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A comparative study of sterols in oil seeds of Solanum species
Phytoc/wmistry, Vol. 35, No. 1, pp. 163-167, 1994 Printed in Great Britain.
A COMPARATIVE
0031~%22/94 S6.00+0.00 Q 1993 Pcrgamon Press Ltd
STUDY
OF STEROLS SPECIES
IN OIL SEEDS OF SOLANUM
J. A. ZYGADLO C&&a de Quimica Organica, Facultad de Ciencias Exactas, Fisicas y Naturales (Instituto Multidisciplinario de Biologia Vegetal CONICET-UNC)
- Av. Velez Sarsfield 2!W5000 CORDOBA, Argentina (Receiued in revised form 10 June 1993)
Key Word Index--Solanwn;
Solanaceae; sterols; chemosystematic.
Abstract-
Cholesterol, campesterol, sitosterol, stigmasterol, As-avenasterol, A’-avenasterol, A’-stigmastenol and a-spinasterol, were identified in the seeds of 13 species of Solarnun. The chemosystematic significance of sterols in these species is discussed.
INTRODUCTION
Sterols have contributed to the assessment of taxonomic schemes in algae [l, 23, fungi [2, 33, ferns [4, 51, mosses [6] and higher plants [7-l 11. The value of sterol patterns in deducing systematic relationships among Solanum species is not well known. Bergmann in his 1953 review of the plant sterols [12], listed several groups of species according to the sterol structure; some species of Solanum show a predominance of AS-sterols or A*(9)-sterols. According to Itoh et al. [13] the Solunaceae seeds that have been examined can be arranged in two groups based on the compositions of the seed sterols; Solanum is included in the group containing sitosterol as the predominant component. For this reason, it seems pertinent to analyse additional species of Solanum to determine the possible chemotaxonomic value of sterols in this genus.
RESULTS AND DISCUSSION
Since the validity of comparing sterol composition for taxonomic purposes depends on the stability of the patterns under varying geographical and ecological conditions, seeds from several geographically separated populations of 13 species were analysed (Table 1). The sterols identified were cholesterol, campesterol, sitosterol, stigmasterol, r-spinasterol, A’-avenasterol, A’stigmastenol and A’-avenasterol (Table 2). Cholesterol (RR, 0.61) had a fragmentation pattern which agreed with that of a reference specimen of cholesterol and also that reported by Knights [ 143, molecular ion [M] + at m/z 386 (calculated for C,,Hh60, M 386) with other principal ions at m/z 371, 368, 353, 301, 275, 273 and 247. A’Stigmastenol (RR, 1.18) had [M] + at m/z 414 accompanied by the ionsm/z 399 [M-Me]+, 396 [M-H,O]+ and 381 [M -Me-HzO]‘. Peaks were also found at m/z 273 [M-side chain]+ and 255 [M-side chain - H,O]+. Moreover, peaks were observed at m/z 246 [M
- side chain - 273 + and 229 [M-side chain-27 - H,O]+. These fragmentation patterns agree with those given by A’-sterols [14-161. A’-Avenasterol (RR, 1.32) showed [Ml+ at m/z 412 with other abundant ions at m/z 397, 379, 314, 296, 279, 273 and 253. a-Spinasterol (RR, 1.03) showed [M] + at m/z 412 (calculated for Cz9H4z0, M 412) with other abundant ions at m/z 397, 394, 379, 369, 351, 273, 271, 255, 246, 231, 229 and 213. The fragmentations giving the ions at m/z 369 [M - C,H,]+ and 351 CM-C3H7-HzO]+ involve the isopropyl group of the side chain and appear to be characteristic for A22-sterols [14,16]. The fragmentation pattern of the mass spectrum of the sterol with RR, 1.03 was basically similar to that of authentic a-spinasterol (24ethylcholesta-722dienol). As-Avenasterol (RR, 1.12) gave [M]’ at m/z 412 (calculated for C,,H,,O, M 412) with other ions at m/z 397,394,379,314,296,281,273 and 271. Campesterol (RR, 0.81); showed [Ml’ at m/z 400, with other abundant ions at m/z 382, 255 and 213. Stigmasterol (RR, 0.88) had m/z 412 [M] + accompanied with ions at m/z 394,379,351,315,300,271,255,229 and 213. Sitosterol gave m/z 414 [Ml’, with other abundant ions at m/z 399,396, 381,324,303,273,255,231,213, 145 and 107 (RR, 1.00). The literature concerning A7-avenasterol and A’-stigmastenol in species of Solanaceae is contradictory [13,15,1fl; for this reason it is interesting to point out that according to this study these A’-sterols were identified in four species of Solanum (Table 2). The results showed a clear separation of three sterol patterns in Solanum (Table 2). Although five sterols (cholesterol, campesterol, stigmasterol, sitosterol and a-spinasterol) appear in the three groups, the first one (with S. palitans and S. incisum) has only the five common sterols already mentioned; As-avenasterol is the additional sterol that characterizes the second group (with S. atriplicijXwn, S. sublobatum, S. chenopodioides, S. riparium, S. pseudocapsicum, S. dijlorum and S. argentinum). 163
J. A. ZYGADW
164
Table I. So/unum populations sampled
Abbreviations
1600 1700 1540 800 1460 2a.m 1200 1200
86 J. A. Zygadlo 74 J. A. Zygadlo 75 J. A. Zygadlo 143 J. A. Zygadlo 104 J. A. Zygadlo 105 J. A. Zygadlo 139 J. A. Zygadlo 140 J. A. Zygadlo 141 J. A. Zygadlo
SPI SP2 SP3 SI4 SI5 S16 S17 S18 S19
600
21 J. A. Zygadlo
SAiO
Cadillal (lbmnin) Agua de Oro (Cbrdoba) Las Tapias (C6rdoba) Ledeama (Jujuy) Rosario de la Frontera (Salta) Cadillal (Tucum&l) Agua de Oro (C6rdoba) L.a Quebrada (C6rdoba) m Mallin (Cbrdoba) 21 Colanchanga (C6rdoba) 22 La Falda (C6rdoba) 23 Agua de Oro (Cbrdoba)
1200 1200 600 600 1200 1200 1200 600 700 900 700 930 600
113 J. A. Zygadlo 114 J. A. Zygadlo 26 J. A. Zygadlo 133 J. A. Zygadlo 162 J. A. Zygadlo 163 J. A. Zygadlo 53 J. A. Zygadlo 40 J. A. Zygadlo 107 J. A. Zygadlo 119 J. A. Zygadlo 108 J. A. Zygadlo 138 J. A. Zygadlo 24 J. A. Zygadlo
SSll SSl2 SC13 SC14 SRl5 SRI6 SR17 SPsl8 SPsl9 SPs20 SPs21 sPs22 SW23
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
800 500 700 Iso0 3ooo 530 860 600 1200 1200 700 800 500 1700 3wo 700 500 530 300 600 600 760 800 800 800 500 500 750 600 890 500
4458 R. Subils 4413 R. Subils 81 J. A. Zygadlo 91 J. A. Zygadlo 93 J. A. Zygadlo 100 J. A. Zygadlo 1 J. A. Zygadlo 12 J. A. Zygadlo 17 J. A. Zygadlo 1I5 J. A. Zygadlo 110 J. A. Zygadlo 4457 R. Subils 47 J. A. Zygadlo 82 J. A. Zygadlo 89 J. A. Zygadlo 95 J. A. Zygadlo 4414 R. Subils 25 385 A. T. Hunziker 4463 R. Subils 18 J. A. Zygadlo 3 J. A. Zygadlo 16 J. A. Zygadlo 25 395 A. T. Hunziker 25 396 A. T. Hunziker 25403 A. T. Hunziker 4412 R. Subils 25 386 A. T. Hunziker 42 J. A. Zygadlo 1I7 J. A. Zygadlo I1 8 J. A. Zygadlo 97 J. A. Zygadlo
SD24 SD25 SD26 SD27 SD28 SD29 SY30 SY31 SY32 SY33 SY34 SL35 SL36 SL37 SL38 SL39 SE40 SE41 SE42 SE43 SE44 SE45 SE46 SE47 SE48 SJ49 SJSO SJ5l SJ52 SJ53 SJ54
Populations
S. palitans MORTON
1 2 3 4 5 6 7 8 9
S.
in&urn
GRISEB.
S. atriplijiijlium GILLIES ex MEYEN S. sublobatum WILLD. ex ROEM et SCHULT. S. chenopodioides LAM. S. riparium PERSOON
S. pseudocapsicum L.
S. diporum VELL. S. argentinwn BITTER et LILLO
S. sisymbriijoliwn
LAM.
S. palinacanthum
DUNAL
S. elaeagnifolium CAV.
S. juwnale THELLUNG
Altitude (m.s.n.m)
Collection data
Speciea
Chorrillos (Salta) Quijano (Salta) Yacones (Salta) Los Gigantes (C6rdoba) Bosque Alegre (Cbrdoba) Copina (C6rdoba) Cerro Horcosum (Cbrdoba) Puesto Guz m8n (Cbrdoba) Los Pozos (C6rdoba)
10 Agua de Oro (C6rdoba)
11 Cadillal (Tucumzin)
12 13 14 I5 16 17 18 19
El Alto (Catamarca) Frias (Santiago del Estero) El Zapallar (Salta) Alemania (Salta) Chorrillos (Salta) Soto (C%rdoba) Flor Serrana (C6rdoba) Santa Rosa de Calamuchita (Cbrdoba) Mina Clavero (C6rdoba) Cadillal (TucumBn) La Quebrada (C6rdoba) El Alto (Catamarca) Alta Gracia (Cbrdoba) Yacones (Salta) Chorrillos (Salta) El Zapallar (Salta) Frias (Santiago del Eatero) Soto (Cbrdoba) Embarcadero 977 (Catamarca) Carlos Paz (C6rdoba) Las Tapias (C6rdoba) Santa Rosa de Calamuchita (C6rdoba) Ruta hacia Patquia (La Rioja) Ruta hacia Patquia (La Rioja) Ruta hacia Patquia (La Rioja) Frias (Santiago de1 Eatero) Pichanas (C6rdoba) Villa General Belgrano (C6rdoba) Orilla de1 Lago San Roque (C6rdoba) Cosquin (C6rdoba) Pichanas (C6rdoba)
3ooo
Oil seals of Sohmumspecies Table 2. Sterol compositions of seed oil from Sohm
SpXiol S. p&tans SPl SP2 SP3 S. inciswn S14 SIS SI6 s17 S18 s19 S. otriplicifolium SAlO S. sublobatum SSll SSl2 S. cherwpodioides SC13 SC14 S. riparium SR15 SR16 SR17 S. pseudocapshm SPsl8 SPsl9 SPs20 sPs21 sPs22 S. dijlorum SW23 S. argentinum SAr24 SAr25 SAr26 SAr27 SAr28 SAr29 S. sisymhriffolium SY30 SY31 SY32 SY33 SY34 S. palinacanthum SL35 SL36 SL37 SL38 SL39 S. el~agnfolium SE40 SE41 SE42 SE43 SE44 SE45 SE46 SE47 SE48
I*
2
3
4
Sterols 5
165 species
6
3.2 2.9 2.0
19.5 18.8 17.0
TR. TR. TR.
76.0 77.1 79.7
I.0 0.9 1.0
3.2 2.0 3.2 1.5 TR. 1.0
20.0 17.8 21.0 15.0 22.3 19.5
TR. TR. TR. TR. TR. TR.
76.8 80.1 74.8 828 77.8 79.6
2.2 2.2 3.2 29 2.5 2.2
7.7
8.8
6.8
76.1
TR.
TR.
1.9 6.9
82.0 80.3
TR. TR.
5.2 4.8
10.7 5.7
TR. TR.
7
8
1.7 TR
7.1 TR.
10.1 14.3
78.2 824
TR. TR.
23 2.0
2.0 3.0 1.5
7.0 6.0 5.0
10.1 9.0 8.1
78.0 78.9 80.7
TR. TR. TR.
TR. 0.6 5.0
20.0 15.5 10.0 21.5 19.8
11.2 6.7 16.2 10.2 11.4
7.0 6.0 3.0 6.5 6.7
46.6 55.7 56.7 50.6 46.9
TR. TR. TR. TR. TR.
6.0 6.8 4.7 20 6.0
9.2
10.0
19.2
65.6
1.0
6.0
6.0 TR. 7.8 6.0 4.3 6.0
17.0 12.0 14.0 18.8 15.5 17.0
6.1 6.9 5.0 5.3 5.0 3.1
620 71.9 61.8 64.9 720 73.2
TR TR TR. TR. TR. TR.
8.4 8.5 10.7 5.3 3.0 TR.
2.6 1.2 3.3 1.5 TR.
4.5 29 5.7 3.1 4.1
8.0 3.3 11.3 7.2 10.3
70.7 87.9 66.4 81.1 65.0
1.0 0.6 21 TR. 2.3
11.2 3.5 9.3 6.3 121
0.7 0.6 0.7 TR. 3.4
13 TR. 1.1 TR. 27
1.0 2.0 0.9 1.3 0.8
3.9 4.0 1.5 5.2 6.0
3.5 4.0 5.1 3.7 3.5
m.0 720 79.3 84.1 74.1
TR. TR 2.1 0.6 TR.
16.0 l.l.7 7.8 2.1 123
25 3.1 1.1 TR. 3.1
1.5 28 21 28 TR.
3.0 29 1.6 3.2 4.0 TR. 8.1 6.9 5.1
7.0 5.8 5.8 8.6 6.8 3.6 3.3 7.0 3.0
0.8 7.3 9.1 5.4 6.3 25 10.2 8.1 5.7
77.8 78.4 80.0 78.4 77.8 86.9 75.8 74.8 80.3
0.8 TR. 1.1 0.9 1.3 2.1 0.6 0.8 2.0
TR 0.8 0.9 TR 1.5 3.0 0.9 TR 21
1.0 1.2
3.0 3.4 0.7 3.2 2.3 TR TR 1.9 TR.
E. TR. 1.7 1.1 TR. 1.6
J.
166
A. ZYGADLO
Table 2. Continued Sterols Species
I*
2
3
4
5
6
7
8
0.8 2.5 TR.
1.2 3.1
1.0 TR. 1.3
1.0 2.1 1.7
S. juuenale
SJ49 SJ50 SJSl SJ52 SJ53
4.0 3.9 6.1 3.7 10.2
4.0 5.1 4.6 4.8 2.3
8.5 1.2 8.0 6.6 3.3
70.1 77.2 73.1 13.5 73.1
1.0 TR. 1.0 1.5 0.7
SJ54
7.7
8.1
5.8
70.1
2.9
‘1 Cholesterol: 8 A’-avenasterol;
2 camoesterol: 3 stiemasterol; TR., tracks
The third group (with S. sisymbriijolium, S. palinacanthum, S. elaeagni$olium and S. juvenale) is characterized by having A’-avenasterol and A’-stigmastenol as an addition to the sterols that are present in the first and second groups. However, more extensive sampling is needed in this large genus before comparative sterol data can be applied taxonomically or phylogenetically.
7.7 TR. 8.0 7.9 8.1 2.4
4 sitosterol; 6 As-avenasterol;
7 A’-stigmastenol;
GC used an OV-17 (2%) column, carrier gas Nz at 30 ml min-‘, column temp. programmed 180”-290”, 10” min-‘, FID. GC-MS used an OV-17 column (3%), 200”-290”, 10” min- I., N, carrier gas, 28 ml min - ‘; ionizing voltage, 70eV; trap current, 6OpA; accelerated high voltage 3500 v. Acknowledgements-The
EXPERIMENTAL
The seed oils were prepared from the corresponding seeds by Soxhlet extraction using CHCI,. The sterols occurring in the oils examined in this work were identified by comparing their RR, with those in the literature [15-193. The RR, is expressed by the ratio ofthe retention time for substance under examination to the retention time for sitosterol(30 min). Specimens of pure cholesterol, campesterol, stigmasterol, and sitosterol were supplied by Sigma. The A’-avenasterol, A’-avenasterol, A’-stigmastenol and a-spinasterol were obtained from poppy [15], castor [ 151, safflower [ 151, sunflower [ 16,203 and alfalfa [20] seed oils. These specimens were used as reference substances for the identification of the respective sterols occurring in the oils examined in this work. Estimations of sterol compositions were based upon area per cent values obtained from GC peaks using the triangulation method. Differences in GC response, if any, for different sterols were calculated. Identification of sterols was also carried out by GC-MS. Oil (2 g) in 30 ml alcoholic 1.0 M KOH was refluxed for 1 hr. The reaction mixt. was diluted with 60 ml H,O and the unsaponifiable material was extracted with Et,0 (3 x 30 ml). The combined Et,0 extract was washed with H,O and dried over dry Na,SO,, the solvent was removed under red. pres. (30”). Unsaponifiable material was fractionated on prep. TLC, developed with nhexane-Et,0 (7:3). After developing, a part of the plate was sprayed with a 2,7-dichlorofluorescein soln in EtOH and observed under UV light (3600 A). Four separated zones containing less polar compounds, triterpene alcohols, Cmethylsterols and sterols, respectively, were removed and extracted with Et,O. The sterol fr. was purified further by repeated prep. AgNO,-silica gel TLC (2: 8) for subsequent GC analysis.
author is deeply indebted to Professor A.T. Hunziker for identification of the species and providing some samples of plant material; Dr R. Subils for collection of some species. I also thank Dr C. A. Guzman for his helpful comments; CONICET, CONICOR for financial assistance and to the firma ‘Garcia e Hijos S.A.’ for providing freely n-hexane used in this research. REFERENCES
1. Patterson, G. W. (1971) Lipids 6, 120. 2. Weete, J. D. (1976) in Chemistry and Biochemistry of Natural Waxes (Kolatukudy, P. E., ed.), chap. 9. Elsevier, New York. 3. Senatore, F. (1990) Biochem. Syst. Ecol. 18, 103. 4. Chiu, P. L., Patterson, G. W. and Salt, T. A. (1988) Phytochemistry 27, 8 19. 5. Sunder, R., Ayengar, K. N. N. and Ragaswami, S. (1974) Phytochemistiy 13, 1610. 6. Matsuo, A. and Sato, A. (1991) Phytochemistry 30, 2305.
7. Adams, R. P., Tomb, A. S. and Price, S. C. (1987) Biochem. Syst. Ecol. 15, 395. 8. Mirallos, J., Noba, K., Ba, A. T., Gaydou, E. M. and Kornprobst, J. M. (1988) Biochem Syst. Ecol. 16,475. 9. Rafii, Z. A., Zavarin, E. and Pelleau, Y. (1991) Eiothem. Syst. Ecol. 19, 249. 10. Patterson, G. W., Xu, S. and Salt, T. A. (1991) Phytochemistry 30, 523.
11. Akihisu. T., Kokke, W. C. H. C. and Tamura, T. (1993) in Physiology and Biochemistry of Sterols (Patterson, G. W. and Nes, W. D., eds), p. 172. American Oil Chemists Society, Champaign. 12. Bergmann, W. (1953) A. Rev. Plant Physiol. 4, 383. 13. Itoh, T., Tamura, T. and Matsumoto, T. (1977) Steroids 30, 425.
Oil seeds ofSolanw
14. Knights, B. A. (1967) J. Gas Chromatogr. 5, 273. 15. Jeong, T. M., Itoh, T., Tamura, T. and Matsumoto, T. (1974) Lipids 9, 921. 16. Itoh, T., Tamura, T. and Matsumoto, T. (1974) Lipids 9, 173. 17. Dasso. I., Gros, E. G. and Cattaneo, P. (1980) An. Asoc. Quim. Argentina 66, 109.
species
167
18. Farines, M., Cocallene, S. and Soullier, J. (1988) Lipids 23, 349. 19. Frega, N., Bocci, F., Conte, L. S. and Testa, F. (1991) J. Am. Oil Chem. Sot. 68,29. 20. Itoh,T., Tamura, T. and Matsumoto, T. (1973) J. Am. Oil Chem. Sot. 50, 122.
A COMPARATIVE
0031~%22/94 S6.00+0.00 Q 1993 Pcrgamon Press Ltd
STUDY
OF STEROLS SPECIES
IN OIL SEEDS OF SOLANUM
J. A. ZYGADLO C&&a de Quimica Organica, Facultad de Ciencias Exactas, Fisicas y Naturales (Instituto Multidisciplinario de Biologia Vegetal CONICET-UNC)
- Av. Velez Sarsfield 2!W5000 CORDOBA, Argentina (Receiued in revised form 10 June 1993)
Key Word Index--Solanwn;
Solanaceae; sterols; chemosystematic.
Abstract-
Cholesterol, campesterol, sitosterol, stigmasterol, As-avenasterol, A’-avenasterol, A’-stigmastenol and a-spinasterol, were identified in the seeds of 13 species of Solarnun. The chemosystematic significance of sterols in these species is discussed.
INTRODUCTION
Sterols have contributed to the assessment of taxonomic schemes in algae [l, 23, fungi [2, 33, ferns [4, 51, mosses [6] and higher plants [7-l 11. The value of sterol patterns in deducing systematic relationships among Solanum species is not well known. Bergmann in his 1953 review of the plant sterols [12], listed several groups of species according to the sterol structure; some species of Solanum show a predominance of AS-sterols or A*(9)-sterols. According to Itoh et al. [13] the Solunaceae seeds that have been examined can be arranged in two groups based on the compositions of the seed sterols; Solanum is included in the group containing sitosterol as the predominant component. For this reason, it seems pertinent to analyse additional species of Solanum to determine the possible chemotaxonomic value of sterols in this genus.
RESULTS AND DISCUSSION
Since the validity of comparing sterol composition for taxonomic purposes depends on the stability of the patterns under varying geographical and ecological conditions, seeds from several geographically separated populations of 13 species were analysed (Table 1). The sterols identified were cholesterol, campesterol, sitosterol, stigmasterol, r-spinasterol, A’-avenasterol, A’stigmastenol and A’-avenasterol (Table 2). Cholesterol (RR, 0.61) had a fragmentation pattern which agreed with that of a reference specimen of cholesterol and also that reported by Knights [ 143, molecular ion [M] + at m/z 386 (calculated for C,,Hh60, M 386) with other principal ions at m/z 371, 368, 353, 301, 275, 273 and 247. A’Stigmastenol (RR, 1.18) had [M] + at m/z 414 accompanied by the ionsm/z 399 [M-Me]+, 396 [M-H,O]+ and 381 [M -Me-HzO]‘. Peaks were also found at m/z 273 [M-side chain]+ and 255 [M-side chain - H,O]+. Moreover, peaks were observed at m/z 246 [M
- side chain - 273 + and 229 [M-side chain-27 - H,O]+. These fragmentation patterns agree with those given by A’-sterols [14-161. A’-Avenasterol (RR, 1.32) showed [Ml+ at m/z 412 with other abundant ions at m/z 397, 379, 314, 296, 279, 273 and 253. a-Spinasterol (RR, 1.03) showed [M] + at m/z 412 (calculated for Cz9H4z0, M 412) with other abundant ions at m/z 397, 394, 379, 369, 351, 273, 271, 255, 246, 231, 229 and 213. The fragmentations giving the ions at m/z 369 [M - C,H,]+ and 351 CM-C3H7-HzO]+ involve the isopropyl group of the side chain and appear to be characteristic for A22-sterols [14,16]. The fragmentation pattern of the mass spectrum of the sterol with RR, 1.03 was basically similar to that of authentic a-spinasterol (24ethylcholesta-722dienol). As-Avenasterol (RR, 1.12) gave [M]’ at m/z 412 (calculated for C,,H,,O, M 412) with other ions at m/z 397,394,379,314,296,281,273 and 271. Campesterol (RR, 0.81); showed [Ml’ at m/z 400, with other abundant ions at m/z 382, 255 and 213. Stigmasterol (RR, 0.88) had m/z 412 [M] + accompanied with ions at m/z 394,379,351,315,300,271,255,229 and 213. Sitosterol gave m/z 414 [Ml’, with other abundant ions at m/z 399,396, 381,324,303,273,255,231,213, 145 and 107 (RR, 1.00). The literature concerning A7-avenasterol and A’-stigmastenol in species of Solanaceae is contradictory [13,15,1fl; for this reason it is interesting to point out that according to this study these A’-sterols were identified in four species of Solanum (Table 2). The results showed a clear separation of three sterol patterns in Solanum (Table 2). Although five sterols (cholesterol, campesterol, stigmasterol, sitosterol and a-spinasterol) appear in the three groups, the first one (with S. palitans and S. incisum) has only the five common sterols already mentioned; As-avenasterol is the additional sterol that characterizes the second group (with S. atriplicijXwn, S. sublobatum, S. chenopodioides, S. riparium, S. pseudocapsicum, S. dijlorum and S. argentinum). 163
J. A. ZYGADW
164
Table I. So/unum populations sampled
Abbreviations
1600 1700 1540 800 1460 2a.m 1200 1200
86 J. A. Zygadlo 74 J. A. Zygadlo 75 J. A. Zygadlo 143 J. A. Zygadlo 104 J. A. Zygadlo 105 J. A. Zygadlo 139 J. A. Zygadlo 140 J. A. Zygadlo 141 J. A. Zygadlo
SPI SP2 SP3 SI4 SI5 S16 S17 S18 S19
600
21 J. A. Zygadlo
SAiO
Cadillal (lbmnin) Agua de Oro (Cbrdoba) Las Tapias (C6rdoba) Ledeama (Jujuy) Rosario de la Frontera (Salta) Cadillal (Tucum&l) Agua de Oro (C6rdoba) L.a Quebrada (C6rdoba) m Mallin (Cbrdoba) 21 Colanchanga (C6rdoba) 22 La Falda (C6rdoba) 23 Agua de Oro (Cbrdoba)
1200 1200 600 600 1200 1200 1200 600 700 900 700 930 600
113 J. A. Zygadlo 114 J. A. Zygadlo 26 J. A. Zygadlo 133 J. A. Zygadlo 162 J. A. Zygadlo 163 J. A. Zygadlo 53 J. A. Zygadlo 40 J. A. Zygadlo 107 J. A. Zygadlo 119 J. A. Zygadlo 108 J. A. Zygadlo 138 J. A. Zygadlo 24 J. A. Zygadlo
SSll SSl2 SC13 SC14 SRl5 SRI6 SR17 SPsl8 SPsl9 SPs20 SPs21 sPs22 SW23
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
800 500 700 Iso0 3ooo 530 860 600 1200 1200 700 800 500 1700 3wo 700 500 530 300 600 600 760 800 800 800 500 500 750 600 890 500
4458 R. Subils 4413 R. Subils 81 J. A. Zygadlo 91 J. A. Zygadlo 93 J. A. Zygadlo 100 J. A. Zygadlo 1 J. A. Zygadlo 12 J. A. Zygadlo 17 J. A. Zygadlo 1I5 J. A. Zygadlo 110 J. A. Zygadlo 4457 R. Subils 47 J. A. Zygadlo 82 J. A. Zygadlo 89 J. A. Zygadlo 95 J. A. Zygadlo 4414 R. Subils 25 385 A. T. Hunziker 4463 R. Subils 18 J. A. Zygadlo 3 J. A. Zygadlo 16 J. A. Zygadlo 25 395 A. T. Hunziker 25 396 A. T. Hunziker 25403 A. T. Hunziker 4412 R. Subils 25 386 A. T. Hunziker 42 J. A. Zygadlo 1I7 J. A. Zygadlo I1 8 J. A. Zygadlo 97 J. A. Zygadlo
SD24 SD25 SD26 SD27 SD28 SD29 SY30 SY31 SY32 SY33 SY34 SL35 SL36 SL37 SL38 SL39 SE40 SE41 SE42 SE43 SE44 SE45 SE46 SE47 SE48 SJ49 SJSO SJ5l SJ52 SJ53 SJ54
Populations
S. palitans MORTON
1 2 3 4 5 6 7 8 9
S.
in&urn
GRISEB.
S. atriplijiijlium GILLIES ex MEYEN S. sublobatum WILLD. ex ROEM et SCHULT. S. chenopodioides LAM. S. riparium PERSOON
S. pseudocapsicum L.
S. diporum VELL. S. argentinwn BITTER et LILLO
S. sisymbriijoliwn
LAM.
S. palinacanthum
DUNAL
S. elaeagnifolium CAV.
S. juwnale THELLUNG
Altitude (m.s.n.m)
Collection data
Speciea
Chorrillos (Salta) Quijano (Salta) Yacones (Salta) Los Gigantes (C6rdoba) Bosque Alegre (Cbrdoba) Copina (C6rdoba) Cerro Horcosum (Cbrdoba) Puesto Guz m8n (Cbrdoba) Los Pozos (C6rdoba)
10 Agua de Oro (C6rdoba)
11 Cadillal (Tucumzin)
12 13 14 I5 16 17 18 19
El Alto (Catamarca) Frias (Santiago del Estero) El Zapallar (Salta) Alemania (Salta) Chorrillos (Salta) Soto (C%rdoba) Flor Serrana (C6rdoba) Santa Rosa de Calamuchita (Cbrdoba) Mina Clavero (C6rdoba) Cadillal (TucumBn) La Quebrada (C6rdoba) El Alto (Catamarca) Alta Gracia (Cbrdoba) Yacones (Salta) Chorrillos (Salta) El Zapallar (Salta) Frias (Santiago del Eatero) Soto (Cbrdoba) Embarcadero 977 (Catamarca) Carlos Paz (C6rdoba) Las Tapias (C6rdoba) Santa Rosa de Calamuchita (C6rdoba) Ruta hacia Patquia (La Rioja) Ruta hacia Patquia (La Rioja) Ruta hacia Patquia (La Rioja) Frias (Santiago de1 Eatero) Pichanas (C6rdoba) Villa General Belgrano (C6rdoba) Orilla de1 Lago San Roque (C6rdoba) Cosquin (C6rdoba) Pichanas (C6rdoba)
3ooo
Oil seals of Sohmumspecies Table 2. Sterol compositions of seed oil from Sohm
SpXiol S. p&tans SPl SP2 SP3 S. inciswn S14 SIS SI6 s17 S18 s19 S. otriplicifolium SAlO S. sublobatum SSll SSl2 S. cherwpodioides SC13 SC14 S. riparium SR15 SR16 SR17 S. pseudocapshm SPsl8 SPsl9 SPs20 sPs21 sPs22 S. dijlorum SW23 S. argentinum SAr24 SAr25 SAr26 SAr27 SAr28 SAr29 S. sisymhriffolium SY30 SY31 SY32 SY33 SY34 S. palinacanthum SL35 SL36 SL37 SL38 SL39 S. el~agnfolium SE40 SE41 SE42 SE43 SE44 SE45 SE46 SE47 SE48
I*
2
3
4
Sterols 5
165 species
6
3.2 2.9 2.0
19.5 18.8 17.0
TR. TR. TR.
76.0 77.1 79.7
I.0 0.9 1.0
3.2 2.0 3.2 1.5 TR. 1.0
20.0 17.8 21.0 15.0 22.3 19.5
TR. TR. TR. TR. TR. TR.
76.8 80.1 74.8 828 77.8 79.6
2.2 2.2 3.2 29 2.5 2.2
7.7
8.8
6.8
76.1
TR.
TR.
1.9 6.9
82.0 80.3
TR. TR.
5.2 4.8
10.7 5.7
TR. TR.
7
8
1.7 TR
7.1 TR.
10.1 14.3
78.2 824
TR. TR.
23 2.0
2.0 3.0 1.5
7.0 6.0 5.0
10.1 9.0 8.1
78.0 78.9 80.7
TR. TR. TR.
TR. 0.6 5.0
20.0 15.5 10.0 21.5 19.8
11.2 6.7 16.2 10.2 11.4
7.0 6.0 3.0 6.5 6.7
46.6 55.7 56.7 50.6 46.9
TR. TR. TR. TR. TR.
6.0 6.8 4.7 20 6.0
9.2
10.0
19.2
65.6
1.0
6.0
6.0 TR. 7.8 6.0 4.3 6.0
17.0 12.0 14.0 18.8 15.5 17.0
6.1 6.9 5.0 5.3 5.0 3.1
620 71.9 61.8 64.9 720 73.2
TR TR TR. TR. TR. TR.
8.4 8.5 10.7 5.3 3.0 TR.
2.6 1.2 3.3 1.5 TR.
4.5 29 5.7 3.1 4.1
8.0 3.3 11.3 7.2 10.3
70.7 87.9 66.4 81.1 65.0
1.0 0.6 21 TR. 2.3
11.2 3.5 9.3 6.3 121
0.7 0.6 0.7 TR. 3.4
13 TR. 1.1 TR. 27
1.0 2.0 0.9 1.3 0.8
3.9 4.0 1.5 5.2 6.0
3.5 4.0 5.1 3.7 3.5
m.0 720 79.3 84.1 74.1
TR. TR 2.1 0.6 TR.
16.0 l.l.7 7.8 2.1 123
25 3.1 1.1 TR. 3.1
1.5 28 21 28 TR.
3.0 29 1.6 3.2 4.0 TR. 8.1 6.9 5.1
7.0 5.8 5.8 8.6 6.8 3.6 3.3 7.0 3.0
0.8 7.3 9.1 5.4 6.3 25 10.2 8.1 5.7
77.8 78.4 80.0 78.4 77.8 86.9 75.8 74.8 80.3
0.8 TR. 1.1 0.9 1.3 2.1 0.6 0.8 2.0
TR 0.8 0.9 TR 1.5 3.0 0.9 TR 21
1.0 1.2
3.0 3.4 0.7 3.2 2.3 TR TR 1.9 TR.
E. TR. 1.7 1.1 TR. 1.6
J.
166
A. ZYGADLO
Table 2. Continued Sterols Species
I*
2
3
4
5
6
7
8
0.8 2.5 TR.
1.2 3.1
1.0 TR. 1.3
1.0 2.1 1.7
S. juuenale
SJ49 SJ50 SJSl SJ52 SJ53
4.0 3.9 6.1 3.7 10.2
4.0 5.1 4.6 4.8 2.3
8.5 1.2 8.0 6.6 3.3
70.1 77.2 73.1 13.5 73.1
1.0 TR. 1.0 1.5 0.7
SJ54
7.7
8.1
5.8
70.1
2.9
‘1 Cholesterol: 8 A’-avenasterol;
2 camoesterol: 3 stiemasterol; TR., tracks
The third group (with S. sisymbriijolium, S. palinacanthum, S. elaeagni$olium and S. juvenale) is characterized by having A’-avenasterol and A’-stigmastenol as an addition to the sterols that are present in the first and second groups. However, more extensive sampling is needed in this large genus before comparative sterol data can be applied taxonomically or phylogenetically.
7.7 TR. 8.0 7.9 8.1 2.4
4 sitosterol; 6 As-avenasterol;
7 A’-stigmastenol;
GC used an OV-17 (2%) column, carrier gas Nz at 30 ml min-‘, column temp. programmed 180”-290”, 10” min-‘, FID. GC-MS used an OV-17 column (3%), 200”-290”, 10” min- I., N, carrier gas, 28 ml min - ‘; ionizing voltage, 70eV; trap current, 6OpA; accelerated high voltage 3500 v. Acknowledgements-The
EXPERIMENTAL
The seed oils were prepared from the corresponding seeds by Soxhlet extraction using CHCI,. The sterols occurring in the oils examined in this work were identified by comparing their RR, with those in the literature [15-193. The RR, is expressed by the ratio ofthe retention time for substance under examination to the retention time for sitosterol(30 min). Specimens of pure cholesterol, campesterol, stigmasterol, and sitosterol were supplied by Sigma. The A’-avenasterol, A’-avenasterol, A’-stigmastenol and a-spinasterol were obtained from poppy [15], castor [ 151, safflower [ 151, sunflower [ 16,203 and alfalfa [20] seed oils. These specimens were used as reference substances for the identification of the respective sterols occurring in the oils examined in this work. Estimations of sterol compositions were based upon area per cent values obtained from GC peaks using the triangulation method. Differences in GC response, if any, for different sterols were calculated. Identification of sterols was also carried out by GC-MS. Oil (2 g) in 30 ml alcoholic 1.0 M KOH was refluxed for 1 hr. The reaction mixt. was diluted with 60 ml H,O and the unsaponifiable material was extracted with Et,0 (3 x 30 ml). The combined Et,0 extract was washed with H,O and dried over dry Na,SO,, the solvent was removed under red. pres. (30”). Unsaponifiable material was fractionated on prep. TLC, developed with nhexane-Et,0 (7:3). After developing, a part of the plate was sprayed with a 2,7-dichlorofluorescein soln in EtOH and observed under UV light (3600 A). Four separated zones containing less polar compounds, triterpene alcohols, Cmethylsterols and sterols, respectively, were removed and extracted with Et,O. The sterol fr. was purified further by repeated prep. AgNO,-silica gel TLC (2: 8) for subsequent GC analysis.
author is deeply indebted to Professor A.T. Hunziker for identification of the species and providing some samples of plant material; Dr R. Subils for collection of some species. I also thank Dr C. A. Guzman for his helpful comments; CONICET, CONICOR for financial assistance and to the firma ‘Garcia e Hijos S.A.’ for providing freely n-hexane used in this research. REFERENCES
1. Patterson, G. W. (1971) Lipids 6, 120. 2. Weete, J. D. (1976) in Chemistry and Biochemistry of Natural Waxes (Kolatukudy, P. E., ed.), chap. 9. Elsevier, New York. 3. Senatore, F. (1990) Biochem. Syst. Ecol. 18, 103. 4. Chiu, P. L., Patterson, G. W. and Salt, T. A. (1988) Phytochemistry 27, 8 19. 5. Sunder, R., Ayengar, K. N. N. and Ragaswami, S. (1974) Phytochemistiy 13, 1610. 6. Matsuo, A. and Sato, A. (1991) Phytochemistry 30, 2305.
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Oil seeds ofSolanw
14. Knights, B. A. (1967) J. Gas Chromatogr. 5, 273. 15. Jeong, T. M., Itoh, T., Tamura, T. and Matsumoto, T. (1974) Lipids 9, 921. 16. Itoh, T., Tamura, T. and Matsumoto, T. (1974) Lipids 9, 173. 17. Dasso. I., Gros, E. G. and Cattaneo, P. (1980) An. Asoc. Quim. Argentina 66, 109.
species
167
18. Farines, M., Cocallene, S. and Soullier, J. (1988) Lipids 23, 349. 19. Frega, N., Bocci, F., Conte, L. S. and Testa, F. (1991) J. Am. Oil Chem. Sot. 68,29. 20. Itoh,T., Tamura, T. and Matsumoto, T. (1973) J. Am. Oil Chem. Sot. 50, 122.