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Ind. Eng. Chem. Res. 1996, 35, 326-331
Unique Occurrence of Unusual Fatty Acids and Their Industrial Utilization Kallappa M. Hosamani Department of Chemistry, Janata Shikshana Samiti’s (J.S.S.) College, Vidyagiri, Dharwad 580 004, India
Ebenaceae plant family consists of 7 genera and more than 320 species. The present investigation describes the unique occurrence of hitherto unknown 9-keto-cis-13-octadecenoic acid (29.0%) and the cyclopropenoid fatty acids (malvalic acid 12.7% and sterculic acid 8.7%) in the seed oil of Diospyros melanoxylon as well as in the Ebenaceae plant family. Other normal fatty acids are also detected. The identification and characterization were based on FTIR, 1H NMR, 13C NMR, MS, and GLC techniques and chemical degradations. Introduction
Table 1. Analytical Values of Diospyros melanoxylon Seed Oila
In continuation of research (Hosamani, 1994b) on unusual fatty acids and their industrial utilization, Diospyros melanoxylon seed oil has been examined for its industrial utilization. This seed oil has been shown to be the most interesting finding in the unique occurrence of a hitherto unknown keto fatty acid and cyclopropenoid fatty acids along with other normal fatty acids. Diospyros melanoxylon is a moderate-sized tree, and all parts of the plant are used for various medicinal purposes. It is found throughout India (CSIR, 1952). An exhaustive survey of the literature reveals that no work has been reported about the seeds of Diospyros melanoxylon as well as in the Ebenaceae plant family for the unique occurrence of novel keto fatty acid and cyclopropenoid fatty acids. The present investigation describes 29.0% of novel 9-keto-cis-13-octadecenoic acid and 21.4% of cyclopropenoid fatty acids and their possible industrial utilization. The new and interesting unusual fatty acids present in high concentrations of certain seed oils are being exploited for industrial utilization. These fatty acids of unusual structures are highly important to the production of oleochemicals. The seed oil of Licania rigida (Swern, 1979) has attained commercial status as it contains an enormous amount of the 4-ketoeleostearic acid (70-80%). This acid is popular for its drying properties, and hence it is used as an ingredient of paints and varnishes. In the production of nylon-11, Castor oil is transesterified with methyl alcohol to form methyl ricinoleate and glycerol. The seed oils containing novel fatty acids are industrially important as they are used in protective coatings, plastics, urethane derivatives, surfactants, dispersants, cosmetics, lubricant additives, a variety of synthetic intermediates as stabilizers in plastic formulations, and the preparation of the other long-chain compounds. The ethoxylated hydroxy fatty acid containing seed oils are used as stabilizers of hydrophobic substances in industries such as perfumes and cosmetics. The epoxidized seed oils have some of the properties of a polymeric plasticizer but with some aging properties, e.g., soya and linseed oils. The cyclopropenoid fatty acids have a number of unusual properties including high dipole moment, high reactivity toward addition reactions driven by a 26 kcal/ mol reduction in strain energy upon conversion, a tendency to complex with metals, and ring-opening reactions which sometimes involve vinylcarbenes as reactive organic intermediates. The main impetus which led to the discovery of cyclopropenoid fatty acids came from the food and agriculture industries and the 0888-5885/96/2635-0326$12.00/0
oil content in seeds unsaponifiable matter iodine value saponification value Halphen test 2,4-dinitrophenylhydrazine (2,4-DNPH) TLC test picric acid TLC test Durbetaki titration at 55 °C
6.0% 2.1% 85.0 200.0 + + 21.6%
a + indicates a positive response to the test. - indicates a negative response to the test.
Table 2. Component Fatty Acids of Diospyros melanoxylon Seed Oil fatty acids
percentage
palmitic stearic oleic linoleic keto acid (9-keto-cis-13-octadecenoic acid) malvalic [7-(2-octylcyclopropenyl)heptanoic acid] sterculic [8-(2-octylcyclopropenyl)octanoic acid]
13.7 5.5 18.5 11.9 29.0 12.7 8.7
need to detect the adulteration of more expensive oils with cotton seed oil. Diospyros melanoxylon is a rich source of unusual fatty acids and shows sufficient promise for its exploitation for industrial utilization. The seed oils rich in lauric, myristic, palmitic, stearic, and oleic acids have been developed to replace part or all of the cocoa butter in chocolate-flavoured products. These fats fall into two basic catagories commonly known as cocoa butter substitutes and cocoa butter equivalents, e.g., coconut, palm, corn, and cotton seed oils. Cocoa powder production today is an important part of the cocoa and chocolate industry because of the increased consumption of chocolate-flavored products. Cocoa powder is the basic flavoring ingredient in the most chocolateflavored cookies, biscuits, cakes, and ice cream. It is also used extensively in the confectionary coatings for candy bars (Mark et al., 1978). Experimental Section The systematic solvent fractionation of air-dried seeds of Diospyros melanoxylon with light petroleum ether (bp 40-60 °C) extraction in a Soxhlet extractor for 24 h yielded 6.0% of reddish-yellow oil. The ether extracts were dried over anhydrous sodium sulfate, and the solvent was removed in vacuo at 40 °C. The analytical values of the oil so obtained were determined according to the standard American Oil Chemists’ Society (AOCS) Methods (AOCS, 1973) and are listed in Table 1. The saponification of the oil was effected by stirring overnight at room temperature (27 °C) with 0.8 N © 1996 American Chemical Society
Ind. Eng. Chem. Res., Vol. 35, No. 1, 1996 327
Figure 1.
Figure 2.
alcoholic KOH. The unsaponifiable matter was removed. The methyl esters were prepared by Fischer esterification, and direct TLC showed two distinct spots. The first spot is for oxygenated (keto) fractions, and the second spot is for the nonoxygenated fractions. The oxygenated and nonoxygenated fractions were separated by preparative TLC, and each was separated into its components of fatty acids. The yield of a keto acid was 29.0%. A concentrate of pure keto fatty acid (28.9%) was obtained by chromatographic techniques. The methyl esters of nonoxygenated fraction (200 mg)
were treated with 60 mL of absolute methanol saturated with silver nitrate (Schneider et al., 1968). The reaction was carried out at room temperature with constant stirring for 24 h. The cyclopropenoid derivatives (ketone and ether) and the normal methyl esters were recovered by the usual ether extraction. The ether extracts were dried over anhydrous sodium sulfate. The solvent was removed in a stream of nitrogen. The GLC analysis was carried out using Sterculia foetida methyl esters as a reference standard. The results are summarized in Table 2.
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Figure 3.
Figure 4.
Instrumentation. UV spectra of the methyl esters of the oil were taken on a Hatachi Model 270-30 instrument in methanol with 0.001% concentration. IR spectra were also recorded on a Fourier transform infrared (FTIR) Bomem Michelson series model instrument as liquid films. 1H NMR spectra were recorded from deuterochloroform (CDCl3) solvent on a Varian Model T-60 instrument. The chemical shifts (δ) were measured in parts per million (ppm) downfield from
internal TMS at δ 0. The 13C NMR spectra were recorded at 75 MHz on a Varian VXR-300 (FT mode) spectrophotometer using CDCl3 as the solvent (δ 77.2). Mass spectra were recorded on a Finnigan Mat with a PDP Micro Computer 810 at 70 eV with a source temperature of 150 °C. The quantitative examination of the methyl esters was carried out on a Perkin-Elmer Model Sigma Unit instrument stainless steel column using 15% diethylene glycol succinate (DEGS) on Chro-
Ind. Eng. Chem. Res., Vol. 35, No. 1, 1996 329 Scheme 1. Mass Spectral Fragmentation of 9-Keto-cis-octadec-13-enoate [M+ ) 310]
mosorb W 354-250 µm (45-60 mesh). Temperatures at the injection port, detector port, and oven were 240, 240, and 190 °C, respectively. The nitrogen flow and chart speed were 30 mL/min 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 similar conditions. The melting points were recorded on a Thomas-Hoover capillary melting point apparatus. Chromatography. Analytical TLC was performed on glass plates coated with 0.25 or 1.0 mm layers of silica gel G using 20 or 30% diethyl ether in hexane as the solvent system. The preparative TLC was effected on 20 × 20 cm plates with 1.0 mm layers of silica gel. When the plates were sprayed with dichlorofluoroscein, the separated bands were clearly visible under ultra-
violet (UV) light. The fatty acids from silica were extracted with ether. Results and Discussion The seed oil of Diospyros melanoxylon gave a positive Halphen test (Halphen, 1897) and 2,4-dinitrophenylhydrazine (2,4-DNPH) test (Davis et al., 1969), indicating the presence of cyclopropenoid and keto fatty acids, respectively. The direct TLC test (Hosamani, 1994a) and picric acid TLC test (Fioriti, 1968) revealed the absence of hydroxy and epoxy fatty acids, respectively. Infrared spectra of the oil and its methyl esters (Figures 1 and 2) showed the characteristic absorption bands at 1740, 1705, and 1010 cm-1 for the ester carbonyl, chain carbonyl, and cyclopropenoid functional groups, respectively. The infrared spectrum also showed character-
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Figure 5.
Scheme 2. Ether and Ketone Derivatives of Cyclopropenoid Fatty Acids (Malvalic Acid and Sterculic Acid)
istic absorption bands at 715 and 1620 cm-1, indicating the presence of a cis double bond. However, IR and UV spectra of the oil showed no evidence for trans unsaturation or the presence of conjugation. The methyl esters of the oil had a typical NMR singlet signal for the cyclopropene hydrogens at δ 0.72. The Durbetaki titration (Harris et al., 1963) of the oil at 55 °C indicated 21.6% of the total cyclopropenoid fatty acids. The unsaturated keto fatty acid on hydrogenation (Vogel, 1956) with 10% Pd/C in ethanol (5 mL) gave 9-ketooctadecanoic acid (mp 43-44 °C). The unsaturated keto acid on oxidation (von Rudloff, 1956) with KMnO4/NaIO4 in tert-butanol gave azelaic acid (mp 106-107 °C) and valeric acid (p-bromophenacyl ester; mp 75-76 °C). The GLC analysis of the resulting products as their methyl esters showed the cleavage fragments were azelaic acid and valeric acid. The infrared spectrum of unsaturated keto fatty ester showed characteristic absorption bands at 1740 and 1705 cm-1
for ester carbonyl and chain carbonyl, respectively. The infrared spectrum also showed absorption bands at 715 and 1620 cm-1 for a cis double bond. The 1H NMR spectrum (Figure 3) of unsaturated keto fatty ester exhibited multiplet signals at δ 5.8 (2H, -CHdCH-), 3.2 (2H, -CH2COO-), and 2.3 (4H, -CH2CdCCH2-). The singlet signals were observed at δ 3.8 (3H, -COOCH3), 1.2 (18H, chain -(CH2)9-), and 0.89 (3H, terminal -CH3). The 1H NMR spectrum of saturated keto fatty ester (9-keto-octadecanoate) showed signals at δ 0.89 (s, 3H, terminal -CH3), 3.2 (m, 2H, -CH2COO-), 1.22 (bs, 26H, shielded methylene protons -(CH2)13-), and 3.8 (s, 3H, -COOCH3). The 13C NMR spectrum (Figure 4) showed sharp singlet signals at δ 173 for carbonyl carbon, at δ 127 and 129 for sp2hybridized carbon atoms, at δ 102 and 104 for the ester functional group, and at δ 13-51 for sp3-hybridized carbon atoms. The solvent CDCl3 exhibited a signal at δ 77.2. The structure of an unsaturated keto fatty acid was further confirmed by mass spectrometry (Figure 5). The mass spectrum of a keto fatty ester showed a fragment ion peak at m/z 310 [M]+, indicating a C18 chain with keto group and unsaturation. This fragment ion peak also corresponded to a molecular formula obtained by combustion data. The two significant fragment ions obtained as a result of R-cleavages on either side of a keto group were present at m/z 185 (M+ - 157, M+ 143, M+ - 129, M+ - 115, M+ - 101, M+ - 87, M+ 73, and M+ - 59) and 153 (M+ - 125, M+ - 111, and M+ - 97). Another set of fragment ions resulted from β-cleavage on both sides of a keto group due to γ-hydrogen abstraction (McLafferty rearrangement) (McCloskey, 1970) appeared at m/z 200 and 168. These four fragment ions placed the keto group at the C9 position. The position of a double bond was determined by the allylic cleavage fragment ions which were observed at m/z 267 and 97. The other important fragment ions were observed at m/z 199, 168, and 43. The McLafferty rearrangement (McCloskey, 1970) due to γ-hydrogen abstraction for an ester carbonyl group was observed at m/z 74 (base peak). The fragmentation pattern is given in Scheme 1. The molecular ion peak of a saturated keto fatty ester was also observed at m/z 312. The McLafferty rearrangement (McCloskey, 1970) of γ-hydrogen abstraction, on either side of a keto group gave fragment ion peaks at m/z 168 and 200:
Ind. Eng. Chem. Res., Vol. 35, No. 1, 1996 331
where R ) -(CH2)3CHdCH(CH2)3CH3
The cyclopropenoid fatty acid characterization was determined by their ether and ketone derivatives as given in Scheme 2 (structures I-VIII). The cyclopropenoid fatty acids were converted into their methyl esters by transesterification and were converted into their ether and ketone derivatives with treatment of an excess of absolute methanol saturated with silver nitrate. These ether and ketone derivatives were submitted to the GLC analysis and compared with the derivatives of methyl esters of Sterculia foetida seed oil as a reference standard. Thus, the gas-liquid chromatographic analyses of ether and ketone derivatives of cyclopropenoid fatty acids have been characterized as 7-(2-octacyclopropen1-yl)heptanoic acid (malvalic acid) and 8-(2-octacyclopropen-1-yl)octanoic acid (sterculic acid). Literature Cited
where R ) -(CH2)7COOCH3. The McLafferty rearrangement (McCloskey, 1970) due to the γ-hydrogen abstraction of the ester carbonyl group was observed at m/z 74 (base peak):
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 16-48. CSIR. The Wealth of India: Raw Materials; Council of Scientific and Industrial Research: New Delhi, India, 1952; Vol. 3, pp 81-84. Davis, E. N.; Wallen, L. L.; Goodwin, J. C.; Rohwedder, W. K.; Rhodes, R. A. Microbial Hydration of cis-9-Alkenoic Acids. Lipids 1969, 4, 356-362. Fioriti, J. A.; Sims, R. J. A spray reagent for the identification of epoxides on thin layer plates. J. Chromatogr. 1968, 32, 761763. Halphen, G. J. Pharm. Chim., Ser. 6 1897, 6, 390. Harris, J. A.; Magne, F. C.; Skau, E. L. Cyclopropenoid fatty acidsII; A step-wise hydrogen bromide titration method for the cyclopropenoid and epoxy derivatives. J. Am. Oil Chem. Soc. 1963, 40, 718-720. Hosamani, K. M. Terminalia chebula seed oil: A minor source of 12-Hydroxy-octadec-cis-9-enoic acid: Natural products as a source for the food and agricultural industries. J. Sci. Food Agric. 1994a, 64, 275-277. Hosamani, K. M. A rich source of novel 9-keto-octadec-cis-15-enoic acid from Cassia absus seed oil and its possible industrial utilization. Ind. Eng. Chem. Res. 1994b, 33, 1058-1061. Mark, H. F.; Othmer, D. F.; Overberger, C. G.; Seaberg, G. T. In Encyclopedia of Chemical Technology: Chocolate and Cocoa, 3rd ed.; John Wiley & Sons: New York, 1978; pp 1-19. McCloskey, J. A. In Topics in Lipid Chemistry; Gunstone, F. D., Ed.; Logos Press: London, 1970; Vol. 1, Chapter 6, pp 369370. Schneider, E. L.; Loke, S. P.; Hopkins, D. T. Gas liquid chromatographic analysis of cyclopropenoid acids. J. Am. Oil Chem. Soc. 1968, 45, 585-590. Swern, D., Ed. Bailey’s Industrial Oil and Fat Products; John Wiley & Sons: New York, 1979; Vol. 1, pp 42-48. Vogel, A. I. In Textbook of Practical Organic Chemistry, 3rd ed.; Longmanns Green & Co., Ltd.: London, 1956; pp 866 and 950. von Rudloff, E. Oxidations of lipids in media containing organic solvents. Can. J. Chem. 1956, 34, 1413-1418.
Received for review September 22, 1994 Revised manuscript received March 20, 1995 Accepted September 22, 1995X
where R ) -(CH2)4C(O)(CH2)3CHdCH(CH2)5CH3. Thus, the structure of hitherto unknown keto acid isolated from Diospyros melanoxylon seed oil has been characterized as 9-keto-cis-13-octadecenoic acid.
IE940557L X Abstract published in Advance ACS Abstracts, December 1, 1995.
