY hl. KATHMANN AND LOUISE R.MORROW
5648
Vol. 72
TABLE I
PRELIMINARY FRACTIONATION OF CORNGLUTEN SITOSTEROLS Yield, %C
5.3 9.5 4.4 9.7 10.9 26.0 11.5 12.4
Acetate fractions [a]"D
(CKC1r)d
Sterol fractioxisn
"5. C.p..
131-133 130-133 125-130 -37 - 33 118-125 120-124 - 30 - 28 121-124 -26 123-126 25 120-126 5.9 -22 126-127 107-117 0.3 - 13 a First crop from hydrolysis of acetate. of starting material. Average values. from sterol fraction. 0 Decomposes.
Yield,
%$
Tosyfatc fractiotlsb
M.p.,
[pl"D
(CHClr)
"C.
Y,$l$ .O,
PSD
( HCla)
M.
p.,
OC.
r-
-41
-40
137-138 ( ( 69 -36.5 -35.8 123-124 69 -35.2 71 137-1 39 -36.9 125-127 60 -32.4 '70 -36.4 136-137 126-12s -27.5 70 133-136 68 -29.1 126-131 133-135 -23.8 70 .... . . . . 67 134-137 .... -24.3 .. ...... 135-137 -19.5 129-130 35 -17.9 79 -15.8 135 68 9.0 117-123 39 -13.6 62 133-136 112-124' 47 -10.5 21 -10.6 130-133 .. .... ...... First crop after adsorption and recrystallization. Based on sterol content Based on theoretical yield from acetate fraction. Based on theoretical yield . I
-
-
e
'
-23.7'; crop B, 9.670, m. p. 133-134', [ L Y ] * ~ D-25.5"; 10.24; S , 5.62. Found: C,75.50; 75.72; H, 9.70, 9.76; and crop 4%, m. p. 129-132', [ a I s ~ - 0.6". S, 5.49. Although the melting paint was lower than the Crop A contained 0.7% of volatiles, 1 . 3 d of total fatty decomposition point of 154r155' reported by Stoll' for sitoacid (as oleic) and had a sitosterol content of 95.23% by stanyl tosylate, it agreed with that reported for stigmasthe digitonin method and 92.2Yo by the Liebermann-Burtanyl tosylate.s This tosylate fraction reacted with chard method. The bromine number was 415 as compared methanol containing potassium acetate t o yield IVA and V. with 385 calculated for y-sitosterol. An attempt t o deterSitosteryl p-To1uanesalfonate.-Recrystallization of the tosylate fraction of highest specific rotation yielted colormine the sitostanol content directly by the method of less platelets, m. p. 123-124.5", [ c x ] * ~ D-34.3 . Anal. Schoenheimerg gave an inconclusive result: the weight of sterol precipitated by digitonin after bromination was in Calcd. for CS&I&S: C, 76.00; H, 9.92; S, 5.64. Found: excess of the amount equivalent to the digitonin added. C, 75.70; H, 9.71; S, 5.94. The specific rootation is in A very low content of stigmasterol was indicated by the good agreement with the value, [a]=D -36 , calculated failure to isolate stigmasteryl acetate 22,Sdibromide for y-sitosteryl tosylate by the method of Bernstein, et from the products of the reaction of sodium iodide' with U Z . , ~ and differs markedly from [ c x ] * ~ D-47" reported for the crude bromositosteryl acetate (m. p. 109-115", [ a ] " ~ stigmasteryl tosylate, m. p. 148-150°.3 This tosylate fraction reacted with methanol containing potassium ace-29.6'). Preparation and Fractionation of the Acetates.-A solu- tate t o form I. tion of equal weights of the crude sitosterols and acetic Preparation and Fractionation of the Methyl Ethers.anhydride in 2 volumes of glacial acetic acid was refluxed The i-sitosteryl methyl ether was prepared by refluxing a for 5.5 hours and then allowed to stand overnight at room mixture of equal weights of the tosylate fraction and freshly temperature. The yield of crude acetates was 95yo (based fused potassium acetate in 50 volumes of methanol for 011the sterol content of the starting material). The acetwenty-four hours. About half of the methanol was distates were recrystallized once from ethylene dichloride tilled off before the reaction product was isolated by the containing ethyl alcohol and nine times from a 1 : 1 mix- addition of water and extraction with ether. The crude ture of benzene and isopropyl alcohol, giving the fraction5 methyl ethers were obtained as oils in almost quantitative listed in Table I. yields (based on the weights of the tosylate fractions). Hydrolysis of the Acetate Fractions.-A solution of 1 part The crude product dissolved in 25 volumes of hexane by weight of the acetate in 5 t o 6 volumes of 10% potaswas adsorbed on a column of aluminalo which was then sium hydroxide in 95% ethyl alcohol plus 1.5 volumes of washed with hexane until no more material was eluted and benzene was refluxed under nitrogen for 2.5 hours. The then with hexane containing methanol to elute more sterols were isolated in the usual manner and were recrys- strongly adsorbed compounds. The ratio of the weight tallized from a 1 : 1 mixture of benzene-isopropyl alcohol. of methyl ether t o alumina in this preliminary adsorption Some properties of the first crops of crystals are summarized was about 1: 15. The eluate fractions were rechromatoin Table I for comparison with those of the acetate frac- graphed by a similar procedure, using an approximate weight ratio of the ether to alumina of 1:50, until the tions from which they were obtained. Preparation of the p-Toluenesulfonates .-The p - specific rotation was lint 3ignificantlv changed hv readtoluenesulfonate was prepared by the reaction of an equal sorption. The sole exception t o this method of fractionation was weight of thc sterol fraction and purified p-toluenesulfonyl chloride in 3 volumes of anhydrous pyridine for forty- the case of the product from the tosylate of low negative three hours a t room temperature. The product was specific rotation. This consisted OF a wax from which a crystalline mixture (11') having [ a ]2 L f18" ~ was obtained usually isolated in 90% crude yield. The crude product was purified by adsorption on alumina from a hexane solu- by slow cooling of a solution in hexane. The hexane tion and some fractionation of the tosylates was simultane- mother liquor yielded only an oil which was finally chroously achieved. F'roperties of the first crops obtained by matographed in the usual manner. Final results of the recrystallization from d r y acetone are summarized in Table fractionations of the methyl ethers are summarized in I . In agreement with the results of other workers, it Table 11. The following fractions were identified: was found that the conversion of a sterol to its tosylate does I. i-y-Sitosteryl Methyl Ether.-The oily methyl ~ t o 46" could not be crystallized from not have a marked effect on the specific rotation. Further ether of [ a ] %+43 recrystallizations yielded two distinctly different tosybtes. hexane, acetone or methanol, and the specific rotation Sitostanyl p-Tohrenesulfonate .-R lization of the could not be changed significantly by readsorption on tosylate fraction of lowest specific from ethyl ( 8 ) stoii, z fittysioi c ~ I G ? a~ , ~ 1 (1937). , wetate yielded a white powder, m. p. 146-14S0, [also IU) Bcrnstt-iii Kauzmann .m
