Reprinted from LIPIDS, November, 1969, Vol. 4, No.6, Pages: 450-453 New Sources of 9-D-Hydroxy-cis-12-octadecenoic Acid 1
R. G. POWELL, R. KLEIMAN and C. R. SMITH, JR., Northern Regional Research Laboratory,2 Peoria, Illinois 61604
ABSTRACT gl V cerid e mixt ure isolated from Nerium ;l~ander L. seed oil. 9-D -Hydroxy-cis-12-octadecenoic acid has been isolated from 3 seed oils of the family Apocynaceae: Holarrhena anti PROCEDURES AND DATA dysenterica (73%), Nerium oleander Infrared (IR) spectra were determined on a (II %) and Nerium indicum (8%). The Perkin-Elmer 137 instrument as liquid films or known occurrence of this acid was as I % solutions in carbon tetrachloride or car previously limited to the genus bon disulfide. Nuclear magnetic resonance Strophanthus (9-15%). A mixture of (NMR) spectra were recorded on Varian A-60 unusual tetra-acid glycerides was isolated or HA-IOO spectrometers with deuteriochloro from N. oleander oil by thin layer chro form solutions containing 1% tetramethylsilane matography. Pancreatic lipase hydrolysis as an internal standard (unless otherwise indi of the glyceride mixture showed that cated). Optical rotatory dispersion (ORO) 9 -D-acetoxy-cis-12-octadecenoic acid is measurements were made on a Cary Model 60 esterified exclusively at an Cl'-glycerol recording spectropolarimeter at 26° in absolute position and that normal fatty acids methanol and in 0.5 dm cells. Melting points occupy the remaining 2 glycerol posi were determined with a Fisher-Johns melting tions. A portion of the hydroxy acid in point apparatus and are uncorrected.. N. indicum oil was also acetylated, how Thin layer chromatography (TLC) was per ever. no acetate was found in either H. formed on plates coated with 0.25 mm or 1.0 antidysenterica or Strophanthus hispidus mm layers of Silica Gel G or 20% silver nitrate oils. impregnated Silica Gel G with 20% or 30% ether in hexane as the solvent. Bonc ac!d INTRODUCTION impregnated plates were used for analysis of lipolysis products. Spots on analytical plates Gunstone (1) first reported the occurrence were visualized by charring with sulfuric acid of 9-hydroxy-cis-12-octadecenoic acid in chromic acid. After preparative plates were Strophanthus sarmentosus seed oil and later sprayed with dichlorofluorescein, bands were showed that it probably is of general occur visualized under ultraviolet light. rence (9-15%) in seed oils of the genus Gas liquid chromatography (GLC) of methyl Strophanthus, family Apocynaceae (2). The esters was conducted as described by Miwa et structure has been confirmed by synthesis (3) al. (7). Triglycerides were analyzed on an F &M a nd the naturally occurring "Strophanthus Model 5750 instrument equipped with a 24 X acid" has been assigned the D configuration (4). 1/8 in. metal column packed with 3% OV-l. The Strophanthus acid may also be obtained by The instrument was linearly temperature-pro partial diimide reduction of naturally occ~rring grammed at 4 deg/min from 200-400 C. 9 -D-h y d r 0 xy t ra m-I O,cis-I 2-octadecad!enOlC Oxidative cleavages were according to the acid (5,6). method of von Rudloff (8). Iodine values were Strophanthus oils are convenient sources of determined by the AOCS method (9). Pan min or amounts of 9-D -hydroxy-cis-12-octa creatic lipase hydrolyses, silylations and subse decenoic acid. however. the low hydroxy acid quent GLC of silylated lipolysis products were content and u'navailability of the oils suggested as described by Tallent and Kleiman (10). a search for additional sources among the closely related genera Holarrhena and Nerium. Isolation and Purification of Hydroxy Acids In tltis paper we report the characterization Seeds were ground and extracted overnight of 9-D-hydroxy-cis-12-octadecenoic acid from 3 in a Soxhlet apparatus with petroleum ether new sources. One, Holarrhena antidysenterica (30-60 C); solvent was removed from the oils (Roth) Wall ex. DC. seed oil, contains this acid on a rotary evaporator. Both Nerium oleander as the major component (73%). We also report and Naium indicum oils exhibited bands in the the characterization of an unusual tetra-acid IR at 1235 cm-! and 1020 cm-! (acetate) which were not present in spectra of H. anti ! Presented at the AOCS Meeting, San Francisco, dvsenterica or Strophanthus hispidus oils. April, 1969. . Methyl esters were prepared by refluxing the 2No. Utiliz. Res. Dev. Div., ARS, USDA. oils for 2 hr with 5% hydrochloric acid in
[I] 9-HYOROXY-CIS-OCTAOECENOIC ACID
anhydrous methanol. Esters were recovered by ...'"
Concentrates of the hydroxy esters were ob >, ,..., tained by preparative TLC. The concentrates ~ '" ~ >0 U 2 - ,..., r-: were saponified by refluxing for 1 hr with 1N "'0 0 t"- o -l >,-" t"- potassium hydroxide in ethanol, and unsaponi v :: - >, fiables were removed by extracting dilute ,:;;; aqueous solutions of the soaps with ether. After .s'"en acidification, the free hydroxy acids were ,..., ,..., "": N recovered by ether extraction. The acids were '"~ - " - 0 0 esterified with diazomethane, and the resulting '""'" - esters were purified by preparative TLC. Overall 2 .5 yields were near 80% in each instance (based on N ~ r-: GLC of the total esters). "t"- t"- .d-.'" ~ "-
hexane, gave methyl 9-hydroxyoctadecanoate. t""-~-oo ~~I.CiM After preparative TLC and recrystallization M~('.ll""'l from hexane, the saturated ester melted at 51-52 C and had a plain negative ORD curve (Table II). Known methyl 9-o-hydroxyocta decanoate gives a similar melting point, 51.5-52.8 C, and a nearly superimposable plain negative ORD curve (11). A 156 mg portion of methyl 9-hydroxy-cis 12-octadecenoate, from Holarrhena oil, was acetylated by allowing to stand overnight in a mixture of pyridine-acetic anhydride (1: I). Column chromatography on 2 g of neutral alumina (hexane) yielded 176 mg of product. The product gave IR and NMR spectra which
[2 ] R. G. POWELL, R. KLEIMAN AND C. R. SMITH, JR. TABLE II Optical Rotatory Dispersion of Hydroxy Esters Specific rotation in degrees Concentration, Ester and source [a] 589 [a1500 [a]400 g/100 ml Methyl 9-0-hydroxy-cis-12-octadecenoate Holarrhena antidysenterica -0.83 -1.18 -2.00 2.88 Nerium oleander -1.76 -2.30 -3.20 1.13 Nerium indicum -1.92 -2.34 -3.40 0.95 Strophan thus lzispidus -1.87 -2.48 -4.12 2.67 Strophanthus kombea -1.17 -1.76 -2.54 1.02 Methyl 9-0-hydroxyoctadecanoate Hydrogenated Holan-hena ester -0.12 -0.16 -0.30 2.29 Hydrogenated methyl dimorphecolatea -0.17 -0.23 -0.37 1.49 aSpecific rotations of these materials were obtained by Applewhite et aJ. (11) and are listed here for comparison. were consistent with those expected for methyl free carboxylic acid, but also had bands at 1724 9-acetoxY-cis-12-octadecenoate. GLC and TLC cm-1 and 1235 cm- l , characteristic of an showed the product to be homogeneous. acetoxy derivative. Treatment with diazo methane gave the methyl ester which was iden Glyceride Characterization tical with known methyl 9-acetoxY-cis-12-octa An unknown component was observed upon decenoate, as demonstrated by IR, NMR, GLC TLC analysis of N. oleander oil, which had an and TLC. Rf slightly lower than normal triglycerides. A portion of lipolysate of the unusual GLC of the oil indicated that acetotriglycerides glyceride mixture was silylated, and GLC and triterpene acetates were absent because no showed no monoglycerides with acetoxy func significant components with carbon numbers tional groups. Similar treatment of a lipolysate less than C52 were observed. A significant peak of the original oil showed that 5% of the mono was eluted as Cs 6, however, even though only glycerides contained acyl groups with hydroxy 2% C20 esters were found in the ester analysis. or acetoxy functions, or both. A portion of the The presence of glycerides containing an whole oil lipolysate was treated with diazo acetylated hydroxy acid constituent was indi methane and subsequent GLC demonstrated 1 cated by a conspicuous 1235 cm- (acetate) that 12% acetoxy and 2% hydroxy methyl band in the IR; only weak hydroxyl absorption esters were present in the mixture. Thus, was present. assuming no acyl migration during lipolysis, all IR of the unusual component, isolated by the acetoxyacyl and a portion of the hydroxy 1 preparative TLC, showed a strong 1235 cm- acyl groups are esterified on the outer glycerol (acetate) band and no hydroxyl. A sharp singlet carbons. was present in the NMR spectrum at 7 8.1 (CC1 solution) characteristic of an acetoxy 4 DISCUSSION group. A multiplet was evident at 75.3, due to a single proton, and a similar multiplet was The hydroxy acids of H. antidysenterica, N. noted in the NMR spectrum of methyl oleander, N. indicum and S. hispidus are clearly 9-acetoxy-12-octadecenoate. This signal was identical, and experimental data are consistent assigned to the proton on a carbon bearing an with only one structure: 9-0-hydroxy-cis-12 acetoxy group (C9). A multiplet, equivalent to octadecenoic acid. The presence of this acid in 4 protons, was present at 7 5.9, indicative of S. hispidus oil was demonstrated previously protons on the outer carbons of a glycerol (12). Assignment of the 0 configuration was moiety. Except for the signals at 7 8.1 and 7 possible as these 4 esters and known methyl 5.3, the spectrum was otherwise consistent with 9-0-hydroxy-cis-12-octadecenoate (11) all gave that of a glyceride. This unusual component, plain negative ORD curves that were nearly when analyzed by GLC, gave peaks having car superimposable. Confirmation of this assign bon numbers of CS2 ,CS4 and CS6 ' ment was obtained by hydrogenating the After the unusual glyceride mixture was sub Holarrhena ester; the product gave an ORD jected to pancreatic lipase hydrolysis, a product curve essentially the same as that for known was isolated that migrated between normal free methyl 9-D-hydroxyoctadecanoate (4,11). acids and diglycerides on TLC. This product Gunstone (1) found that methyl 9-hydroxy had an IR band at 1705 cm- l , associated with l2-octadecenoate gives abnormally high iodine
[ 3 ] 9-HYDROXY-CIS-OCTADECENOIC ACID values, apparently because of involvement of ACKNOWLEDGMENTS the reagent with the hydroxyl group. Similarly, the iodine value determined for Holarrhena oil Preliminary screening results and G LC analyses by (Table I) is considerably higher than that calcu F. R. Earle, J. W. Hagemann and R. W. Miller; NMR spectra by L. W. Tjarks; Holarrhena and Nerium seeds lated from the fatty acid composition. More were furnished by Q. Jones, USDA, Beltsville, Md.. satisfactory results may be obtained after and the Strophanthus seeds by F. D. Gunstone, Uni acetylation (1) and the Nerium oils do not versity of St. Andrews, Scotland. show this discrepancy. Since pancreatic lipase (EC 3.1.1.3) displays REFERENCES a strong specificity for the 1 and 3 (or Q and Q') positions of triglycerides (13), lipolysis with J. Gunstone, F. D.. J. Chern. Soc., 1274-1278 this enzyme has been widely used to determine (1952). the intraglyceride distribution of various acyl 2. Gunstone, F. D.. and L. J. Morris. J. Sci. Food groups. In Strophanthus oils 9-hydroxy-12 Agr. 10:522-526 (1959). octadecenoic acid shows strong preference for 3. Kennedy, J., A. Lewis, N. J. McCorkindale and R. the 2 (or (3) glycerol position, and there is no A. Raphael, J. Chern. Soc., 4945-4948 (1961). evidence for triglycerides with more than 3 acyl 4. Schroepfer, G. J., Jr., and K. Bloch, J. BioI. groups (14). Since lipolysis of the mixture of Chern. 240:54-63 (1965). tetra-acid glycerides from N. oleander gave no 5. Powell, R. G.. C. R. Smith, Jr. and I. A. Wolff, J. monoglycerides with acetoxy functional Org. Chern. 32:1442-1446 (1967). 6. Badami, R. C., and L. J. Morris, JAOCS gr0 ups, evidently 9-acetoxy-12-octadecenoic 42:1119-1121 (1965). acid occurs exclusively at I (or both) of the Q 7. Miwa, T. K., K. L. Mikolajczak, F. R. Earle and I. glyceroi positions. However, 9-hydroxy-12 A. Wolff, Anal. Chern. 32: 1739-1 742 (1960). octadecenoic acid is also present, as shown by 8. Rudloff, E. von, Can. J. Chern. 34:1413-1418 lipolysis of the whole oil, and the major portion (1956). of this acid occurs at the (3 glycerol position. 9. American Oil Chemists' Society. "Official and The tetra-acid glycerides of N. oleander, con Tentative Methods of Analysis," Chicago, 1957. taining an acetylated monohydroxy acid, are 10. Tallent, W. H., and R. Kleiman, J. Lipid Res. 9: 146-148 (1968). apparently unique although similar tetra-acid 1J. Applewhite, T. H., R. G. Binder and W. Gaffield, (I5) and penta-acid (16) glycerides having J. Org. Chern. 32: 1173-11 78 (1967). acetylated di- or trihydroxy acids are also 12. Gunstone, F. D., 1. Sci. Food Agr. 4:129-132 known. (1953). The Holarrhena alkaloids are considered 13. Entressangles, B., H. Sari and P. Desnuelle, Bio possible starting materials for industrial prepa chim. Biophys. Acta 125:597-600 (1966). ration of steroid hormones, including the 14. Gunstone, F. D., and M. I. Qureshi, J. Sci. Food adrenocortical hormone aldosterone (17). Oils Agr. 19:386-388 (1968). from other species of Holarrhena may prove IS. Mikolajczak, K. L., C. R. Smith, Jr. and I. A. equally rich in 9-hydroxy-12-octadecenoic acid, Wolff, Lipids 3:215-220 (1968). 16. Mikolajczak, K. L., and C. R. Smith, Jr., Biochim. and moderate quantities of the oils could Biophys. Acta 152:244-254 (1968). become available as by-products of the pharm 17. Cerny, V., and F. Sorm, "The Alkaloids," Vol. 9, aceutical industry. At present, H. anti Edited by R. H. F. Manske, Academic Press, New dysenterica seed oil is, by far, the richest York, 1967, p. 305. known source of 9-D-hydroxy-cis-12-octa decenoic acid. [Received April 3, 1969]
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