Methods for Isolation and Determination of Anacardic Acids J. L. Gellerrnan and Herrnann Schlenk The Hormel Institute, Unicersity of Minnesota, Austin, Minn. 55912
Anacardic acids from the leaves of ginkgo and from cashew nuts were studied. They were obtained from these sources, together with fatty acids and other lipids. Treatment of the mixtures with CH,OH 3% H2S04yielded fatty esters but did not esterify anacardic acids. The esters and acids were separable from each other by chromatography on SOz. Anacardic acids were converted into methyl esters by reaction with CH2N2under mild conditions. Prolonged treatment with CHzNz in the presence of CH,OH gave dimethyl anacardic ether esters. The dimethyl derivatives can be separated by GLC without decomposition. Reversed-phase liquid-liquid chromatography of phenolic esters served for preparations on a larger scale. Structures of the olefinic chains of anacardic acids were determined by ozonization. A simple method is suggested for analysis of anacardic acids in cashew shell extract.
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ANACARDIC ACIDS are salicylic acids substituted in position 6 with saturated or unsaturated long-chain alkyl groups ( 1 , 2 ) . The variety of alkyl chains in anacardic acids encountered in nature suggests a notation similar to that commonly accepted for fatty acids. In the following we use the abbreviations Anl3:0, An15:3, Anl7:1, etc., to specify n-tridecyl, n-pentadecatrienyl, n-heptadecenyl, etc., as alkyl chain in individual anacardic acids. Anacardic acids
cardium occidentale are known to be rather resistant to pests. Antimold, antibacterial, and antiviral activity has been ascribed to constituents of Ginkgo biloba, and the phenolic components are responsible at least for some of such effects (8). It is apparent from the literature that systematic study of anacardic and similar phenolic lipids was hampered by lack of efficient preparative and analytical methods. This may be caused by the combination of phenolic and lipid character which is hybrid in terms of current fields of techniques and interests. Anacardic acids are obtained as mixtures with fatty acids and other lipids by extraction of the source materials or by saponification of the extracted lipids. They can be separated from most of the other lipid classes by adsorption chromatography. However, an efficient method for separation of anacardic from fatty acids has not previously been devised. Mixtures of anacardic acids have been fractionated by crystallization (9) and, more recently, argentation chromatography has been applied (10, 11) for separation according to their degree of unsaturation. Gas-liquid chromatography (GLC) for separation and identification of anacardic acids had not met with success (12). This report describes the separation of anacardic acids from fatty acids and the fractionation of the former by chromatographic methods. EXPERIMENTAL AND RESULTS
R
urushiol of poison ivy and must be thoroughly removed or destroyed by roasting or fermenting when ginkgo or cashew nuts are prepared for human consumption. When cashew shell liquid is prepared by roasting, the acids are, to a great extent, decarboxylated and the resulting alkylphenols are essential for the properties of lacquers and varnishes for which the liquid is used (5-7). Ginkgo biloba and Ana-
Isolation. Lipids were extracted from ginkgo leaves or whole cashew nuts in a Waring Blendor with CHC1, C H 3 0 H , 2 : 1 (v/v). After saponification and removal of nonsaponifiables, phenolic acids were recovered together with fatty acids. The latter were selectively esterified by reflux for 30 minutes in 10 volumes of anhydrous C H 3 0 H containing 3 HzSOa. Phenolic acids and fatty acid methyl esters were recovered after addition of 10 volumes H 2 0 by extraction with petroleum ether. They were separated by chromatography on a column of 250 grams of SiOz(Mallinckrodt AR, 100 mesh). The adsorbent had been suspended and washed in C H 3 0 H and diethyl ether (13) and had been activated at 100” C for 4 hours. Figure 1 represents a chromatogram which yielded 9.2 grams of fatty esters and 0.6 of a gram of phenolic acids from 12.5 grams of the partially esterified lipids of ginkgo leaves. In a similar chromatogram, 6.2 grams of fatty esters and 4.0 grams of anacardic acids were obtained from 11 grams of partially esterified material prepared from whole cashew nuts.
(1) H. J. Backer and N. H. Haack, Rec. Trati. Chim., 60, 678
(8) F. W. Eichbaum, Mem. Inst. Butantan (Suo Paulo), 19, 71
(1941). ( 2 ) P.T. Izzo and C. R. Dawson, J. Org. Chem., 14,1039 (1949). (3) S. Furukawa, Sei. Papers Itzsr. Phys. Chem. Res. (Tokyo), 24, 304 (1934). (4) H. A. Bokenoogen, Chem. Ind. (London), 1967,p 387. (5) F. M. Daunitz in “Protective and Decorative Coatings,” Vol. I, Wiley, New York, 1941, p 74 (6) M. T. Harvey and S . Caplan, Ind. Etig. Chem., 32, 1306 (1940). (7) C. C. Jordan and D. R. Coone, Foreign Commerce Weekly, U. S. Dept. of Commerce, August 8, 1942, p 4.
(1946). (9) V. J. Paul and L. M. Yeddanapalli, Nature, 174,604 (1954). (10) H. P. Kaufmann and J. Barve, Fette, Seifen, Anstrichmittel, 69, 437 (1967). (11) J. H. P. Tyman and L. J. Morris, J. Chromatog., 27, 287 (1967). (12) L. J. Morris, in “New Biochemical Separations,” Van Nostrand, London, 1964, p 295. (13) J. Hirsch and E. H. Ahrens, Jr., J. Biol. Chem., 233, 311 (1958).
R
=
R
=
An13 :O
-(CHz)iz-CHa
-(CH~X-CH=CH-CH~--CH=CH-CH-CHZCHzCH2
R
=
8,11,14-An15:3
-(CH2)11-CH=CH-(CH2)3-CH3 12-An17 :1
The best known source of anacardic acids is cashew nuts (Anacardium occidentale), but they occur also in leaves and nuts of the ginkgo tree (Ginkgo biloba) (3) and in other plant materials (4). Anacardic acids are skin irritants similar to
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VOL. 40, NO. 4, APRIL 1968
739
n B
ACN. 7% H20,30
1.0
-
0.75
-
0.5
-
-I
0 0
-
\
2
a
a 0.25
I
-
(3
98 %). G L C on a low-polarity phase separated anacardic ether esters mainly according t o chain length. Beckman GC-2A instruments were used with flame ionization detectors, or with a thermal conductivity detector when fractions were collected (17). The stationary phase was 3% SE-30 on silicone-treated Chromosorb W, 6@80 mesh, in a n aluminum column, 100 cm by 0.5-cm i d . , or 3 z Apiezon J on the same support in a column, 100 cm by 0.15-cm i.d. Column temperature was 236' C with 3.1 atmosphere inlet pressure of He. The procedure was particularly useful for preparative fractionation of anacardic ether esters from ginkgo. G L C on polar phase separated the ether esters according t o chain length and unsaturation. The stationary phase was 5 cycloheptaamylose acetate (19). Figure 3 shows a chromatogram from a column, 100 cm by 0.5-cm i d . , under conditions as described for G L C on low-polarity phase. Equivalent chain lengths (20) of anacardic ether esters are listed in Table 11 as ECLF, in reference t o fatty acid methyl esters and as ECLA, in reference t o saturated dimethyl anacardic ether esters. Degradation of Olefinic Chains. The positions of double bonds were determined by ozonization procedures as described for unsaturated fatty esters (17,21-24). Ozonizationreduction yielded aldehydes which were identified by G L C in reference t o C3 t o CS aldehydes. The position of the last double bond in the chain was deduced from them, except for acids with a terminal double bond. The position of other
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GLC on polar phase, cycloheptaamylose acetate.
Table 111. Anacardic Acids from Ginkgo Leaves and from Cashew Nuts Cashew nuts Ginkgo leaves Position of Position of Acid double bonds %" double bonds Xb An13:O 8 An15:O An15:l As > A l a 52 A8 34 An15:2 AS,ll 25 An15:3 AS, 11,14' 40 An17:O An17:l A12 40 An17:2
34, 1529 (1962). (20) T. K. Miwa; K. L. Mikolajczak, F. R. Earle, and F. A. Wolff, Zbid., 32, 1739 (1960). (21) D. M. Sand, N. Sen, and H. Schlenk, J . Am. Oil Chemists' SOC.,42, 511 (1965). (22) H. Schlenk and J. L. Gellerman, Zbid., p 504. (23) R. A. Stein and N. Nicolaides, J. Lipid Res., 3,476 (1962). (24) H. Schlenk, J. L. Gellerman, and D. M. Sand, Biochim. Biophys. Acta, 137, 420 (1967).
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Percentages according to GLC areas. Gravimetric determinations from liquid-liquid column chromatograms. Structure was verified by IR absorption of the terminal methylene group at 908 and 988 cm-'. a
aliphatic double bonds was determined by ozonizationoxidation of dimethyl anacardic ether esters. The resulting acids were esterified by CH2N2 and then analyzed by GLC. This way the phenolic moiety with the proximal part of the chain and the fragments between double bonds were identified. Fatty acid methyl esters were separated and analyzed as previously reported (22, 25). Tables I11 and IV list the structures and compositions of anacardic and fatty acids. Simplified Analysis of Cashew Shell Extract. The oil extracted from cashew nut shells contains anacardic and fatty acids virtually all in free form. Therefore, the material was subjected to complete methylation without prior saponification. Anacardic ether esters were separated from fatty esters and other components by preparative TLC. A layer of Silica Gel H , approximately 2.5 mm thick, was streaked with 560 mg of methylated cashew shell oil and developed diethyl ether acetic acid, 84:15:1. with Skellysolve B Zones were indicated by dichlorofluorescein in UV light, Five fractions were scraped off the plate and extracted with CHC13. The total recovery of lipids (97%) included 100 mg (18%) fatty esters and, well separated from them, 378 mg (6873 anacardic ether esters. The former were analyzed by G L C on ethylene glycol adipate, the latter on cycloheptaamylose acetate as described above. The results are given in Table V where percentages refer t o the total of esterified oil.
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(17) J. L. Gellerman and H. Schlenk, J. Protozool., 12, 178 (1965). (18) H. Schlenk and J. L. Gellerman, J. Am. Oil Chemists' SOC., 38, 555 (1961). (19) H. Schlenk, J. L. Gellerman, and D. M. Sand, ANAL.CHEM.,
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(25) J. L. Gellerman and H. Schlenk, Experieritia, 19, 522 (1963). VOL 40, NO. 4 APRIL 1968
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~
~
~~
Table IV. Fatty Acids from Ginkgo Leaves and from Cashew Nuts Chain length: Number of double bonds 14:O 16:O 16:l 16:2 16:3 18:O 18:l 18:2 18:3
Ginkgo leaves” Structure of chain
Cashew nuts* Structure of chain
%c
zc
1.5 0.2 19.3 10.9 AQ< Ai, A I 1 , trans-A3 1.5 AQ>> Ai, A I 1 0.7 A7,10 AQtlZ 0.3 AQ.12>> Ai,lO 0.1 ~ 7 , 1 0 , 1 3> AO11Z115d 4.9 1.6 7.3 AQ > A I 1 > A13 Ag 58.1 4.8 A9,lZ 8.2 AS,lZ >> A6,Q 22.3 A93 1 2 - 1 5 48.2 A9,1Z115 > All1l4.17d AQ,12716118-20:4,3.477. 0 , traces of ‘ In addition, the sample contained A 6 ~ l 1 -> A11-14-20:2,0.5%; A6s11.14-20:3,1.77. A6q11 20:O to 27:O. * In addition, the sample contained traces of 20:0, 22:0, and 24:O. c Percentages according to GLC peak areas. d Structure was verified by IR absorption of the terminal methylene group, at 908 and 988 cm-1. 0 ,
Table V. Anacardic and Fatty Acids from Cashew Shell Extract by GLC of TLC Fractions Anacardic acids Fatty acids Chain length: hydrogenated Chain length: Double bonds Z“ %* Double bonds Zc An13 :O 0.3 1.4 14:O An13:l An13:2 An15:O 0.9 61.3 16:O 2.3 An15:l 14.3 16: 1 0.8 An15:2 10.8 16:2 0.4 An15:3 38.4 16:3 2.0 An17:O 0.3 2.3 18:O 0.6 An17 : 1 0.8 18:l 8.6 An17:2 0.3 18:2 3.2 An17:3 1.o 18:3 0.4 Correction factors for accurate quantification by weight were determined from model mixtures of pure compounds. They were 1.1 for An15:2 and 1.8 for An15:3, relative to An15:1, with a flame ionization detector. * Peak areas of compounds not identified were approximately 2% of total areas (See Discussion). c Area percentages.
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DISCUSSION
Methylation of the phenolic hydroxyl group must be avoided in preparing anacardic acids for further investigations because demethylation of the methyl ethers meets with difficulty, On the contrary, complete methylation of anacardic acids is necessary for their analysis, in particular by GLC. Accordingly, different procedures are used for the preparative and for the analytical purposes. In preparations, advantage is taken of the distinct reactiv3% H2S04 where fatty acids are rapidly ities in CHaOH esterified but only traces of anacardic methyl esters are formed. Anacardic acids and fatty esters are then separated by chromatography on S O z (Figure 1). The fatty esters contained less than 1 of anacardic compounds. These are detectable as ether esters by rechromatographing the fraction of fatty esters after treatment with CH2N2under conditions of complete methylation of anacardic esters. Differential methylation of anacardic acids can be accomplished with CH2N2. Phenolic esters are the first product of methylation and they are of importance for separation of relatively large amounts by partition chromatography (Figure 2). When CHzNz is activated by a n appreciable
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ANALYTICAL CHEMISTRY
14117-
amount of CH30H, dimethyl anacardic ether esters are formed. They are the derivatives best suitable for G L C analyses (Table I1 and Figure 3) and the chromatograms d o not indicate any products of decomposition. Diazomethane was the only satisfactory reagent found for preparing the mono- or dimethyl derivatives of anacardic acids, in particular for preparing them selectively. Dimethyl sulfate (26) gave ether esters of anacardic acids but never in yields of more than 50%. Esterification catalyzed by BF3 in methanol was incomplete even after refluxing for 2 hours. Although this reagent esterifies fatty acids very rapidly (27), the distinction of anacardic from fatty acids is superior when esterifying with H2S04in methanol. Difficulties were also encountered in demethylation of anacardic ethers. Quantitative analyses of methoxy groups according t o Zeisel (28) gave low values with dimethyl anacardic ether esters even when reaction periods were extended to 80 minutes. Their identity as dimethyl derivatives was established, however, by mass spectra which showed molecule ions of the expected mass, and by nuclear magnetic resonance spectra which showed 3 protons attached to a benzene ring. These criteria rule out the possibility that the nucleus had been methylated under the rigorous conditions (29). TLC enabled evaluation of reaction products which were to be used for further separations. However, common fatty acids and anacardic acids (or the respective methyl esters) have virtually the same Rfvalues. Furthermore, one must be aware in interpretation of chromatograms that the R, of derivatives of anacardic acids are greatly influenced by the proximity of substituents at the phenyl ring. For example, etherified anacardic compounds migrate on SiO, more slowly than the phenolic compounds (Table I). More rapid procedures can be used for analyses and they are exemplified with cashew shell lipids. The quality of the shell extract permitted to forego saponification and the material was subjected directly to complete methylation. Ether esters and fatty esters were separated by TLC and the fractions were analyzed by GLC (Table V). Very minor peaks (
