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Analysis of Cassia marginata and Cassia corymbosa seed oils: An approach for the industrial utilization
Industrial Crops and Products 17 (2002) 57 /60 www.elsevier.com/locate/indcrop
Analysis of Cassia marginata and Cassia corymbosa seed oils: An approach for the industrial utilization Kallappa M. Hosamani , Raghavendra M. Sattigeri P.G. Department of Studies in Chemistry, Karnatak University, Pavate Nagar, Dharwad 580 003, India Accepted 1 August 2002
Abstract Cassia marginata , Roxb and Cassia corymbosa , Linn seed oils contain unusual fatty acids like vernolic acid (8.5 and 9.2%), malvalic acid (3.5 and 3.2%) and sterculic acid (2.6 and 2.8%). They also contain palmitic acid (17.3 and 17.2%), palmitoleic (trace and 7.4%), stearic acid (4.5 and 4.2%), oleic acid (14.2 and 14.8%) and linoleic acid (49.4 and 41.2%), respectively. These fatty acids were determined and characterized by UV, FTIR, 1H NMR, MS, GLC-techniques and chemical degradations. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Leguminosae; Cassia marginata ; Cassia corymbosa ; Seed oils; Unusual fatty acids: vernolic, malvalic, sterulic; Industrial utilization
1. Introduction The new and interesting unusual fatty acids present in high concentrations in certain seed oils are being exploited for the industrial utilization. These fatty acids of unusual structures are highly important to the production of oleochemicals. The seed oils containing unusual fatty acids are industrially important as they are used in protective coatings, plastics, urethane derivatives, surfactants, dispersants, cosmetics, plasticizers, lubricant additives, pharmaceuticals, soaps, detergents, textiles and a variety of synthetic intermediates.
Corresponding author. Fax: /91-836-747884 E-mail address: [email protected] Hosamani).
(K.M.
Seed oils containing epoxy fatty acids are potential interest as stabilizers in plastic formulations and in the preparation of other long chain compounds e.g. Vernonia anthelmintica seed oil. The seed oils containing cyclopropenoid fatty acids have attracted much attention owing to their biological effects in animals and their co-carcinogenic properties. Cassia marginata and Cassia corymbosa belong to the Leguminosae plant family which consists of 400 genera and more than 7000 species. Cassia marginata is a medium-sized tree (Cooke, 1967). Cassia corymbosa is a shrub (Bailey, 1927). The present investigation describes the occurrence of unusual fatty acids in Cassia marginata and Cassia corymbosa seed oils. An exhaustive survey of the literature reveals that no work has been reported
0926-6690/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 6 - 6 6 9 0 ( 0 2 ) 0 0 0 7 1 - 7
K.M. Hosamani, R.M. Sattigeri / Industrial Crops and Products 17 (2002) 57 /60
58
about component fatty acids in the seed oils of Cassia marginata and Cassia corymbosa .
2. Experimental section The air-dried seeds of Cassia marginata and Cassia corymbosa were extracted thoroughly with light petroleum ether (b.p. 40/60 8C) in a Soxhlet extractor for 24 h separately. The solvent was removed in a vacuum at 40 8C. The analytical values of the seed oils were determined according to the standard American Oil Chemists’ Society methods (Link, 1973) and are listed in the Table 1. The direct t.l.c. test and 2,4 dinitrophenyl hydrazine (2,4-DNPH) t.l.c. test (Davis et al., 1969) revealed the absence of hydroxy and keto fatty acids, respectively. However, the seed oils responded to the Halphen test and picric-acid t.l.c. test (Fioriti and Sims, 1968), thereby indicating the presence of cyclopropenoid and epoxy fatty acids, respectively. The Durbetaki titration (Harris et al., 1963) of seed oils at 3 and 55 8C temperatures indicated the presence of 8.5 and 9.3% of epoxy and 6.2 and 6.1% of cyclopropenoid fatty acids, respectively. The infrared spectra of methyl esters of seed oils showed the characteristic absorption bands at 820 and 1015 cm 1 for the epoxy and cyclopropenoid Table 1 Analytical values of Cassia seed oils Cassia marginata
Cassia corymbosa
Oil content in seeds (%) Non-saponifiable matter (%) Iodine value Saponification value Halphen test Picric-acid t.l.c. test 2,4-DNPH t.l.c. test Direct t.l.c. test
4.5 2.2 110.0 201.0 /vea /vea /veb /veb
4.8 2.4 85.0 200.0 /vea /vea /veb /veb
Durbetaki titration At 3 8C (%) At 55 8C (%)
8.6 6.2
9.3 6.1
a b
Indicates positive response to the test. Indicates negative response to the test.
functional groups, respectively. The presence of conjugation and trans unsaturation was ruled out on the basis of ultraviolet and infrared spectral analyses. The seed oils gave typical NMR singlet signals for the presence of cyclopropene hydrogens at d 0.72. 2.1. Acetolysis of epoxides A portion of the oils (20.0 g) were stirred overnight at room temperature (27 8C) with 80 ml of 10% sulphuric acid in 200 ml of glacial acetic acid (Wilson et al., 1961), separately. The acetolysed products were saponified with 0.8 N alcoholic KOH at room temperature (27 8C), separately. After careful acidification to pH 5 with 0.5 N sulphuric acid and the liberated mixed fatty acids were extracted with solvent ether. The ether extracts were washed thoroughly with distilled water until neutral and the solvent was removed in a stream of nitrogen. Direct t.l.c. of mixed fatty acids were revealed the presence of dihydroxy fatty acids and showed infrared absorption bands at 3400 cm 1 for the hydroxyl functional groups, respectively. 2.2. Isolation of dihydroxy fatty acids The separation of mixed fatty acids into oxygenated and non-oxygenated was accomplished by the preparative TLC techniques. These fatty acids were examined for the characterization of individual fatty acids. The yield of dihydroxy fatty acids are 8.5 and 9.2%, respectively after allowance. A concentrate of pure dihydroxy fatty acids (8.4 and 9.0%) were obtained by the column chromatographic techniques using neutral alumina. The fatty acids of methyl esters were prepared by the Fischer esterification. 2.3. Chemical degradations The unsaturated dihydroxy fatty acids on hydrogenation (Vogel, 1956) furnished 12,13-dihydroxy stearic acid (m.p. and mixed m.p. 96/97 8C). The unsaturated dihydroxy fatty acids were cleaved with permanganate-periodate (Von Rudloff, 1956). The GLC analyses of the resulting
K.M. Hosamani, R.M. Sattigeri / Industrial Crops and Products 17 (2002) 57 /60
59
products as their methyl esters showed the cleavage fragments were hexanoic acid (p-bromophenacyl ester, m.p. and mixed m.p 70/71 8C) and azelaic acid, m.p. and mixed m.p. 106/107 8C (p -bromophenacyl ester, 131 /132 8C), hence the cleavage points were at C9, C10 and C12, C13. 2.4. Preparation of cyclopropenoid fatty acids derivatives The methyl esters (200 mg) were treated with the absolute methanol (60 ml) saturated with the silver nitrate (Schneider et al., 1968) solution separately. The reactions were allowed to proceed at room temperature (27 8C) with stirring for 24 h and were extracted with the solvent ether. The ether extracts were dried over anhydrous sodium sulphate. The solvent was removed. The GLC analyses were carried out using Cassia siamea (Daulatabad et al., 1988) methyl esters as a reference standard.
3. Results and discussion The infrared spectra of dihydroxy fatty esters showed the characteristic absorption bands at 3400 cm 1 for the free hydroxyl and 1740 cm 1 for the ester carbonyl functional groups, respectively. The infrared spectrum also showed the absorption bands at 715 and 1620 cm 1 for the presence of a cis double bond. However, UV and IR spectra showed no evidence for the trans unsaturation or the presence of conjugation. The unsaturated dihydroxy fatty acids had the same Rf value as threo -12,13-dihydroxy oleic acid obtained by the acetolysis of Vernonia anthelmintica seed oil. The 1H NMR spectra of each unsaturated dihydroxy ester gave signals at d 5.4 (m, 2H, /CH /CH / and coupling constant J /7.5 Hz for a cis double bond), 5.0 (m, 2H, 2 / /CH /OH, which is disappeared on D2O addition), 3.6 (s, 3H, /COOCH3), 3.5 (m, 2H, 2 / /CH OH), 3.3 (m, 2H, /CH2COOCH3). 2.0 (m, 4H, /CH2CH /CH / CH2 /), 1.2 (s, 18H, /(CH2)9 /, shielded methylene protons) and 0.9 (t, 3H terminal /CH3). The saturated dihydroxy methyl ester did not exhibit
Scheme 1. Mass spectral fragmentation of methyl 12,13diacetate-octadec-cis -9-enoate.
signals at d 5.4. The other signals were observed at d 5.0 (m, 2H, 2 / /CHOH, which is disappeared on D2O addition), 3.6 (s, 3H, /COOCH3), 3.5 (m, 2H, 2 / /CHOH), 3.3 (m, 2H, /CH2COOCH3), 1.2 (s, 28H, /(CH2)14 /, shielded methylene protons) and 0.9 (s, 3H, terminal CH3). The mass spectra of each diacetyl derivative of unsaturated dihydroxy methyl esters showed small molecular ion peak at m /z 412. The allylic cleavage (m /z 197) established double bond at C9 and C10. The alpha cleavage on the either side of two acetate groups gave signals at m /z 341, 269, 215, 143 and placed the acetate groups at C12 and C13 (Scheme 1). The McLafferty rearrangement (McCloskey, 1970) due to the hydrogen abstraction of ester carbonyl group was observed at m /z 74 (Scheme 2). Thus the structure of isolated epoxy fatty acids as their dihydroxy fatty acids obtained from Cassia marginata and Cassia corymbosa seed oils have been characterized as 12,13-epoxy-octadeccis -9-enoic (vernolic) acid.
Scheme 2. McLafferty rearrangement due to g-hydrogen abstraction of ester carbonyl group.
K.M. Hosamani, R.M. Sattigeri / Industrial Crops and Products 17 (2002) 57 /60
60
Table 2 Component fatty acids of Cassia seed oils Fatty acids
Cassia marginata (%)
Cassia corymbosa (%)
Palmitic Palmitoleic Stearic Oleic Linoleic Vernolic Malvalic Sterculic
17.3 Trace 4.5 14.2 49.4 8.5 3.5 2.6
17.2 7.4 4.2 14.8 41.2 9.2 3.2 2.8
The cyclopropenoid fatty acids characterization was determined by their ether and ketone derivatives. These derivatives were submitted to the GLC analyses along with the other normal fatty esters. The individual peaks were recorded directly by the weight per cent and were identified by comparing their retention times with those of standard reference samples under the similar conditions. Thus, the GLC analyses of ether and ketone derivatives of cyclopropenoid fatty acids have been characterized as 7-(2-octacyclopropen-1yl)heptanoic acid (malvalic acid) and 8-(2-octacyclopropen-1-yl)octanoic acid (sterculic acid). The other normal fatty acids have been characterized as myristic, palmitic, palmitoleic, stearic, oleic and linoleic. The results are summarized in the Table 2.
Acknowledgements This research work is financially supported by the Department of Science and Technology (DST),
New Delhi. (Ref. No: No. SP/S1/G-15/99 dated 14-03-2000).
References Bailey, L.H., 1927. The Standard Encyclopedia of Horticulture, vol. I. McMillan & Co., Ltd, London, pp. 682 /683. Cooke, T., 1967. The Flora of Presidency of Bombay, vol. I. Botanical Survey of India, Calcutta, pp. 304 /456. Daulatabad, C.D., Hosamani, K.M., Mirajakar, A.M., 1988. Cassia siamea seed oil: a minor source of vernolic and cyclopropenoid fatty acids. J. Am. Oil Chem. Soc. 65, 952 / 953. Davis, E.N., Wallen, L.L., Goodwin, J.C., Rohwedder, W.K., Rhodes, R.A., 1969. Microbial hydration of cis -alkenoic acids. Lipids, 4, 356 /362. Fioriti, J.A., Sims, R.J., 1968. A spray reagent for the identification of epoxides on thin layer plates. J. Chromatogr. 32, 761 /763. Harris, J.A., Magne, F.C., Skau, E.L., 1963. Cyclopropenoid fatty acids-II: a step-wise hydrogen bromide titration method for the cyclopropenoid and epoxy derivatives. J. Am. Oil Chem. Soc. 40, 718 /720. Link, W.E. (Ed.), Official and Tentative Methods of the American Oil Chemists Society, third ed. (Methods Da 15 /48 and DA 16 /48). AOCS, Champaign, IL 1973. McCloskey, J.A., 1970. In: Gunstone, F.D. (Ed.), In Topics in Lipid Chemistry, vol. I. Logos Press, London, pp. 369 /370. Schneider, E.L., Loke, S.P., Hopkins, D.T., 1968. Gas liquid chromatographic analysis of cyclopropenoid acids. J. Am. Oil Chem. Soc. 45, 585 /590. Vogel, A.I., 1956. In a Text Book of Practical Organic Chemistry, third ed.. Longmanns, Green and Company, London, England, pp. 866, 950. Von Rudloff, E., 1956. Oxidation of lipids in media containing organic solvents. Can. J. Chem. 34, 1413 /1418. Wilson, T.L., Smith, C.R., Jr., Mikolajczak, K.L., 1961. Characterization of cyclopropenoid acids in selected seed oils. J. Am. Oil Chem. Soc. 38, 696 /698.
Analysis of Cassia marginata and Cassia corymbosa seed oils: An approach for the industrial utilization Kallappa M. Hosamani , Raghavendra M. Sattigeri P.G. Department of Studies in Chemistry, Karnatak University, Pavate Nagar, Dharwad 580 003, India Accepted 1 August 2002
Abstract Cassia marginata , Roxb and Cassia corymbosa , Linn seed oils contain unusual fatty acids like vernolic acid (8.5 and 9.2%), malvalic acid (3.5 and 3.2%) and sterculic acid (2.6 and 2.8%). They also contain palmitic acid (17.3 and 17.2%), palmitoleic (trace and 7.4%), stearic acid (4.5 and 4.2%), oleic acid (14.2 and 14.8%) and linoleic acid (49.4 and 41.2%), respectively. These fatty acids were determined and characterized by UV, FTIR, 1H NMR, MS, GLC-techniques and chemical degradations. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Leguminosae; Cassia marginata ; Cassia corymbosa ; Seed oils; Unusual fatty acids: vernolic, malvalic, sterulic; Industrial utilization
1. Introduction The new and interesting unusual fatty acids present in high concentrations in certain seed oils are being exploited for the industrial utilization. These fatty acids of unusual structures are highly important to the production of oleochemicals. The seed oils containing unusual fatty acids are industrially important as they are used in protective coatings, plastics, urethane derivatives, surfactants, dispersants, cosmetics, plasticizers, lubricant additives, pharmaceuticals, soaps, detergents, textiles and a variety of synthetic intermediates.
Corresponding author. Fax: /91-836-747884 E-mail address: [email protected] Hosamani).
(K.M.
Seed oils containing epoxy fatty acids are potential interest as stabilizers in plastic formulations and in the preparation of other long chain compounds e.g. Vernonia anthelmintica seed oil. The seed oils containing cyclopropenoid fatty acids have attracted much attention owing to their biological effects in animals and their co-carcinogenic properties. Cassia marginata and Cassia corymbosa belong to the Leguminosae plant family which consists of 400 genera and more than 7000 species. Cassia marginata is a medium-sized tree (Cooke, 1967). Cassia corymbosa is a shrub (Bailey, 1927). The present investigation describes the occurrence of unusual fatty acids in Cassia marginata and Cassia corymbosa seed oils. An exhaustive survey of the literature reveals that no work has been reported
0926-6690/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 6 - 6 6 9 0 ( 0 2 ) 0 0 0 7 1 - 7
K.M. Hosamani, R.M. Sattigeri / Industrial Crops and Products 17 (2002) 57 /60
58
about component fatty acids in the seed oils of Cassia marginata and Cassia corymbosa .
2. Experimental section The air-dried seeds of Cassia marginata and Cassia corymbosa were extracted thoroughly with light petroleum ether (b.p. 40/60 8C) in a Soxhlet extractor for 24 h separately. The solvent was removed in a vacuum at 40 8C. The analytical values of the seed oils were determined according to the standard American Oil Chemists’ Society methods (Link, 1973) and are listed in the Table 1. The direct t.l.c. test and 2,4 dinitrophenyl hydrazine (2,4-DNPH) t.l.c. test (Davis et al., 1969) revealed the absence of hydroxy and keto fatty acids, respectively. However, the seed oils responded to the Halphen test and picric-acid t.l.c. test (Fioriti and Sims, 1968), thereby indicating the presence of cyclopropenoid and epoxy fatty acids, respectively. The Durbetaki titration (Harris et al., 1963) of seed oils at 3 and 55 8C temperatures indicated the presence of 8.5 and 9.3% of epoxy and 6.2 and 6.1% of cyclopropenoid fatty acids, respectively. The infrared spectra of methyl esters of seed oils showed the characteristic absorption bands at 820 and 1015 cm 1 for the epoxy and cyclopropenoid Table 1 Analytical values of Cassia seed oils Cassia marginata
Cassia corymbosa
Oil content in seeds (%) Non-saponifiable matter (%) Iodine value Saponification value Halphen test Picric-acid t.l.c. test 2,4-DNPH t.l.c. test Direct t.l.c. test
4.5 2.2 110.0 201.0 /vea /vea /veb /veb
4.8 2.4 85.0 200.0 /vea /vea /veb /veb
Durbetaki titration At 3 8C (%) At 55 8C (%)
8.6 6.2
9.3 6.1
a b
Indicates positive response to the test. Indicates negative response to the test.
functional groups, respectively. The presence of conjugation and trans unsaturation was ruled out on the basis of ultraviolet and infrared spectral analyses. The seed oils gave typical NMR singlet signals for the presence of cyclopropene hydrogens at d 0.72. 2.1. Acetolysis of epoxides A portion of the oils (20.0 g) were stirred overnight at room temperature (27 8C) with 80 ml of 10% sulphuric acid in 200 ml of glacial acetic acid (Wilson et al., 1961), separately. The acetolysed products were saponified with 0.8 N alcoholic KOH at room temperature (27 8C), separately. After careful acidification to pH 5 with 0.5 N sulphuric acid and the liberated mixed fatty acids were extracted with solvent ether. The ether extracts were washed thoroughly with distilled water until neutral and the solvent was removed in a stream of nitrogen. Direct t.l.c. of mixed fatty acids were revealed the presence of dihydroxy fatty acids and showed infrared absorption bands at 3400 cm 1 for the hydroxyl functional groups, respectively. 2.2. Isolation of dihydroxy fatty acids The separation of mixed fatty acids into oxygenated and non-oxygenated was accomplished by the preparative TLC techniques. These fatty acids were examined for the characterization of individual fatty acids. The yield of dihydroxy fatty acids are 8.5 and 9.2%, respectively after allowance. A concentrate of pure dihydroxy fatty acids (8.4 and 9.0%) were obtained by the column chromatographic techniques using neutral alumina. The fatty acids of methyl esters were prepared by the Fischer esterification. 2.3. Chemical degradations The unsaturated dihydroxy fatty acids on hydrogenation (Vogel, 1956) furnished 12,13-dihydroxy stearic acid (m.p. and mixed m.p. 96/97 8C). The unsaturated dihydroxy fatty acids were cleaved with permanganate-periodate (Von Rudloff, 1956). The GLC analyses of the resulting
K.M. Hosamani, R.M. Sattigeri / Industrial Crops and Products 17 (2002) 57 /60
59
products as their methyl esters showed the cleavage fragments were hexanoic acid (p-bromophenacyl ester, m.p. and mixed m.p 70/71 8C) and azelaic acid, m.p. and mixed m.p. 106/107 8C (p -bromophenacyl ester, 131 /132 8C), hence the cleavage points were at C9, C10 and C12, C13. 2.4. Preparation of cyclopropenoid fatty acids derivatives The methyl esters (200 mg) were treated with the absolute methanol (60 ml) saturated with the silver nitrate (Schneider et al., 1968) solution separately. The reactions were allowed to proceed at room temperature (27 8C) with stirring for 24 h and were extracted with the solvent ether. The ether extracts were dried over anhydrous sodium sulphate. The solvent was removed. The GLC analyses were carried out using Cassia siamea (Daulatabad et al., 1988) methyl esters as a reference standard.
3. Results and discussion The infrared spectra of dihydroxy fatty esters showed the characteristic absorption bands at 3400 cm 1 for the free hydroxyl and 1740 cm 1 for the ester carbonyl functional groups, respectively. The infrared spectrum also showed the absorption bands at 715 and 1620 cm 1 for the presence of a cis double bond. However, UV and IR spectra showed no evidence for the trans unsaturation or the presence of conjugation. The unsaturated dihydroxy fatty acids had the same Rf value as threo -12,13-dihydroxy oleic acid obtained by the acetolysis of Vernonia anthelmintica seed oil. The 1H NMR spectra of each unsaturated dihydroxy ester gave signals at d 5.4 (m, 2H, /CH /CH / and coupling constant J /7.5 Hz for a cis double bond), 5.0 (m, 2H, 2 / /CH /OH, which is disappeared on D2O addition), 3.6 (s, 3H, /COOCH3), 3.5 (m, 2H, 2 / /CH OH), 3.3 (m, 2H, /CH2COOCH3). 2.0 (m, 4H, /CH2CH /CH / CH2 /), 1.2 (s, 18H, /(CH2)9 /, shielded methylene protons) and 0.9 (t, 3H terminal /CH3). The saturated dihydroxy methyl ester did not exhibit
Scheme 1. Mass spectral fragmentation of methyl 12,13diacetate-octadec-cis -9-enoate.
signals at d 5.4. The other signals were observed at d 5.0 (m, 2H, 2 / /CHOH, which is disappeared on D2O addition), 3.6 (s, 3H, /COOCH3), 3.5 (m, 2H, 2 / /CHOH), 3.3 (m, 2H, /CH2COOCH3), 1.2 (s, 28H, /(CH2)14 /, shielded methylene protons) and 0.9 (s, 3H, terminal CH3). The mass spectra of each diacetyl derivative of unsaturated dihydroxy methyl esters showed small molecular ion peak at m /z 412. The allylic cleavage (m /z 197) established double bond at C9 and C10. The alpha cleavage on the either side of two acetate groups gave signals at m /z 341, 269, 215, 143 and placed the acetate groups at C12 and C13 (Scheme 1). The McLafferty rearrangement (McCloskey, 1970) due to the hydrogen abstraction of ester carbonyl group was observed at m /z 74 (Scheme 2). Thus the structure of isolated epoxy fatty acids as their dihydroxy fatty acids obtained from Cassia marginata and Cassia corymbosa seed oils have been characterized as 12,13-epoxy-octadeccis -9-enoic (vernolic) acid.
Scheme 2. McLafferty rearrangement due to g-hydrogen abstraction of ester carbonyl group.
K.M. Hosamani, R.M. Sattigeri / Industrial Crops and Products 17 (2002) 57 /60
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Table 2 Component fatty acids of Cassia seed oils Fatty acids
Cassia marginata (%)
Cassia corymbosa (%)
Palmitic Palmitoleic Stearic Oleic Linoleic Vernolic Malvalic Sterculic
17.3 Trace 4.5 14.2 49.4 8.5 3.5 2.6
17.2 7.4 4.2 14.8 41.2 9.2 3.2 2.8
The cyclopropenoid fatty acids characterization was determined by their ether and ketone derivatives. These derivatives were submitted to the GLC analyses along with the other normal fatty esters. The individual peaks were recorded directly by the weight per cent and were identified by comparing their retention times with those of standard reference samples under the similar conditions. Thus, the GLC analyses of ether and ketone derivatives of cyclopropenoid fatty acids have been characterized as 7-(2-octacyclopropen-1yl)heptanoic acid (malvalic acid) and 8-(2-octacyclopropen-1-yl)octanoic acid (sterculic acid). The other normal fatty acids have been characterized as myristic, palmitic, palmitoleic, stearic, oleic and linoleic. The results are summarized in the Table 2.
Acknowledgements This research work is financially supported by the Department of Science and Technology (DST),
New Delhi. (Ref. No: No. SP/S1/G-15/99 dated 14-03-2000).
References Bailey, L.H., 1927. The Standard Encyclopedia of Horticulture, vol. I. McMillan & Co., Ltd, London, pp. 682 /683. Cooke, T., 1967. The Flora of Presidency of Bombay, vol. I. Botanical Survey of India, Calcutta, pp. 304 /456. Daulatabad, C.D., Hosamani, K.M., Mirajakar, A.M., 1988. Cassia siamea seed oil: a minor source of vernolic and cyclopropenoid fatty acids. J. Am. Oil Chem. Soc. 65, 952 / 953. Davis, E.N., Wallen, L.L., Goodwin, J.C., Rohwedder, W.K., Rhodes, R.A., 1969. Microbial hydration of cis -alkenoic acids. Lipids, 4, 356 /362. Fioriti, J.A., Sims, R.J., 1968. A spray reagent for the identification of epoxides on thin layer plates. J. Chromatogr. 32, 761 /763. Harris, J.A., Magne, F.C., Skau, E.L., 1963. Cyclopropenoid fatty acids-II: a step-wise hydrogen bromide titration method for the cyclopropenoid and epoxy derivatives. J. Am. Oil Chem. Soc. 40, 718 /720. Link, W.E. (Ed.), Official and Tentative Methods of the American Oil Chemists Society, third ed. (Methods Da 15 /48 and DA 16 /48). AOCS, Champaign, IL 1973. McCloskey, J.A., 1970. In: Gunstone, F.D. (Ed.), In Topics in Lipid Chemistry, vol. I. Logos Press, London, pp. 369 /370. Schneider, E.L., Loke, S.P., Hopkins, D.T., 1968. Gas liquid chromatographic analysis of cyclopropenoid acids. J. Am. Oil Chem. Soc. 45, 585 /590. Vogel, A.I., 1956. In a Text Book of Practical Organic Chemistry, third ed.. Longmanns, Green and Company, London, England, pp. 866, 950. Von Rudloff, E., 1956. Oxidation of lipids in media containing organic solvents. Can. J. Chem. 34, 1413 /1418. Wilson, T.L., Smith, C.R., Jr., Mikolajczak, K.L., 1961. Characterization of cyclopropenoid acids in selected seed oils. J. Am. Oil Chem. Soc. 38, 696 /698.