Translation Series No by E. Fedeli, and C. Mariani

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1 _4'\_R^^^IVES FISHERIES AND MARINE SERVICE Translation Series No Complexity of the sterol and triterpene fractions in plant oils - Note III by E. Fedeli, and C. Mariani Original title: Complessitâ della frazione sterolica e triterpenica degli oli vegetali - Nota III From: La rivista italiana delle sbstanze grasse 51(April) : , Translated by-the Translation Bureau(mMM) Multilingual Services Division Department of the Secretary of State of Canada P ^ t Department of the Environment Fisheries and Marine Service Halifax Laboratory Halifax, N:S pages typescript bj

2 D'EPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU MULTILIPGUAL SERVICES DIVISION rek,pg ue7u ocem CANADA SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES TRANSLATED FROM - TRADUCTION DE INTO - EN.Sel ô AUTHOR - AUTEUR Italian E. FEDELI et al. English TITLE IN ENGLISH - TITRE ANGLAIS Complexity of the Sterol and Triterpene Fractions in Plant Oils - Note III. TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTLRES ROMAINS) Complessità della frazione sterolica e triterpenica degli oh i vegetali - Nota III. REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS, RÉFÉRENCE EN LANGUE ÉTRANGÉRE (NOM DU LIVRE OU PUBLICATION), AU COMPLET, TRANSCRIRE EN CARACTÈRES ROMAINS. La rivista italiana delle sostanze grasse. REFERENCE IN ENGLISH - RÉFÉRENCE EN ANGLAIS PUBLISHER - ÉDITEUR The Italian Review of Fatty Substances. Stazione sperimentale per l'industria degli oh i e dei grassi PLACE OF PUBLICATION LIEU DE PUBLICATION Milan, Italy YEAR ANNÉE DATE OF PUBLICATION DATE DE PUBLICATION VOLUME ISSUE NO. NUMÉRO April PAGE NUMBERS IN ORIGINAL NUMÉROS DES PAGES DANS L'ORIGINAL NUMBER OF TYPED PAGES NOMBRE DE PAGES DACTYLOGRAPHIÉES 10 REQUESTING DEPARTMENT MINISTÈRE-CLIENT Fpvirrement TRANSLATION BUREAU NO. NOTRE DOSSIER NO BRANCH OR DIVISION DIRECTION OU DIVISION Fisheries and Marine TRANSLATOR (INITIALS) TRADUCTEUR (INITIALES) MM/4 PERSON REQUESTING DEMANDÉ PAR YOUR NUMBER VOTRE DOSSIER N 0 DATE OF REQUEST DATE DE LA DEMANDE Dr. R.G. Ackman, Halifax Laboratory Not-state_d 3rd August, UNEDITED TRANSLATION For inforrneion orly TRADUCTION NON REVISEE Information seuloment NOV (REV. 2/66)

3 'DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION CANADA SECRE TARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES CLIENT'S 140. DEPARTMENT DIVISION/BRANCH CITY t10 DU CLIENT. MINIST2RE DIVISI ON/DIRECTION VILLE Environment Fisheries and Marine Halifax. BUREAU HO. LANGUAGE TRANSLATOR (INITIALS) ti 0 DU BUREAU LANGUE ' TRADUCTEUR (INITIALES) O N 00\J e261, Italian M MM ctb,.....,.. COMPLEXITY OF THE STEROL AND TRITERPENE FRACTIONS OF PLANT OILS -. NOTE by: E. Fedeli, and C. Mariani. National Research Council - Milan, Italy - Milan Experimental Station for Oils and Fats Complexity of the. sterol and triterpene fractions of plant oils*- - Note III. (129)* The sterol and triterpene fractions obtained from some vegetable oils were analyzed as trimethylsilyl derivatives (TMS)-on two columns with different polarities. The analyses on the more polar column show the presence of some substances that.did not appear in the chromatograms obtained with the lower polarity column. COMPLEXITY OF THE STERDLIC AND TRITERPENIC FRACTIONS IN VEGETABLE OILS - Note III. Sterolic and triterpenic fractions from a number of vegetable oils were analyzed as trimethylsilyl derivatives on two columns of different polarities. Analytical findings cbteined from the more polar column evicienced the presence of constituents that did not show in the lower-polarity column chromatograms. In the two previbus notes (1, 2) we stated that the sterol Ïraction obtained from the insaponifiable fractions of plant oils, shows a greater number of components as compared with those revealed by GLC analysis on non-polar (conventional) columns and that its cdmplexity depends on the Oa U1',IEDITED TRANSLATION * For informalion only Number of page in original text - translator. - TRADUCTION NON REVISEE -. Information seulement SCJI%

4 2 presence of 4-methylsterols, à 7 -sterols, dihydrosterols, partly identified in note II,which is being printed. Therefore, it seemed appropriate to us on the basis of the knowledge required,to verifylthe GLC behaviour of the sterol fraction and of the triterpene fraction, under different experimental conditions. This note refers to the GLC behaviour of the sterol and triterpene fractions obtained by conventional methods from some oils, on two stationary phases, one non-polar and one of greater polarity, in order to acquire new useful data permitting more accurate analyses of fatty substances. substances. EXPERIMENTAL PART Preparation of the unsaponifiable fraction The NGD method was used (7). Separation of the fractions of the unsaponifiable portion 20 x 20 Silica Gel G plates'impregnated with 2% of H 3 PO4 were used and eluted with hexano-ether 60-40(3). GLC analysis of the sterol' and terpene fractions a)..ixr column; glass column; length 2 m; diameter 4 mm; stationary phase 3% MIR; support: P Gas Chrom mesh; C. Erba model GV gas chromatograph; temperature: column 220 C, evaporator 320 C, detector 280 C; carrier gas: N2 40 ml/min. h) 0V-17 column; glass column; length 2 m; diameter 4 mm; stationary phase 3% 0V-17; support: Q Gas Chrom mesh; C. Erba model GV gas chromatograph; temperature: column 250 C, evaporator 320 C, detector 280 C; carrier gas N2 30 ml/min.

5 3 RESULTS Sterol Fraction As may be observed from Table I, the two stationary phases used, in the experimental conditions selected, give different analytical results, probably due to the fact that the greater polarity of the OV-17 also brings about the separation of some substances which are not separable on the. However, we consider that not even the OV-17 completely shows the differences in composition between the sterol fractions of thé various plant oils. We preferred for the moment to leave the structure of some components which we have indicated with an X unknown, until such time as the.new separation procedures which we are presently studying can give us a more in-depth knowledge of the actual structures by means of GLC MS. The sterol fraction of maize oil,shows the appearance on the OV-17 column, of two new peaks, X3 and X4, the first of which has a certain importance quantitatively; the examination of the data further shows that not even peak X3 can be considered as a single one because its 6.3_ percentage value seems partly to come from the diminution of the ^-sitosterol, and partly from the A 7-stigmasterol. For the analysis of the sterol fraction of olive oil,we*considered a refined olive residues oil and also in this case we noted substantial differencès in the GLC patterns of the same sample, obtained with the different columns; we note the presence of small quantities of one sterol may be seen, which has the same retention time as A 7-stigmastenol (and is therefore shown in the table column corresponding to it) but whose structure has yet to be confirmed, and of the sterol previously indicated as X3 in maize oil. Both of these.'sterols evidently derive from the R-s_itosterol peak, as may be shown

6 4 by a simple mathematical calculation and we therefore consider it improbable that the second peak could correspond to the structure of A 7-stigmastenol, which is certainly separable under the analysis conditions used on (J X R), from the (3-sitosterol itself. By inference only, we think it more likely that dihydro-sitosterol, already identified by us in other oils is present in peak X3. The compound indicated as being present (0.5%) under (130) X4, in reality, has a slightly different retention time from the other X4 components shown in the table. The analysis of the sterôls of grape-seed oil, on the two different columns, shows the exchanges of retention times occurring through the simultaneous influence of the two factors which govern the.separation, the polarity of the compounds and their molecular weight. In fact, in the analysis on neither the X5 nor X4 appears and they are probably combined under X3; the two former ones never appeared in the analysis of the grape oil sterols. The analysis pf peanut oil on 0V-17, also reveals a component having the same retention time as A 7 -stigmastenol and which is not present in the analysis on and a quite remarkable concentration of the X3 compenent; comparing the two values of the sitosterol, it must be concluded that these two peaks.derive in part from compounds having a lower retention time (campesterol and stigmasterol). The crucifer oils (crambe and rape) are the only ones whose sterol fraction components include the component indicated by us as X, and present only in the analyses on 0V-17. Also, on this column, we note the components X3 and A 7 stigmastenol for which we must repeat the sanie remarks made above; furthermore,there is daesignificant presence on 0V-17 of the component X2 which is not present in other oils.

7 5 Table I - GLC Analysis of the Sterol Fractions of some plant Oils on Columns with Different Polarities 0 Id ^ t I Jo CG,N o?^ > ;:j ----;- tj Maize Olive Grapeseed eanut rambe tape OV-17 OV-17 OV17 ov-17 OV-17 OV-17 Sunflower OV-17 tr. - tr ,5 0,4 tr, 0,1 0,4 0,6 9,4 8,0 10,9. 10,0 19,0 19,6 2,8 3,2 10,0 9,4 17,9 17,8 36,8 34,3 26,9 25,2 10,4 10,2 21,3 19,3 8,6 6,6 1,3 1,2 11,9 10,2 9,7 7,0 tr. 2,6 tr. 10,6 9,8 9,2 6,1 - pres. - 1,3 69,7 65,2 70,5 71,4 69,4 71,4 72,4 69,3 53,8 53,7 58,7 57,2 68,8 62,3 69,5 49,6 1,6 3,0 4,6 12,1 3,3 1,5 0,8 0,5 0,7 ; - 2,6 0,7 2,4 1,1 0,5 - _ - 0,2 10,2 8,7 3,1 0,8 0, t - 18,8 6,1 17,4 4,0 4,7 4,2 ^. - 0,7 115 Soya R OV-17 0,62 f 0,67 0,53 0,59 20,4 20,6 0,711 0,81 0,65 j 0,78 21,4 21,0 0,86 0,87-0,93 0,89 0,94 54,0 54,7 22 1,00 1,00 1,11 4,2 1,5 1,14 1,17 tr. 1,31 1,45 1,36 1,50 1,54 1,59 In the case of sunflower oil, the GLC on OV-17 allows us to identify a compound to which we attribute the structure of a7-campesterol, a component which is present to a considerable extent only in this oil, of all those examined; the sterol composition of sunflower seed oil with the simultaneous presence of considerable quantities of Xt is remarkably different from that of the other oils. An extremely broad variation in the composition data on the two columns is shown by sesame oil: the content of ^-sitosterol differs greatly in the two analyses (69.5 on, 49.6 on OV-17) both

8 as a result of the presence of a component X6 (sesamolin), and of the relatively high value of the component X3. Also present on 0V-17 are X4, and a sterol which has the retention time of estigmastenol. On the other hand, the sterol fraction of soya showed an important variation only as regards the presence of r-stigmastenol,the value of which drops from 4.2% on to 1.5% on 0V-17. Triterpene Fraction For the components of this extremely complex fraction which are not identifiable, through their relative retention times, as tetra or pentacyclic triterpene alcohols (u, f3-amyrin, cycloarthenol, 24-methylenecycloarthanol) known for some time, we prefer to disregard fication even though data do exist in the literature (4, 5). their identi- In fact, the com-. plexity of the fraction, also in relation to the incomplete separation under the experimental conditions used, renders both the qualitative and quantitative data rather'imprecise (see Table II). However, for some unsaponifiable fractions the qualitative and quantitative validity of the data is similar to that obtained, with the sterol fractions (maize, crambe, rape). On 0V-17, the triterpene fraction of maize oil shows two peaks more (131) than the tracer on, corresponding to retention times which are higher than that of 24-methylenecycloarthanol (24 MCA). The two peaks mentioned above are absent in the analysis on 0V-17 of the triterpene fraction of olive oil which, however, shows a strong peak between (3-amyrin (e-a) and cycloarthenol (CA). The complexity of the fraction in this area is also shown by the analysis on. A situation similar to olive-oil is also found for sunflower seed ogwhere the same corn-. ponent,with a relative retention time of 0.90 (0V-17),is present; furthermore,

9 7 grape-seed oil shows peaks g, h, i, after the 24 MCA,also present in the chromatogram on 0V-17 of the triterpenes of other oils (with the exception of 1). On the colum,the peanut triterpenes show the presence of two unidentified components, eluted after the 24 MCA, presumably different from those shown by other oils on the 0V-17 column only; furthermore present, again on the non-polar column, is a curve on the peak of 24 MCA,which indicates the presence of an unknown component. The GLC (0V-17) of the terpene fraction of sesame oil appears to be quantitatively dominatedhy sesamolin, while the tetra- and pentacyclic triterpenes form a simple mixture of f3-a, CA, 24 MCA. The data obtained on the two columns for sunflower and soya oils are quite different; examples of these differences are shown in table II. Finally, we must consider the remarkable differences of a qualitative, but more especially, of a quantitative nature between the varieties of Brassicaceae, a sample of rape oil and the crambe variety; whilst the former contains CA as the main component of the triterpene fraction (approximately 51%), the latter shows similar contents of CA and 24 MCA. Furthermore, the crambe oil triterpenes show a curve on the CA peak, jndieating an unknown component. DISCUSSION OF THE RESULTS In our opinion, the data given and reported in Tables I and II demonstrate that even small differences in the polarity of the columns used for the analysis of the sterol and triterpene fractions of plant oils provide information on the complexity of the mixtures superior to that shown by conventional analyses (on non-polar columns of SE-30 or ). As regards the sterols, the 0V-17 shows some components which are not revealed

10 8 Table II - GLC analysis of the Triterpene Fractions of some Plant Oils on Columns of Differing Polarity i H H H Cyclo- ri.ri 1 arthenol 4=.1 a 0 b c d 14 e f 4Jo cu g h i I -, 24-BIC thyl c_) 4 enechclo ce Ci :, P U 4,P ^10 cd. arthanol 0 Olive jxr 1,8 6,4.-- 7,8 21,8 p1-es., 28, , ,87 0V-17-1,4 0,9 0,9 13,6 11,7 pres. 35,7-35, Ses ame 5,1 1, , i 35,4 1,1 Ma -- 2, ,1 0V , i ,0 -- 1, ,0 221 Soya -- 3,0 5, ,2 7,8 : 2 9, 8 0,6 24, V ,7 5,7 15,7 21,5 pres. ; 27,9-22,0 5, Sun ,3 2 1,3 25, , ,86 f lower 0V ,2 -- 6,8 30, ,3 2,1 45 1,01 eanut jxr, 3 :.81 1,5 0,8 1, ,2 33,3 3A 23,0 1,8 2, ,45 0V ,2 6,1 : ,3 29, ,7 0,8 9,7 (1 ) rape7 MR ,8 72 8,1 26, , ,85 -eed -0V , ,8 10,2 pres. 28,8-26,2 2,9 1,7 0,2-1,09 faize , , V , , ,7 2,7 1, rambe IXR MA -- 8,0 35, , V ,4 35, ,7 -- 0,82 - 'ape ,0 15,9 - pres. 64, ,35 0V , , , m -- 0,80 0, ,00 1,05 1, , VR n --. 0y5 0,80 0,85 0, ! IMO 1,10 1, VR 0V-17 n - 0,66 0,70 0,76 0,84 0,90 i 0,96 I 1,00-1,10 1,2 1,44 1,59 1,79 -- (1) 2 peaks of 1.8 and 2.4; m = retention times with respect to 8-sitosterol; n = retention times with respect to cycloarthenol. by conventional analysis, lika substance X1, specific to rape oil, X2, specific to the crucifer oils, r-campesterol, specific to sunflower oil. Component X3 is present in all the oils examined but represents an important fraction of sesame oil. Maize, sesame and sunflower show component X4 in the 0V-17 chromatograms which in the latter oil assumes relatively high values (3.1%). Finally, it must be noted that as regards the sterol fractions

11 9 e-stigmastenol, where present, always appears on 0V-17 in lesser quantity than on a non-polar column, as may be seen from table I for sunflower, maize and soya oils; for the two latter oils,the percentage decrease in content is respectively 54 and 64%. The complexity of the sterol fraction is. confirmed once again by the presence, on the 0V-17 columns, of compounds with the retention time of e-stigmastenol, in oils which normally do not contain it, like olive and peanut oils. The results of the analyses of the triterpene fraction are even more complex because of the difficulty in obtaining quantitative data due to the poor resolution of the peaks. However, even with the reser- vations mentioned above, some data obtained seemed to us to be of considerable interest, like the presence of component e) in olive, soya and grape-seed oils for significant fractions. of the mixture. This component is indicated as butyrospermol according to the data supplied by one of us (6). Soya, sunflower, peanut, grape-seed and maize, contain significant quantities of com- ponent c) (VR-OV 0 ' 70); the saine oils have the compound g(vu._ OV 0'70) as a common component and, with the exception of soya, component h as.well. Finally, the triterpene fraction of the two crucifer oils examined show3sig-. the two varieties, both from the quantitative nificant differences between and qualitative viewpoints due to the presence in rape of component d(v 0.85). R- This work is merely a preliminary study for a more complete examination of the sterol and terpene fractions,which most certainly will require new research methods allowing to more accurately resolve the complexity of the mixtures, identifying their components (even those not be- longing to the classes indicated) as a premise for an in-depth know- ledge of. biosynthesis in oil-bearing fruits and seeds and a more rational use of such knowledge for analytical purposes.

12 10 BIBLIOGRAPHY BIBLIOGRAFIA I) E. FEDELI, N. CORTESI, C. MARIANI, A. GOVONI, D. Bmt, Riv. It. Sost. Grasse 49, 105 (1972). 2) E. FEDELI, A. DActlErrA, D. BARON', N. CORTESI, Riv. It. Sost. Grasse 49, 159 (1972). 3) E. FEDELI, N. CORTESI, F. CAMURATI, G. JACINI, J. Am. 011 Chem. Soc. 49, 763 (1972). 4) T. Iron, T. TAMURA e T. NIATSUMOTO, J. Am. Oil Chem. Soc. 50, 122 (1973). 5) T. Itott, T. TAmuRA e T. MATSUMOTO, J. Am. Oil Chem. Soc. 50, 300 (1973). 6) P. CAPELLA, E. FEDELL, M. CIRINIELE, G. JACINI, Ri'. It. Sost. Grasse 40, 296 (1963), 7) Norme Grassi e Derivati, Stazione Sperimentale Oh i e Gras. si, Milano. Metodo Ba-IV 13 (1957). Italian Bibliographic Item 7) Regulations for Fats and their Derivatives, Oils and Fats Experimental Station, Milan.

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