Linear, Steroidal, and Triterpene Esters, and Steryl Glycosides from Festuca argentina

Adriana C. Casabuono and Alicia B. Pomilio*

PROPLAME-CONICET, Departamento de Qumica Orgnica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, 1428 Buenos Aires, Argentina

ABSTRACT: Ester waxes and steryl glycosides of the grass Festuca argentina were studied. Saponication of the waxes from the petroleum ether extract led to n-hexacosanol as the major single linear alcohol, along with pentacyclic triterpenols, such as amyrin, germanicol, isobaurenol, lupeol, hopenol-a and hopeol, and low amounts of sterols, such as cholesterol, campesterol, stigmasterol, sitosterol and dihydrositosterol, identied by gas chromatography/mass spectrometry (GC/MS). Fatty acids were identied as methyl esters as C12:0, C14:0, C16:0, C18:0, C18:2, and C20:0. The occurrence of a wide chainlength range of fatty acids and a single linear alcohol closely matched for other reports on the tribe Festuceae. On the contrary, pentacyclic triterpenols with a variety of skeletons, especially isobauerenol, are not usual as esters of fatty acids in the Gramineae. Low amounts of steryl glycosides were also obtained from the methylene chloride percolate of the methanol extract. Upon acetylation followed by hydrolysis, aglycones were identified by capillary gasliquid chromatography (GLC) and GC/MS. As 7-cholesterol, campesterol, stigmasterol, sitosterol, dihydrositosterol, and the sugars as glucose, xylose, and arabinose by GLC of the respective alditol acetates. This is the rst report on the linear, steryl, and triterpenyl esters of F. argentina. It is noteworthy that 7-steryl glycosides are rare, and steryl monoarabinosides have not been previously reported on the family Gramineae. Lipids 32, 205210 (1997).

Festuca argentina (Speg.) Par. (Gramineae; common name: coirn negro, coirn duro) is a perennial xerophyte Patagonian grass popularly known as toxic to sheep (1) that grows in the pre-Andinian region, and eastward around the Gulf of San Jorge (2) in the Patagonian coast of Argentina. We have previously reported on free sterols and triterpenes from this species (3). The 5-, 7-, and dihydrosterols were identied as well as pentacyclic triterpenones with a variety of skeletons, such as oleanene/ursene (12-oleanen-3-one or amyrenone, 18-oleanen-3-one = germanicone), lupene (lupen3-one), hopene (22(29)-hopen-3-one) and multiflorene (8*To whom correspondence should be addressed at PROPLAME-CONICET, Departamento de Qumica Orgnica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Pabelln 2, Ciudad Universitaria, 1428 Buenos Aires, Argentina. Abbreviations: GC/MS, gas chromatography/mass spectrometry; GLC, gasliquid chromatography; MPLC, medium-pressure liquid chromatography; MS, mass spectra; MW, molecular weight; PE, petroleum ether; Rf, retention front; Rt, retention time; TLC, thin-layer chromatography.

multiorenone = isomultiorenone) and a cyclopropane tetracyclic triterpenone (cycloartanone), the two latter being rare in the Gramineae. However, the expected 4-methylsterols, 3methoxytriterpenes and arborene and/or fernene triterpenoids were not detected in this species, although widespread in the Gramineae (4). In continuation of our work on members of the tribe Festuceae, we want to report in this paper the composition of the ester waxes and steryl glycosides of F. argentina that, in contrast to other grasses, showed to be rich not only in a linear alcohol but also in both bound sterols and triterpenols. Wax esters from other members of the Gramineae usually accounted for esters of fatty acidsn-alcohols, e.g., Bromus inermis, F. ovina (5), Secale cereale (6), three wheat species (79), and Agropyron desertorum and A. intermedium (10), due to interesterication of a wide range of acids with a much smaller chainlength range n-alcohols. Combined alcohols from waxes of festucoid grasses (5,11) generally contain one major component, C26 or C28, with the same chainlength as the major free alcohol of the wax. However, in Agropyron smithii (tribe Triticeae) a wide chainlength range of both acids and alcohols from the ester waxes were obtained without any prevalent long-chain major component (12), the alcohol portion also containing unusual appreciable percentage of - and -amyrins (40%). A wide range of chainlength linear alcohols and triterpenol esters was also typical of species of the subfamily Eragrostoideae (13). Detailed information on the identities of naturally-occurring phytosterols as glycosides is far from complete. So far, up to now these compounds have not been described as characteristic of any family, and they are scarcely distributed, except for sitosterol 3-O-glucoside, in agreement with earlier biosynthetic studies in Triticum aestivum, showing that UDP-glucose is the most active glycosyl donor (14). We have detected in F. argentina several steryl glycosides composed of a variety of steryl and sugar moieties. The occurrence of these compounds may be related to a sterol storage function, instead of simple waste products as considered by some plant physiologists especially interested in the role of free sterols (14). MATERIALS AND METHODS Plant material. Plants of F. argentina (Speg.) Par. (family: Gramineae) were collected by Prof. Ing. Angel Soriano in Ro
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Mayo (EE-INTA Ro Mayo; Patagonia), Province of Chubut, Argentina, and identified by Dr. Elisa Nicora (Instituto de Botnica Darwinion, San Isidro, Argentina); voucher specimen were deposited in the Instituto de Botnica Darwinion (Argentina) SI 28004. This is a monocotyledonous matted grass with blades, roots, and spikelets with minute structures which are important markers of taxa. Spikelets of grass species within a genus differ in the rachilla joints, glumes, lemmas, or paleas. In addition it has a distinct festucoid-type embryo. The plant material used in this study was harvested in February (summer season in southern hemisphere) in the so-called fruit stage/period; it was composed of leaves (blades) and adventitious fibrous roots, and devoid of spikelets. Materials and chromatographic conditions. Analytical thin-layer chromatography (TLC) plates (silica gel G, F-254, 250 m layer), silica gel G and silica gel H were purchased from Merck (Darmstadt, Germany); polyamide for chromatography was purchased from Woelm (Eschwede, Germany). Preparative TLC (20 cm 20 cm; 1000 m layer) were prepared in our laboratory using a suspension of silica gel G (30 g) in distilled water (60 mL). Freshly prepared plates were kept at room temperature for 24 h and further activated at 110120C for 1 h prior to use. Spots were visualized under ultraviolet (UV) light at 254 nm and/or by spraying the plates with H2SO4/AcOH (1:1) and heating at 110120C for 2 min. Medium-pressure liquid chromatography (MPLC) was carried out on silica gel H (Merck) columns with an impulse solvent bomb Prominent-Electronic 1001 SCJ (Heidelberg, Germany) with a pulse damper. Flash dry chromatography was performed under vacuum through a fritted Pyrex glass funnel membrane (Thomas Scientific, Swedesboro, NJ). Organic solvents were obtained from Sintorgan (Buenos Aires, Argentina) followed by distillation, except for petroleum ether (PE) and n-hexane which were freed from olenes in the usual way prior to distillation. All solvent mixtures are expressed in volume (vol/vol). Extraction of the plant material. As soon as it was collected, fresh material was air-dried in the laboratory in an aircirculating oven at 25oC to remove soil adhered to roots and blades; then it was ground and immediately extracted in a Soxhlet with PE (6080C) followed by MeOH. Flash dry chromatography of PE extract (1.6 % relative to dry weight) using gradients of n-hexane/EtOAc as previously reported (3) followed by TLC analysis led to six main fractions, termed 16. TLC (n-hexane/EtOAc, 98:2) of fraction 2 gave a main pink spot at Rf 0.60 typical of waxes, a brown spot at Rf 0.53 due to aldehydes, and a light-orange spot at Rf 0.38 due to terpenoids. Separation of these components (subfractions 2a, 2b, and 2c) was afforded by repeated preparative TLC (nhexane/EtOAc, 97:3). Bands were eluted with chloroform and monitored by analytical TLC, and the waxes were characterized by infrared spectra (KBr) using a Perkin-Elmer 710-B spectrophotometer (Palo Alto, CA). The MeOH extract was further percolated on polyamide with CH2Cl2, H2O, and finally MeOH, rendering 1.0, 2.8, and 2.3% relative to dry plant, respectively. The CH2Cl2 percolate was worked up as deLipids, Vol. 32, no. 2 (1997)

scribed before (15) yielding a subfraction 2 that was purified by MPLC (EtOAc). A Liebermann-Burchard positive (16) fraction that gave a diffuse pink spot of Rf 0.65 by TLC (EtOAc/MeOH, 95:5) contained steryl glycosides (3 mg; 0.005% relative to dry plant) which were acetylated (Ac2O/anhydrous pyridine) to overcome their insolubility in organic solvents (Rf 0.80 and 0.85, high-performance thin-layer chromatography: EtOAc/MeOH, 95:5), and further hydrolyzed. Saponication of waxes. Saponication was performed by stirring the waxes (0.13 g) under reflux with 20% KOH in MeOH for 4 h. The reaction was monitored by TLC. The mixture was then evaporated to dryness. Water was added to the residue and the aqueous layer was further extracted with CHCl3. The organic layers were combined, dried over anhydrous Na2SO4 and the chloroform was evaporated under vacuum to give the alcohols. The remaining aqueous layer was acidified and the acidic compounds were extracted with CHCl3. This chloroform layer was dried over anhydrous Na2SO4 and then evaporated to dryness to give the organic acids. These organic acids were methylated (CH2N2/Et2O) for 14 h at room temperature. Hydrolysis of the steryl glycosides. The acetylated glycosides were hydrolyzed with 6% HCl in MeOH. The reaction mixture was heated until dissolution of the glycosides and drops of water were added until there was slight turbidity. Then it was kept at 75C in a sealed tube for 2 h. After neutralization with NaHCO3 and evaporation of the solvent, the residue was partitioned with CH2Cl2/H2O (1:1). The CH2Cl2 layer was dried with anhydrous Na2SO4 and evaporated at 40C, yielding sterols. The aqueous layer containing the sugars was desalted with Amberlite MB-3/thymolphthalein (Sigma, St. Louis, MO) to prepare the alditol acetates (NaBH4, room temperature, 12 h; Ac2O/pyridine, 1:0.8). Gasliquid chromatography (GLC) and gas chromatography/mass spectrometry (GC/MS) analysis of the compounds. GLC was performed on a Hewlett-Packard 5840 A chromatograph (Palo Alto, CA) with flame-ionization detection (FID) and nitrogen as gas carrier. Capillary GC/MS were performed at 70 eV on a Hewlett-Packard 5890A chromatograph coupled with a selective mass detector 5970B. Mass spectra (MS) data were processed by a Lab Base GC-MS data system (40 to 800 mass scanning). The respective alcohol mixtures from the hydrolysis of the waxes and of the glycosylsterols were chromatographed on a bonded-phase fused silica capillary column (DB-1, 30 m length 0.245 mm id, 0.25 m film thickness; J&W Scientific, Folsom, CA). Oven temperature was set at 180C for 1 min with a 5C/min temperature ramp up to a final temperature of 300C for the alcohols from the ester waxes, and 200300C, rate 10C/min for the aglycones from the glycosylsterols. The identities of n-hexacosanol, amyrin, germanicol, lupeol and hopeol, as well as the sterols were conrmed by capillary GLC with standards. In addition, the mixtures were examined by GC/MS on a 25 m 0.25 mm, 0.33 m lm thickness Ultra-1 capillary column, temperature programmed from 240300C at 3C/min for wax alcohols, and 200300C, rate 15C/min for glycosides aglycones.

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Fatty acids from the wax hydrolysis were separated as methyl esters on a 1.8 m 2 mm 8% NPGS packed column on Chromosorb W-AW-DMCS, 60-80 mesh, at 190C, using authentic fatty acid methyl ester standards. Identification was conrmed by GC/MS spectrometry on a 1.2 m length 2 mm i.d. SP-2340 column (Supelco, Bellefonte, PA) programmed from 60270C at 8C/min. Sugars from steryl glycosides were analyzed by capillary GLC of their alditol acetates against synthetic standards (SP 2330, 30 m length 0.25 mm id, 0.20 m lm thickness; Supelco). RESULTS AND DISCUSSION Ester waxes. Repeated chromatographic purification techniques applied to the PE extract of F. argentina yielded a subfraction with an infrared spectrum typical of high molecular weight (MW) ester waxes [2895 (C-H), 1742 (C=O), 1475 (C-H), 1180 (C-O) and 740 cm1 (C-H)]. Upon hydrolysis, a homologous series of saturated fatty acids with even carbon atom from C12 to C20 was obtained, which showed typical fragmentation pattern (17) by GC/MS analysis of their respective methyl esters with m/z 74 as base peak, and M+ at m/z 214, 242, 270, 298, and 326, respectively. In addition, M+ 294 was shown to be a fatty acid of C18 containing two unsaturations (C18:2) by its mass fragmentation pattern. The acid composition of the ester waxes from F. argentina is shown in Table 1, with hexacosanoic acid being the major component among the seven identied fatty acids. By contrast, the wax alcohols (Table 1; Fig. 1) showed a single major linear alcohol, n-hexacosanol (C26:0), which accounted for 61% of the total alcohol wax composition, along with triterpene alcohols (31.2%) and low amounts of sterols (7.6%) (Fig. 1A). The low levels of triterpenols and sterols were detected by GC/MS in the 15 to 24 min region of the amplied chromatogram (Fig. 1B). The MS of the respective sterols showed M+ 386, 400, 412 and 414, and the fragmentation pattern typical of 5-sterols (Table 2) (18), corresponding to cholesterol, campesterol, stigmasterol, sitosterol, along with another sterol of Rt and M+ higher than that of sitosterol, and an intense ion m/z 233 in agreement with dihydrositosterol (19). The GC/MS of the pentacyclic triterpenoid alcohols were in the range of retention time (Rt) 20.34 to 22.99 min. The compound of Rt 20.34 min, M+ 426, and m/z 218 as
TABLE 1 Composition of Acids and n-Alcohols from Festuca argentina Waxes 12:0 14:0 Esteried acidsa Esteried alcoholsb
a

FIG. 1. Gas chromatography/mass spectrometry of the alcohols from ester wax hydrolysis. (A) general chromatogram; (B) chromatogram of the amplied 15 to 24 min region.

16:0 38.8

18:0 25.7

18:2 19.8

20:0 5.7

26:0 61.0

1.9

4.0

Fatty acids analyzed as methyl esters by gasliquid chromatography (GLC) on 8% NPGS and comparison with standards. Identification confirmed by gas chromatography/mass spectrometry (GC/MS) on a SP-2340 column. For details, see the Materials and Methods section. b Alcohols were analyzed by capillary GLC on DB-1. Identification of nhexacosanol is based on cochromatography with a known standard and GC/MS as indicated in Table 2.

base peak showed a fragmentation pattern (Table 2) of a 12ursene/12-oleanene triterpenol (20), confirmed as -amyrin by capillary GLC with authentic samples, due to the fact that both skeletons are undistinguishable by MS. The compound of Rt 20.52 min (Table 2) presented a M+ 426 followed by loss of 15 amu, and an intense ion at m/z 177 (98.5%) typical of a 18-oleanene or 18-friedelene (20); the low abundance (14.7%) of ion m/z 203, usually very high in 18-friedelene (20) provided evidence for a 18-oleanene, further conrmed by GLC comparison with a germanicol sample. The compound of Rt 20.93 min, M+ 426, m/z 247 as base peak and other ions (Table 2) was characteristic (21) of a D:C-friedooleanane/D:C-friedo-ursane skeleton with one unsaturation either at 7-(multiflorenol/bauerenol) or at 8-(isomultiflorenol/isobauerenol), prevailing the bauerene series (20) by the intense m/z 247 and the low abundant m/z 205. Comparison of relative abundances was required for the 7/8 location of the double bond, e.g., base peak, M+ and M+-Me of isobauerenol (22) and -bauerenol (23), thus leading to isobauerenol (bauer-8-en-3--ol) only once reported in Gramineae (24). Compounds with Rt 21.18, 22.76, and 22.99 min showed similar MS (Table 2) concerning the molecular ion (M+ 426) and the highly abundant m/z 207, arising from hydroxyl-containing rings A/B of saturated triterpenols without directing groups. The fact that m/z 189 is more intense than m/z 191, along with the low intensity or absence of M+
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TABLE 2 Alcohols from the Saponication of Festuca argentina Waxes Alcohola n-Hexacosanol Rt (min) 12.52 Characteristic mass peaks (m/z) (% relative abundance) 364 (M+ H2O, 5.2), 336 (364 CH2 = CH2, 2.5), 308 (0.5), 280 (0.8), 266 (0.7), 252 (0.9), 238 (1.0), 224 (1.2), 210 (1.5), 195 (2.3), 181 (3.3), 167 (4.5), 153 (6.6), 139 (10.6), 125 (19.7), 111 (37.2), 97 (72.9), 83 (83.7), 73 Cholesterol 17.59 (1.5), 71 (56.9), 69 (69.0), 57 (100.0), 55 (62.2), 43 (76.9), 41 (21.9). 387 (M+ + 1, 29.2), 386 (M+, 100.0), 371 (M+ Me, 31.2), 368 (M+ H2O, 39.7), 353 (M+ H2O Me, 32.5), 301 (M+ 85, 49.1), 275 (M+ 111, 37.5), 273 (M+ side chain, 21.0), 247 (M+ 139, 7.4), 231 (M+ side chain 42, 17.9). 401 (M+ + 1, 29.6), 400 (M+, 100.0), 385 (M+ Me, 29.3), 382 (M+ H2O, 39.9), 367 (M+ H2O Me, 28.5), 315 (M+ 85, 43.2), 289 (M+ 111, 27.6), 273 (M+ side chain, 18.9), 261 (M+ 139, 5.5), 231 (M+ side chain 42, 16.8). 413 (M+ + 1, 23.0), 412 (M+, 68.9), 397 (M+ Me, 6.1), 394 (M+ H2O, 5.0), 379 (M+ H2O Me, 7.3), 369 (M+ 43, 8.9), 351 (369 H2O, 15.8), 300 (M+ 112, 20.2), 271 (M+ side chain 2H, 19.6). 415 (M+ + 1, 19.7), 414 (M+, 63.1), 399 (M+ Me, 16.9), 396 (M+ H2O, 24.7), 381 (M+ H2O Me, 17.5), 329 (M+ 85, 24.4), 303 (M+ 111, 12.5), 275 (M+ 139, 3.3), 273 (M+ side chain, 10.9). 417 (M+ + 1, 30.8), 416 (M+, 100.0), 401 (M+ Me, 38.8), 398 (M+ H2O, 2.7), 383 (M+ H2O Me, 14.9), 234 (M+ side chain 41, 48.2), 233 (M+ side chain 41 H, 75.2), 215 (57.8). 427 (M+ + 1, 0.6), 426 (M+, 2.7), 411 (M+ Me, 0.5), 393 (M+ Me H2O, 0.9), 219 (18.7), 218 (RDA, rings D and E, 100.0), 205 (rings D and E or rings A and B, 0.5), 203 (218 Me, 39.3), 189 (207 H2O and/or rings D and E 29, 4.5). 427 (M+ + 1, 8.5), 426 (M+, 31.7), 411 (M+ Me of C 17, 26.0), 393 (M+ Me H2O, 1.9), 218 (rings D and E + C 11 + C 12, ), 207 (rings A and B, 19.7), 206 (15.3), 205 (rings D and E + C 12, 37.6), 204 (rings D and E + C 12, 100.0), 203 (218 Me, 14.7), 191 (18.1), 190 (205 Me, 24.9), 189 (93.8), 178 (21.4), 177 (RDA in ring D + C ring, 98.5). 427 (M+ + 1, 7.8), 426 (M+, 21.9), 411 (M+ Me, 14.1), 393 (M+ Me H2O, 5.1), 259 (rings A + B + C + Me 26, 15.5), 247 (RDA ring C and rupt. C 15 C 16, 100.0), 229 (57.3), 205 (rings D and E, 8.8). 427 (M+ + 1, 12.0), 426 (M+, 38.0), 411 (M+ Me, 13.4), 408 (M+ H2O, 1.7), 393 (M+ Me H2O, 3.2), 383 (M+ isopropyl, 3.8), 218 (rings D and E, 89.3), 207 (rings A and B, 59.3), 204 (28.7), 203 (37.4), 191 (26.8), 190 (7.8), 189 (60.8),135 (64.1), 121 (65.3), 109 (70.9), 108 (47.9), 107 (78.8), 105 (46.5), b.p. 43. 427 (M+ + 1, 18.0), 426 (M+, 47.8), 207 (rings A and B, 85.7), 204 (24.2), 191 (35.4), 190 (43.5), 189 (207 H2O and/or rings D and E, 100.0). 427 (M+ + 1, 11.9), 426 (M+, 36.6), 218 (16.9), 217 (rings D and E + C 11 + C 12, 7.2), 208 (RDA, rings A and B, 20.6), 207 (79.6), 205 (22.8), 204 (24.3), 203 (22.9), 191 (32.5), 190 (33.4), 189 (RDA, rings D and E H, 89.1), 135 (60.0), 121 (70.3), 109 (86.6), 107 (76.5), 105 (33.4), b.p. 43.

Campesterol

18.10

Stigmasterol

18.68

Sitosterol

19.79

Dihydrositosterol

19.99

-Amyrin

20.34

Germanicol

20.52

Isobauerenol

20.93

Lupeol

21.18

Hopenol-a Hopeol

22.76 22.99

a Identication conrmed on a 25-m capillary column Ultra-1 by GC/MS (EI-70 eV). For details, see the Materials and Methods section. See Table 1 for abbreviation.

43, is indicative of an isopropenyl group in the molecule, thus supporting the occurrence of either lupane or hopane skeletons, which usually lack a characteristic fragmentation pattern. Consequently, the identity of compounds with Rt 21.18 and 22.99 min was confirmed by capillary GLC comparison with authentic samples of lupeol and hopeol (hopenol-b = hop-22(29)-en-3--ol), respectively. The MS of the minor compound of Rt 22.76 min showed only two fragments in the high-mass region suggesting a difference in the double-bond location of the isopropenyl group relative to the two latter compounds, thus leading to hopenol-a.

Steryl glycosides. Successive MPLC purification steps of the MeOH extract of F. argentina gave rise to a LiebermannBurchard positive fraction (16) composed of steryl glycosides, which accounted for 0.005% relative to dry weight. Its chromatographic behavior (TLC, Rf region) was in agreement with nonacylated steryl monoglycosides. No changes were observed upon O-methylation (ethereal CH2N2), indicating the absence of uronic acids (25). After acid hydrolysis of the acetate derivatives, both sterols (aglycones) and sugars (as alditol acetates) were identified by capillary GLC and capillary GC/MS analysis. Glucitol (16.4%), xylitol (28.9%), and

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arabinitol (54.7%) were characterized and further confirmed by capillary GLC comparison with synthetic standards. Accordingly, 3-O-monoglycosides of cholesterol (11.3%), 7cholesterol (14.2%), campesterol (13.9%), stigmasterol (14.5%), 7-sitosterol (15.3%), sitosterol, and 5,6-dihydrositosterol (30.8%) occur in F. argentina, some of them as 3-Omonoglucosides, 3-O-monoxylosides, and 3-O-monoarabinosides. Although sitosterol 3-O-glucopyranoside is widespread in nature (2628), only few other 5-steryl 3-O-monoglycosides have been reported, such as stigmasterol 3-O-glucopyranoside (29), sitosterol 3-O--D-xylopyranoside (30), sitosterol 3-O--D-riburonofuranoside (25), sitosterol 3-O--Dxyluronofuranoside (31), and sitosterol 3-O--D-glucuronopyranoside (32). To the best of our knowledge, the isolation and identication of monoglycosides of dihydrosterols and 7-sterols from natural sources is rare, such as 3-O--Dglucopyranoside and 3-O--D-galactopyranoside of (24R)stigmast-7,22(E)-dien-3--ol (epichondrillasterol) (33). Moreover, D-glucose is the usual sugar moiety (34) of monoglycosides of a variety of 5-phytosterols, steryl 3-Omonoarabinosides being so rare in nature that stigmasterol L-arabinopyranoside has only been isolated once, from Wallenia yunquensis (35). In summary, free (3), esterified and glycosylated 4demethylsterols were found in F. argentina, whereas pentacyclic triterpenols merely occurred as esters of high-MW fatty acids. These ndings closely resemble earlier reports on Sorghum vulgare (36). Thus, our results show that 5-sterols and dihydrositosterol are found in free (3), esteryl and glycosyl forms, while 7-sterols are present only free (3) and/or as glycosyl derivatives. Nevertheless, the relative percentage of the sterols in free and bound forms are distinctly variable, e.g., free cholesterol (1.3%) (3), glycosyl cholesterol (11.3%), and only traces of esteried cholesterol. Conversely, the proportion of free sitosterol and dihydrositosterol appears to be higher (56.3%) than the glycosylated (30.8%) and esterified (6.46%) forms.

ACKNOWLEDGMENTS
Thanks are due to CONICET (Argentina) and Universidad de Buenos Aires (Argentina) for financial support; to Prof. Ing. A. Soriano and Dr. Dubcovsky for collection of the plant material; to Dr. E. Nicora (Instituto de Botnica Darwinion, Argentina) for identification of the plant material, and to LANAIS-EMAR (Argentina) for GC/MS facilities.

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1. Parodi, L.R. (1950) Las Gramneas Txicas para el Ganado en la Repblica Argentina, Rev. Arg. Agron. 17, 163229. 2. Nicora, E.A. (1978) Gramneas, in Flora Patagnica (Correa, M.N., ed.) Vol. 3, pp. 118119, Coleccin Cientca del Instituto Nacional de Tecnologa Agropecuaria, INTA, Buenos Aires. 3. Casabuono, A.C., and Pomilio, A.B. (1996) Festuca argentina and Festuca hieronymi: Chemical Relationships of Nonpolar Constituents, Biochem., Syst. Ecol. 24, 247254.

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[Received April 15, 1996, and in nal revised form November 19, 1996; Revision accepted December 4, 1996]

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