Reprinted from LIPIDS, Vol. 13, No.4, Pages: ~70-273 (1978)

Cyclopropene Fatty Acids of Selected Seed Oils from Bombacaceae , Malvaceae, and Sterculiaceae

M.B. BOHANNON and R. KLEIMAN, Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois 61604

ABSTRACT Fatty acid compositions of seed oils from three species of Bombacaceae, eleven from i\lalvaceae. and six from Sterculiaceae were determined. Each of the seed oils contains varying amounts of both malvalic and sterculic acids accompanied by one or both of the corresponding ;yclopropane fatty aCids. In addltlOn, the seed oil of Paclzira aqllaric Aub!. (Bombacaceae) contains 12.8% o:-hvdroxv- sterculic acid. . .

INTRODUCTION treated with silver nitrate by the procedure of Schneider et al. (10) to convert the cyclo­ Cyclopropene fatty acids have been reported propene acids to derivatives amenable to chro­ as constituents in the seed oils from many matography. Epoxy acids, when present, were species of the Malvaceae, Sterculiaceae, Tilli;­ converted to methoxy-hydroxy derivatives by ceae, and Bombacaceae families (I). A number treating with boron trifluoride (II). Equivalent of oils from these families have been investi­ gated and found to contain both malvalic and chain lengths (ECL) of methyl esters and the sterculic acids, frequently accompanied by reaction products were determined by GLC as smaller proportions of one or both of the analo­ previously described (6). The normal methyl gous cyclopropane acids (2,3). In addition, seed esters were separated from the reaction prod­ oils containing malvalic acid usually contain ucts of the cyclopropene and hydroxy acids by measurable amounts of epoxy acids (4). thin layer chromatography (TLC) on l.()..mm It is evident from these earlier investigations layers of Silica Gel G developed with n-hexane­ that seed oils containing such an array o~f COm­ ethyl ether (70:30). The normal methyl esters pounds are difficult to quan titatively analyz were separated according to degree of unsatura­ by traditional gas liquid chromatography (GLC) tion on Silica Gel G (containing 20% silver and hydrogen bromide (HBr) titration proce­ nitrate) developed with benzene. Mass spectral dures. Recourt et al. (5) have shown that the data were obtained with a Dupont 21-491-2 c yc!opropene acids te~d to isomerize and m ass spectrometer interfaced to a Bendix decompose as they pass through the GLC Model 2600 gas chromatograph (GC-MS). The column. In addition. G LC data show that the column, 6 ft x Y, in. packed with 5% Silar (5 malvalic acid peak is masked bv the linoleic cp) was held isothermally at 195 C. acid peak (3) and that the corresponding cyclo­ Nuclear magnetic resonance (NMR) spectra propane acid may also be obscured by the were determined with a Varian HA-I 00 spectro­ presence of oleic acid (6). The HBr tit~ation meter. Samples were dissolved in deutero­ methods most often used to determine the chloroform; tetramethylsilane was used as an cyclopropene and epoxy acids present are internal standard. Infrared (IR) spectra were recognized as being su bject to interference by measured on liquid films with a Perkin-Elmer conjugated dienols (7). Model 337 spectrometer. A Beckman DK-2A The present investigation was undertaken to spectrophotometer was used to record ultra­ determine the fatty acid composition of a series violet (UV) spectra. Hydroxyl groups were of seed oils that contains a mixture of normal silylated with bis(trimethylsilyI)-trifluoro­ fatty acids and fatty acids with epoxy, acetamide in pyridine. h y d ro x y, cyclopropene, and cyclopropane functional groups. RESULTS AND DISCUSSION

Preliminary Identification of Methyl Esters EXPERIMENTAL PROCEDURES and Derivatives Oil was obtained from the ground seed as GLC analyses of mixed methyl esters con­ previously described (8). Fatty ;cids in the oils taining only normal esters and derivatized reacting with hydrogen bromide at 55 C were cyc!opropene esters were consistent with the determined by the titration method of Harris et peaks previously reported for the esters from al. (9). Methyl esters for G LC were prepared by derivatized Sterculia foetida oil (10). However, sodium methoxide catalyzed methanolysis and G LC of those mixed esters also containing

270 TAIlLE I

C'ollljlosilioll of S\,'L'd Oils

J'-utly acid l'olllplJ~ilioll hy (:I.e (area %)

._-_.,.,-,._._~_.~-_.- C'yclo- Cyclo- Spectral data (lil ill !l_~!l~~!~::,_ ~!J.~~~,~_:__ HBr reactive ------Sample seed (t)r;) 16:0 16: I 17:0 17: I 18:0 IH: I 18 2 20:0 20: I I H 19 IH 19 Epoxy:l Hydroxyh acids at sse «(XJ) uv lR

/lomhI1CllCl'iH' Chot/sia St. lIiL 21.7 I H.7 0,5 0.2 0.2 2.R II.'I 4'1.7 0.7 (), I 0.5 12.4 10.0 O.H 21.0 Pllcllirll a(fuaticll Alibi 55,5 bO.S 0.3 2.9 7.6 4.7 0.'1 0.1 1.6 8.6 J 2.8 26.6 011 .\'almaJiIl mil/aharicll (DC') SdlOll and End!. 23,6 .lO.O 0.6 0.2 O.H 5.0 16,7 25.0 Ir 0.6 7.5 11.0 1.0 15.5 L3" 1.0 () >-<: A1£1JI'1l/'CI1C () t-< A It!locullin'/{ 1a L. 12.2 1.1.0 0.5 0.3 0.'1 4.'1 7.5 52.8 O.H 0.'1 0.2 16.5 1.6 22.4 ... 0 /lilli,w'l/.\' gramJij1oru,\J.I I 1.7 I H.2 0.4 2.0 6.0 15.5 46.0 0 . .1 O.H 1..1 4.0 .1.0 1.6 7.2 ... OB "tJ l1ihisCII,\' syril1£'II,Vd 27.7 20.0 Ir Ir 0.7 2.2 10.2 42.6 0 . .1 Ir 1.0 1J.4 3.0 2.7 2.B 19.9 ... all :>:l K itaiht'lia vitifolia 0 "tJ Will" 22.6 H.O 0.3 Ir 0.3 2.5 14.2 64.4 0.5 0.2 0.1 7.7 1.5 10.5 OB tTl I.agullarfa pater,WHlff Z C;. DOll I H.t) 23.0 .1.9 Ir 0.4 4.7 21.5 22.3 Ir I.t 7.7 3.9 1.4 14.1 ... OB tTl llva/era kas1lmirfanll 'Tj Comh. 19.1 15.0 Ir Ir 0,6 .1.6 10.H 50.5 0.5 0 . .1 0.5 Ir 16.4 1.2 ... -.. 14.4 ... > iHlllvl111/0nfatW Forsk 18.4 15.7 0.2 Ir 0.4 .1 ..1 10.3 55.5 Ir 0.2 12. I 1.0 ... 1.0 14.4 ...... >-J ...... >-J Malva parviJlora L. 11.0 15.4 0.2 1.1 .1.0 9.0 5.1.'1 O.B 0.2 0.4 12.0 1.7 1.0 1.2 14.8 --< Malva 100UIU'fOl'tiIl1l11 L. 17.0 12.4 0.5 1.6 2.1 .1.5 11.0 4.1.B 2.3 0.2 Ir 0.3 17.H 1.5 2.0 '" 23.7 ...... Ala/ope {rlfltIa 11axl. 18.0 0.3 .1.7 6.8 17.7 0.2 1.6 44.3 0.2 0.2 0 . .1 11.5 3.1 6.5 1.2 24.9 L2" 5.4 OH ()> PUl'fJ1lil1 sf'plufll SI. Ilil 26.6 .11.0 1.0 Ir D.2 2.0 1SA 34.2 0.5 Ir Ir Ir 2.3 1.3 5.1 5.7 17.6 L2 3.6 OH a (fJ S'!erculit1ct'(U' Ffrmitlllll pldtallifo1itl

Schott and End!. 24.3 17.2 1.2 Ir 0.2 2.4 18.2 36.0 0.7 Ir 0 ..1 1.6 20.5 2.0 '" 24.3 Ptt'rygolll a/aId Hoxh, 47.H 24.4 1.6 0.2 0.3 .1.:1 9.9 .18.5 0.6 0.1 0.6 12.2 2.5 2.7 1.4 19.5 ... OB Pterospermlllll (leerifoliulJI (L.)lVilid 21.5 17.0 Ir Ir 0 . .1 2.9 8.6 .12.3 1.0 .12.2 3.8 ...... 39.1 •\'U'J'clilia ftH.'tida L. 5.1.5 14.7 Ir Ir Ir 1.4 4.9 4.5 I.B 0.2 Ir 0.4 6.3 65.1 ...... 64.6 'J'llrric{iulifiliJI..1 29.7 29.2 0.7 0.1 0.2 2,0 20..1 0.6 Ir 0.2 6,8 ...... t-< 18.9 Ir 20.2 25.7 ~ UEpoxy acid is verl1IJlic hy GLC evidcllce. t:! h'-Iydroxy add is a conjugated dicilol hy GLC' L'vidt~I1CC except ill P. aqllatic(J andH. syriaclJs which Wt~re identified as a-hydroxystcrculic. •(fJ <: q-:l conjugaled (dene; % in falty acids. 0 tlAHthorilY no! available. r q~2 conjugated dicne; % ill fatty acids. ~- Z l~ 9 --..I -I> 272 M.B. BOHANNON AND R. KLEIMAN epoxy, hydroxy, or conjugated unsaturation from the isolated component were not as were difficult to interpret. Esters shown to con­ clearly defined. However, the appropriate mole­ tain the epoxy function were further deriva­ cular ions at m/e = 412 for the ketone deriva­ tized by converting this group to the methoxy­ tive and m/e = 428 for the methoxy derivative hydroxy derivative followed by conversion of were apparent. Also, the ion at m/e = 161 re­ the hydroxyl group to the trimethylsiloxy sulting from cleavage alpha to the carbon (TMS) ether (11). The resulting TMS-deriva­ bearing the TMS group was observed. tized esters were reanalyzed by GLC. These Characterization of the Cyclopropene Acids data (Table I) indicated that the seed oils con­ tained varying amounts of epoxy and/or The silver nitrate derivatives obtained from hydroxy acids in addition to the cyclopropanes, the methyl esters of the cyclopropene acids cyclopropenes, and normal fa tty acids. The were found in fraction I! from TLC. The migra­ varying amounts and wide distribution of the tion pattern and GLC elution pattern of these epoxy and hydroxy acids in these oils were as components were identical to the corre­ expected (1,4,12). The mixed esters derived sponding derivatives of malvalic and sterculic from each oil were separated into three frac­ acids from the seed oil of Sterculia [oetida by tions by preparative TLC on Silica Gel G plates. Recourt et al. (5). The MS fragmentation pat­ GLC data indicated that fraction I was a mix­ terns are also identical to those reported by ture of normal long chain methyl esters and Eisele and co-workers (16). possibly the cyclopropane methyl esters. Frac­ Characterization of the Cyclopropane Acids tion I! contained the esters of the cyclopropene derivatives, and fraction III contained the The cyclopropane acids were contained in h y d roxy methyl esters and the methoxy­ fraction I from the preparative TLC along with hydroxy derivatives obtained from the epoxy the normal long chain fatty acids. These acids methyl esters. De Bruin et al. (13) reported that have retention characteristics similar to those of Pachira aquatic oil possibly contained an acid the analogous monounsaturated methyl esters similar to sterculic, but which might also con­ (6). Therefore, it was necessary to further tain hydroxyl group(s). Therefore, the hydroxy separate fraction I according to degree of un­ acid component was isolated and characterized. saturation by AgN03-TLC prior to identifica­ tion of the cyclopropane acids. The fraction collected as the saturated methyl esters from Characterization of the Hydroxy Acid in P. Aquatica this mixture was shown by GLC to contain The migration pattern of the component(s) small amounts of dihydromalvalic acid with in fraction II! isolated from P. aquatica was ECLs of 17.7, (APL column) and 18.4 (R446 compared to that of a known mixture of column) and/or dihydrosterculic acid with a:-hydroxy octadecanoates and methyl sternl­ ECLs of 18.7 (APL column) and 19.4 (R-446 late. The major component in the isolated column). Mass spectra of these acids are similar material remained near the origin along with to those of the parent monoenoic esters (17). the a:-hydroxy acids whereas authentic stercu­ The only apparent difference is that the cyclo­ late migrated nearer to the solvent front. propane acids show a molecular ion 14 mass Distinctive IR absorption bands at 1265, 1210, units greater than the parent monoenoic ester. and 1110 em-I are indicative of a:-hydroxy esters (14). Absorption bands at 1850 and 1010 Description of Seed Oils cm- I were also present (cyclopropene ring). A Oil content of the seeds varied from 11 % in pronounced signal (singlet) in the Nr.lR spec­ Mall'a parl'iflora to 56% in P. Aquatica. The trum at 04.12 suggested that a methine proton seed oils, except for three (Table I) showed no was on the carbon adjacent to the carboxyl UV absorption indicative of conjugated un­ function. Signals at 02.3-2.4 associated with a saturation. Epoxy acids, if detected, ranged methylene group in the alpha position were not from 0.8';-;' in Chorisia speciasa to 6.5% in observed. After conversion of the hydroxyl Alalope triji'da. Cyclopropene acids ranged from group of the major component to a TMS deriva­ 3.6% in Pal'onia sepium to 71.4';j in S. [oetida. tive, ECLs of 18.6 (APL column) and 20.6 In general, the presence of a cyclopropene ring (R-446 column) were observed. The ECLs were was established in the seed oils by HBr titration similar to those of authentic methyl sterculate at 55 C and by the presence of absorption (6). Mass spectral data of the isolated com­ bands in the IR spectrum at 1010 and 1850 ponent were identical to those of a:-hydroxy­ cm- I . GLC analyses for the cyclopropene acids sterculic acid of Pachira insignis as reported by are probably more reliable than the HBr titra­ Morris and Hall (IS). The fragmentation pat­ tion because of uncertainties in the titration terns of the silver nitrate derivatives obtained method pointed out by Feuge et al. (18). Also

LIPIDS, VOL. 13, NO.4 CYCLOPROPENE FATTY ACIDS 273 observed in the spectra was absorption at 3550 VanEtten, and LA. Wolff, J. Am. Oil Chern. Soc. cm- I (hydroxyl) from many of the oils. The 36:304 (1959). 9. Harris, J.A., LC. Magne, and E.L. Skau, J. Am. seed oil of P. aquatica contained 12.8% Oil Chem. Soc. 40:718 (1963). a-hydroxysterculic acid. 10. Schneider, E.L., S.P. Loke, and D.T. Hopkins, J. Am. Oil Chem. Soc. 45:585 (1968). 11. Kleiman, R., G.F. Spencer, and F.R. Earle, Lipids REFERENCES 4:118(1969). 12. Mikolajczak, K.L., R.M. Freidinger, C.R. Smith, 1. Smith, C.R., Jr., in "Progress in the Chemistry of Jr., and LA. Wolff, Lipids 3:489 (1968). Fats and Other Lipids," Vol. XI, Part I, Pergamon 13. de Bruin, A., J .E. Heesterman, and M.R. Mills, J. Press, New York, NY, 1970, pp. 139-177. Sci, Food Agric. 14:757 (1963). 2. Smith, C.R., Jr., T.L.Wilson, and K.L. Miko­ 14. Bohannon, M.B., and R. Kleiman, Lipids 10:703 lajczak, Chern. Ind. (London) 256 (1961). (1975). 3. Wilson, T.L., C.R. Smith, Jr., and K.L. Miko­ 15. Morris, L.J., and S.W. Hall, Chern. Ind. (London) lajczak, J. Am. Oil Chern. Soc. 38:696 (1961). 32 (1967). 4. Hopkins, C.Y., and M.J. Chisholm, J. Am. Oil 16. Eisele, T.A., L.M. Libbey, N.E. Pawlowski, J.E. Chem. Soc. 37:682 (1960). Nixon, and R.O. Sinnhuber, Chern. Phys. Lipids 5. Recourt, J.H., G. Jurriens, and M. Schmitz, J. 12:316 (1974). Chromatogr. 30: 35 (1967). 17. Christie, W.W., and R.T. Holman, Lipids 1:176 6. Miwa, T.K., J. Am. Oil Chern. Soc. 40:309 (1965). (1963). 18. Feuge, R.O., Z.Zarins, J.L. White, and R.L. 7. Smith, C.R., Jr., M.C. Burnett, T.L. Wilson, R.L. Holmes, J. Am. Oil Chern. Soc. 44: 548 (1967). Lohmar, and LA. Wolff, J. Am. Oil Chern. Soc. 37:320 (1960). 8. Earle, F.R., E.H. Melvin, L.H. Mason, C.H. [Received December 5, 1977]

LIPIDS. VOL. 13. NO.4