Ind. Eng. Chem. Res. 1994,33, 1058-1061
1058
RESEARCH NOTES A Rich Source of Novel 9-Ketooctadec-cis-15-enoic Acid from Cassia absus Seed Oil and Its Possible Industrial Utilization Kallappa M. Hosamani Department of Chemistry, Janata Shikshana Samiti's (J.S.S.) College, Vidyagiri, Dharwad 580 004, India Cassia absus seed oil is a rich source of hitherto unknown keto acid (52.0%) and characterized as 9-ketooctadec-cis-15-enoicacid by UV, FTIR, 'H NMR, 13C NMR, MS, and GLC techniques and chemical transformations. I t also contains palmitic acid (12.35% 1, stearic acid (2.5 % ), arachidic acid
(1.4%), oleic acid (7.0%), and linoleic acid (24.8%).
Introduction
Table 1. Analytical Values of Cassia absus Seed Oil
The occurrence of keto fatty acids in natural seed oils is rare, although naturally occurring long-chain hydroxy acids are widely distributed in plants. Licania rigidas has attained commercial status as it contains an enormous amount of 4-ketoeleostearic acid (70-80%). This acid is popular for its drying properties, and hence it is used in paints and varnish industries. The new and interesting novel fatty acids present in high concentration in certain seed oils are being exploited for industrial utilization. These fatty acids of unusual structures are highly important to the production of oleochemicals. Seed oils containing novel fatty acids are industrially important as they are used in protective coatings, plastics, urethane derivatives, surfactants, dispersants, cosmetics, lubricants, a variety of synthetic intermediates, and stabilizers in plastic formulations, and in the preparation of other long-chain compounds. The ethoxylated hydroxy fatty acids containing seed oils are used as stabilizers of hydrophobic substances in industries such as perfumes in cosmetics. Epoxidized oils have some of the properties of a polymeric plasticizer but with some aging properties, e.g., soya and linseed oils. Cassia absus is an erect, annual plant 1-2 f t high, distributed throughout India. The leaves are bitter acrid and astringent. The seeds are used in the treatment of ophthalmia and skin infections and as a cathartic.2 The seeds are also used in syphilitic ulcers and leukoderma.' An exhaustive survey of the literature reveals that no work has been reported about Cassia absus seed oil. The present investigation now reported describes the new and rich source of a keto acid (9-ketooctadec-cis-15-enoicacid, 52.0%) along with the other normal fatty acids in Cassia absus seed oil.
oil content of air-dried seeds 6.0 % unsaponifiable matter 2.2% iodine value 99.0 saponification value 198.0 2,4-dinitrophenylhydrazine(2,4-DNPH)TLC testa +vea picric acid TLC test4 -vea Halphen tests -vea direct TLC test6 -vea infrared characteristic absorption bands chain carbonyl group 1705 cm-l 1740 cm-1 ester carbonyl group a +ve indicates positive response to the test; -ve indicates negative response to the test.
Experimental Section The UV spectra were taken on a Hitachi 270-30 instrument using 0.001% concentration of methanol as solvent. The infrared spectra were recorded on a Fourier transform infrared (FTIR) Bomem Michelson series instrument as liquid films. The 'H NMR spectra were recorded on a Varian VXR-300 (FT mode) spectrophotometer using CDCl3 as solvent. The chemical shifts were measured in parts per million downfield from internal 0888-588519412633-1058$04.50/0
Table 2. Component Fatty Acids of Cassia absus Seed Oil
fatty acids
%
palmitic stearic arachidic oleic linoleic keto acid (9-ketooctadec-cis-15-enoic acid)
12.3 2.5 1.4 7.0 24.8 52.0
standard tetramethylsilane at 6 = 0. The 13CNMR spectra were recorded at 75 MHz on a Varian VXR-300 (FTmode) spectrophotometer using CDCl3 as solvent (6 = 77.2). For mass spectral analysis and gas-liquid chromatographic (GLC) analysis, the samples were introduced as their methyl esters using direct-entry probe. The mass spectra were taken on a Finnigan Mat with PDP Micro Computer 810,at 70 eV with a source temperature 150 OC. The GLC analysis was carried out on a Perkin-Elmer Model Sigma unit using a 15% DEGS column (2 m X 3 mm) on Chromosorb, W,354-250 Fm (45-60 mesh). The temperatures at the injection port, detector port, and oven were 240, 240, and 190 OC, respectively. The nitrogen flow and chart speed were 30 mLlmin and 1 cm/min, respectively. The machine recorded directly the weight percent of individual peaks. The peaks were identified by comparing their retention times with those of standard reference samples under the same conditions. The melting points were recorded on a Thomas-Hoover capillary melting point apparatus. Analytical thin-layer chromatography (TLC) was performed on glass plates coated with 0.25-or 1.0-mm layers of silica gel "G" using 20 or 30% ether in hexane as the solvent system. Preparative TLC was effected on 20 cm 0 1994 American Chemical Society
Ind. Eng. Chem. Res., Vol. 33, No. 4,1994 1059 Scheme 1 bC-OCH3 -14
c------)
1 m/z
O=hCH3
59
tH2-COOCH3 -14
C H3 -C H2-8H-CH= C ti2
m/z 73
m/z 69
5 CH~-CH~-CH=CH-~H~ -14 m/z 6 9
~ H ~ - ( c H COOCH -14 2-m/z
CH~-CH~-CH=CH-CH~-~H~
tH2-(
-14
0
t
m/z 83
m/z 153
!
,
allyliy’cleavege
-.
181
CH2)3-COOCH m/z l a 5 T-14
e- ( CH2) 7-COOCH3
4
m/z 105 ,,’I a -cleavage / -, a -cleavage
I
Ii
I
T
I
ifi
‘
CH3iCH2-CH=CH-CH 21A ( C H 2 ) Z - C H 2I k H 21k 4 !C H 21LCH2-( CH2)5-COOCH3 I
1
9-Keto-octadec-~-15-~noicacid ;( M + ,= 310)
CH~-CH~-~H-CH=CH~
c H2C.C-
6-H m/z 43
\”I
4.
-( CH30H)
m/z 1 6 8 CH2-CHZ-C-( CH2 ) ,-COOCH3 m/z 2 1 3
I X 20 cm plates with a 1.0-mm layer of silica gel. When the plates were sprayed with dichlorofluorescein, the separated bands were clearly visible under UV light. The acids from silica were extracted with ether. The systematic solvent fractionation of Cassia absus seeds with light petroleum ether (bp 40-60 “C) extraction in a Soxhlet extractor for 24 hours yielded 6.0% yellow oil. The oil responded to the 2,4-dinitrophenylhydrazine(2,4DNPH) TLC test? thereby indicating the presence of keto fatty acids. The direct TLC test! picric acid TLC test? and Halphen tests revealed the absence of hydroxy, epoxy, and cyclopropenoidfatty acids, respectively. The infrared spectra of oil and ita methyl esters showed characteristic double carbonyl peaks a t 1740and 1705 cm-1 for the ester
m/z 199
carbonyl and chain carbonyl, respectively. The analytical values of the oil so obtained were determined according to the standard American Oil Chemists’ Society (AOCS) methods’ and are listed in Table 1. The saponification of oil was effected by stirring overnight at temperature (27OC)with 0.8NalcoholicKOH. The unsaponifiable matter (2.2%) was removed. The mixed fatty acids were separated into oxygenated and nonoxygenated fractions by preparative TLC. The yield of keto fatty acid was 52.0% after the allowance. A concentrate of pure keto fatty acid (51.9% ) was obtained by the chromatographic techniques. The nonoxygenated fraction (200mg) was esterified, and the normal methyl esters were recovered by the usual ether extraction. The
1060 Ind. Eng. Chem. Res., Vol. 33, No. 4, 1994
ether extract was dried over anhydrous sodium sulfate, and the solvent was removed in a stream of nitrogen. GLC analysis was carried out, and the results are summarized in Table 2. Results and Discussion The isolated unsaturated keto acid on hydrogenation" with 10% Pd/C in ethanol (5mL) gave 9-ketooctadecanoic acid, mp 43-44 "C. The unsaturated keto acid on oxidation12with KMn041NaI04 in tert-butyl alcohol gave azelaic acid, mp 106-107 "C @-bromophenacyl ester, mp 131-132 "C); adipic acid, mp 150-151 "C @-bromophenacyl ester, mp 154-155 "C); and propionic acid @bromophenacyl ester, mp 63-64 "C). Spectral Studies. The infrared spectra of unsaturated keto ester showed the characteristic double carbonyl peaks at 1740 and 1705 cm-l for ester carbonyl and chain carbonyl,respectively. The infrared spectrum also showed absorption bands at 715 and 1620 cm-l for a cis double bond. However, IR and UV spectra of a keto ester showed no evidence for a trans unsaturation or the presence of a conjugation. The 1H NMR spectrum of a methyl ester exhibited multiplet signals at 6 5.3 (2H, -CH=CH- and coupling constant 7.5 Hz), 3.5 (2H, -CH2COO-), and 2.3 (4H, -CH2C=CCH2-). The other singlet signals were observed at 6 3.7 (3H, -COOCH3), 3.3 (4H, -CH2COCHz-), and 1.3 (16H, chain-(CH2)8-). The triplet signal was observed at 6 0.89 (3H, terminal -CH3) for unsaturated ester. The lH NMR spectrum of saturated 9-ketooctadecanoic ester showed a triplet signal at 6 0.89 (3H, terminal -CH3). The singlet signals were observed at 6 1.3 (18H, chain -(CH&-), 3.3 (4H, -CH2COCHz-), and 3.7 (3H, -COOCH3). The multiplet signal was observed at 6 3.5 (2H, -CHzCOO-). The mass spectrum showed a fragment ion peak at rnlz 312. Carbon-13 nuclear magnetic resonance (l3C NMR) spectroscopy, on the other hand, has recently been shown to be a powerful physical technique for determining the structure, configuration, and confirmation of organic compounds. In particular, l3C NMR applications in the biosynthetic studies and in the structure elucidation of natural products have received attention. The chemical shifts of l3C NMR are influenced by a number of factors includinghybridization at a carbon, inductive effects, steric effects and anisotropic effects. The chemical shifts of 13C nuclei are expressed in units of parts per million (ppm) downfield from tetramethylsilane. The l3C NMR spectrum showed sharp singlet signals at 6 173 for carbonyl carbon, at 6 127 and 129 for sp2-hybridizedcarbon atoms, at 6 102 and 104 for the ester functional group, and at 6 13-51 for sp3-hybridizedcarbon atoms. The solvent CDC13 exhibited a signal at 6 77.2. To furnish conclusive support to the structure of keto ester, it was subjected to mass spectrometry. Its mass spectrum gave a fragment ion peak at rnlz 310 [M+l indicating a CIS chain with a keto group and a double bond. This fragment ion peak also corresponds to the molecular formula obtained by combustion data. Two significant fragment ions obtained as a result of a-cleavages on either side of a keto group were present at rnlz 185 (M+ - 157, M+ - 143, M+ - 129, M+ - 115, M+ - 101, M+ - 87, M+ - 73, and M+ - 59) and 153 (M+ - 125, M+ - 111, M+ - 97, M+ - 83, and M+ - 69). Another set of fragment ions resulting from P-cleavage on both sides of a keto group due to y-hydrogen abstraction (McLafferty rearrangementg)appeared at rnlz 200 and 168. These four fragment ions placed keto group at the Cg position. The position
of a double bond was determined by the allylic cleavage fragment ions which were observed at rnlz 295 and 69. The other important fragment ions were observed at mlz 199,168, and 43. The McLafferty rearrangement due to y-hydrogen abstraction of ester carbonyl group was observed at rnlz 74 (base peak). The fragmentation pattern is given in Scheme 1. McLafferty Rearrangement of 7-Hydrogen Abstraction of a Keto Group.
rnlz 168
where R = -(CHZ)&H=CHCH~CH~.
1
~CH,=CH(CH,)~H=CHCH~HJl
m/z200
where R' = -(CHz),COOCH3. McLafferty Rearrangement of 7-Hydrogen Abstraction of Ester Carbonyl Group.
.+/ H
+/
H
:0. I
w 0II.Y
mlz 74 (base peak)
where R = -(CHz)&O(CH2)&H=CHCH&Hs. Thus, the structure of the hitherto unknown keto acid isolated from Cassia absw seed oil has been characterized as 9-ketooctadec-cis-15-enoic acid. The seed oil of Cassia absus was found to contain a rich source of 9-ketooctadec-cis-15-enoicacid (52.0 % ) along with other normal fatty acids. Palmitic acid (12.3%) is a major component among the saturated fatty acids with smaller amounts of stearic acid (2.5%) and arachidic acid
Ind. Eng. Chem. Res., Vol. 33, No. 4,1994 1061
(1.4%). The unsaturated fatty acids are oleic (7.0%) and linoleic (24.8%). The results are summarized in Table 2.
Acknowledgment The author is grateful to Chairman Sri. D. Veerendra Heggade and Principal Prof. N. Vajrakumar, Janata Shikshana Samiti’s (J.S.S.) College, Vidyagiri, Dharwad4,for their encouragement. Literature Cited (1) AOCS. In Official and Tentative Methods of American Oil Chemists’ Society, 3rd ed.; Link, W. E., Ed.; American Oil Chemists’ Society: Champaign, IL, 1973, Da 15-48 and Da 16-48. ( 2 ) CSIR. In The Wealth of India: Raw Materials; Council of Scientific & Industrial Research New Delhi, 1969;Vol. 11,pp 93-95. (3) Davis, E. N.; Wallen, L. L.; Goodwin, J. C.; Rohwedder, W. K.; Rhodes, R.A. Microbial Hydration of cis-9-AlkenoicAcids. Lipids 1969,4,356-362. (4) Fioriti, J. A.; Sims, R. J. A spray reagent for the identification of epoxides on thin layer plates. J. Chromatogr. 1968,32,761-763. ( 5 ) Halphen, G. J. Pharm. Chim. 6th Ser. 1897,6,390.
(6) Hwamani, K. M. Terminalia chebula seed oil: A minor source of 12-hydroxy-octedec-ciss-9-enoic acid Natural products as a wurce for the food and agricultural industries. J. Sci. Food. Agric. 1994, 64, in press. (7) Kirtikar, K. R.; Basu, B. D. In The Indian Medicinul Plants; Lalit Mohan Basu: Allahabad, India, 1933; Vol. 11, pp 873-876. (8) Levy, G. C.; Lichter, R. L.; Nelson, G. L. In Carbon-13Nuclear Magnetic Resonance Spectroscopy, 2nd ed.; John Wiley & Sons: New York, 1980; pp 50-96 and 136-166. (9) McClogkey, J. A. In Topics in Lipid Chemistry;Gunatone, F. D., Ed.; Logos Press: London, 1970; Vol. I, Chapter 6, pp 369-370. (10) Swern, D., Ed. In Bailey’s Industrial Oil and Fat Products; John Wiley & Sons: New York, 1979; Vol. I, pp 42-48. (11) Vogel, A. I. In Text Book of Practical Organic Chemistry, 3rd ed.; Longmans Green & Co., Ltd.: London, 1956, pp 866 and 950. (12) von Rudloff, E. Oxidations of lipide in media containing organic solvents. Can J. Chem. 1966,34,1413-1418.
Received for review May 13, 1993 Revised manwrcript received October 22, 1993 Accepted December 16, 1993. 0 Abstract published in Advance ACS Abstracts, January 15, 1994.
