Plant Cell Reports (1984) 3:226-229 Plant Cell Reports © Springer-Verlag 1984

Sterols in seeds and leaves of oats (Arena sativa L.)

Waldemar Eichenberger and Birgit Urban

Department of Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland

Received August 29, 1984 - Communicated by I. Potrykus

ABSTRACT

In seeds and leaves of oats (Avena sativa L.) 12 different sterols (cholesterol, chole- stanol, ~ -cholestenol, campesterol, campe- stanol stigmas~erol lophenol _sitosterol stlgm~stanol, ~ -avenasterol, A-avenasterol and ~--stigmastenol) have been identified. The sterol pattern is qualitatively the same, but the relative composition is different in leaves and in seeds. Leaves contain mainly sitosterol, stigmasterol, cholesterol and campesterol, but only minor portion~ of aven~- sterols. Seeds contain sitosterol,A-- and ~'- avenasterol, campesterol, but only minor amounts of stigmasterol and cholesterol. In leaf lipids l-hexaecsanol (2.35 wt % of total lipid) has also been identified.

INTRODUCTION

The sterol composition of oat (Arena sa- tiva L.) seeds has previously been investigat- ed (Knights 1965; Knights 1967a, K~ights 1968, Youngs etTal. 1977). Sitosterol, A-avenaste- rol and A--avenasterol were found to be the major components. In addition, cholesterol, b~assicasterol, campesterol, stigmasterol,

-stigmastenol_ cholestanol campestanol,_ • ,/ ,

stlgmastanol, ~-cholestenol, 24-methyl-~/- cholestenol_ 24-methylenecholesterol and 24- ,! methylene-~-cholenstenol were detected as minor compounds. Therefore, oat seeds contain the C__ C.^ and C~^ sterol series. From

z/' ZU Z t%ese, the saturate~ compou~d~,~ - and A'~mgg99~gs as well as the A ....... - and the

' " -dienes are present.

In green leaves, in contrast, choleste- rol, sitosterol and stigmasterol, but no avenasterols were found as major components (Eichenberger 1982). In order to clarify the sterol composition of oat leaves, which seems to be different from that of seeds, we ex- tended our investigations also to the minor components using capillary gas chromatography and mass spectrometry as analytical tools.

MATERIALS AND METHODS

Oat plants were cultivated as described previously (Eichenberger 1982). Frozen leaves were extracted with methanol. The extract was saponified by alkali and the sterols were precipitated by digitonin (Eichenberger and

Menke 1966). Seed sterols were obtained by extraction of either oat seeds or of commer- cially available oat flour. 4-Methylsterols were separated from 4-demethylsterols on Silicagel 60 (Merck) plates using chloroform- ethanol 100:2 or hexane-acetone 85:15 (Lich- tenthaler et al. 1982) as solvents. Sterols were silylated (Eichenberger 1982) and the TMS-derivatives separated on a capillary co- lumn (0.25 mm I.D., i0 m of length) coated with SE-52 as a stationary phase. The tem- perature was 250°C and the flow 1 ml H 2 per min. Mass spectra were obtained on line on a Varian MAT 445 mass spectrometer.

RESULTS AND DISCUSSION

Sterols, isolated from green leaves of oats by digitonin precipitation and TLC puri- fication, yielded 19 peaks when separated by GLC. 12 of them could be identified by their retention time and by mass spectrometry. The data are given in Table i.

Cholesterol, campesterol, stigmasterol and sitosterel: The identity of these four components has been reported previously (Eichenberger 1982) and is demonstrated by their molecular ions m/e 458, 472, 484 and 486, respectively. They all produce the prom- inent signals due to the fragmentations 2, 4 and 9, which are typical of A unsaturation. The spectra were identical to those obtained from reference sterols.

A7-Cholestenol: The molecular ion m/e 458 suggests a C.., monoene. Fragments ii and 14 show that theZ~osition of the double bond is nuclear. The A ~unsaturation is strongly suggested by the fact that signals ii and 14 are of higher intensity, signals 2, 4, 9, i0 and 13 of lower intensity than the correspond- ing fragments of A sterols.

Lophenol ( 4 ~ - m e t h y l - ~ 7 - c h o ! e s t e n o l ) : The molecular ion m/e 472 suggests a C2R monoene. Fragments ii and 16 show that a douDle bond and an additional methyl group are present in the nucleus. The A-unsaturation is strong- ly suggested by the prominent signals ii and 14 and the low intensity~of signals 2, 4, 9, i0 and 13, compared to ~ sterols. The spec- tra were identical to those obtained from the reference compound. Since lophenol does not separate from stigmasterol in GLC under the

Table i.

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Identification of sterols from oat leaves. (Relative intensity of signals in brackets; R = SiMe3; SC = side chain; RRT = retention time relative to cholesterol- TMS) . The signals were attributed to fragments according to Knights (1967b) and Mercer and Bartlett (1974).

Fragmentation

~ o o o ~ o

,-~ ¢ ,-4 0 ® -~ @ 0

O O~ O ~ ~= 0 O~ ~ ~o ~< O I E -,d D., -.~ ,,-I I I I -e r"-- n~ 4J O -H 4J kO r'--.~ r--

M +

i M-Me

2 M-ROH

3 M-[Me+ROH]

4 M-[Cl-C3+RO] 5 M-SC

6 M-[SC+2H]

7 M-[C25-C27+ROH]

8 M-[CI5-C17+SC]

9 M-[C3-C7+ROH]

i0 M-[CI-C7+ROH]

ii M-[SC+ROH]

12 M-[SC+ROH+2H]

458(17) 458(32) 472(30) 484(26) 472(45) 486(26) 488(5) 484(4) 486(19) 484(2)

443(9) 443(14) 457(13) 469(7) 457(11) 471(10) 473(10) 469(3) 471(6) 469(3)

368(62) 368(10) 382(69) 394(50) 382(14) 396(74) 398(6) 394(9) 396(4) 394(2)

353(47) 353(21) 367(55) 379(18) 367(25) 381(44) 383(11) 379(6) 381(10) 379(5)

329(100) 329(5) 343(100) 355(21) 343(5) 357(100) 355(5) 357(4)

345(2) 345(8) 345(5) 345(2) 345(6)

345(5) 343(4)

351(22)

303(2) 303(5) 303(3) 317(4) 303(12) 305(13)

301(9) 315(13) 329(3) 329(10)

275(16) 275(4) 289(16) 301(3) 301(5)

343(100)

255(55) 255(100) 255(72) 255(100) 269(100) 255(71) 257(6) 255(31) 255(98) 255(19)

253(33) 253(75)

13 M-[Cl-C7,CI0,1018+ROH] 247(50) 247(9)

14 M-[CI6+CI7+SC+ROH]

15 M-[C23-C29+H+CI-C3+RO]

16 M-[CIs-CI7+SC+ROH]

17 M-[CIs-CI7+2H+SC+ROH]

18 M-[Cm3-C29+H]

19 M-[Me+C23-Cm9+H]

20 M-[C23-C29+H+ROH]

21 M-[Me+C23-C29+H+ROH]

261(59) 273(7) 247(11) 275(37) 275(3)

229(15) 229(50) 229(25) 229(15) 243(35) 229(17) 229(47)

257(38)

213(62) 213(94) 213(72) 213(63) 227(88) 213(62) 215(100)1213(45) 213(100) 213(55)

281(13)

211(67)

386(45) 386(30)

371(14) 371(8)

296(100) 296(13)

281(76) 281(21)

RRT sample 1.00 1.14 1.28 1.39 1.40 1.60 1.63 1.66 1.82 1.89

reference 1.00 1.12- 1.28 1.39 1.40 1.60 1.63 1.65" 1.82 1.85"

* Values reported by Homberg (1977) on SE-30 as stationary phase

conditions used, it had to be separated from the latter by TLC.

Stigmastanol: M + 488 indicates a satu- rated C^^-sterol and fragment ii the nucleus

z~ of a demethylsterol. However, fragment ii is of lower intensity ~han the corresponding fragment of ~ or ~ monoenes.

~5-Avenasterol: M + 484 indicates the C29 diene. Fragments ii, 12, 16 and 17 suggest one nuclear doubl~ bond and fragment 15 strong- ly suggests its ~ ~position. Fragments 15 and 18-21 are typical of the 24-ethylidene struc- ture.

A7Avenasterol: M + 484 indicates the C29

diene and fragments ll, 127and 16 suggest one nuclear double bond. The A -structure is shown by the prominent fragment 6 a~d by the absence of fragment 15. As in the A zsomer, the fragments 15 and 18-21, which are typical of the 24-ethylidene structure, are present.

• A7-Stigmastenol: M + 486 suggests a C_^ monoen-e7 ~ i-i and 12 show that th~ 9 double bond is nuclear. The ½ntense fragments ii, 12 and 14 indicate the A- unsaturation. The spectra were identical to those obtained from the reference compound isolated from spinach (Eichenberger and Menke 1966).

All the sterols isolated from oat leaves gave the same retention time as the cortes-

228

ponding reference compounds, as shown in Table 1. Cholestanol (RRT 1.02) and campe- stanol (RRT 1.30) were identified by their retention data only.

The sterol pattern of seeds or seed flour is qualitatively the same as in leaves, as shown in Table 2. However, the relative

Table 2. 8terols of seeds and leaves of oat

weight % of total sterols

leaves seeds* oat flour

Cholesterol 12.8 2.8 2.4

Cholestanol 0.2 trace 0.4

A7-Cholestenol 1.7 0.6 0.9

Campesterol 5.4 6.4 i0.i

Campestanol trace trace trace

Stigmasterol 28.4 3.9 4.6

Lophenol 1.8 0.6

Sitosterol 42.6 42.9 30.6

Stigmastanol 0.8 trace trace

A5-Avenasterol 2.4 26.1 23.2

~7-Stigmastenol 1.7 1.0 2.0

A7-Avenasterol 0.6 9.0 2.9

not identified 1.8 7.3 17.2

* A sample of oat seed sterols was obtained from Dr. J. Kesselmeier, Cologne (Germany).

amounts are different. Leaves contain much larger amounts of both cholesterol and stig- masterol, but lower proportions of avena- sterols than seeds or seed flour.

A possible reason for such differences mgy be the fact that avenasterols as well as A -stigmastenol are considered to be precur- sors of the common C9. sterols (sitosterol and stigmasterol) in~[he general biosynthetic pathway of plant sterols (Goad and Goodwin 1972). Therefore, the accumulation of avena- sterols in seeds could be due to the inhibi- tion of one or several steps leading from these precursors to sitosterol and stigma- sterol. Furthermore, the presence of lophenol, which has not been detected before in oat plants, is in agreement with the general view that this sterol is an intermediate in the biosynthesis of cholesterol (Grunwald 1975). However, lophenol, in contrast to avenasterols, does not accumulate in oat seeds.

In oat $1our, the percentage of sito- sterol and ~--avenasterol is lower, but the amount of unidentified sterols is consider- ably higher than in seeds. This difference could be explained by alterations of the sterol pattern at elevated temperatures dur- ing the processing of seeds to flour.

Besides the free sterols, oat leaves al- so contain steryl glycosides and acylated glycosides. Their quantity and sterol compo- sition as well as the distribution of sterol derivatives in the leaf are presently under investigation.

It is of interest to note that leaf li-

pids also contain considerable amounts of a very long chain alcohol which could be identified as l-hexacosanol. It accounts for 2.35 wt % of ether-soluble leaf lipids com- pared to 0.53 % total free sterols. On Sili- ca gel G (Merck) with chloroform-ethanol 100:2 as a solvent, l-hexacosanol gave a Rf value of 0.38 compared to 0.29 for the 4-de- methylsterols. The TMS derivative gave a retention time of 0.63 (relative to chole- sterol-TMS) . Mass spectra showed a very weak signal at m/e 454 (M+), but an intense sig- nal at m/e 439 (M-Me). The free alcohol gave a weak signal at m/e 382 (M+) , but intense signals at m/e 364 and 336, which could be attributed to M-H?O and M-[HgO + CgHa], res- pectively. The exact mass of-the mTe-364 fragment is 364.40~ and corresponds to the ~ormula of C26H~2.-~C-NMR (25.2 MHz CDCI~ H-broadband decoupling) gave slgnals at

~63.1 (C-l), ~32.9 (C-2), d25.8 (C-B), ~29.7 (C-4 to C-23], ~31.9 (C-24), W25.8 (C-25) and @14.0 (C-26). l-Hexacosanol (Fluka) used as r~erence gave identical data in GC, MS and ~C-NMR.

l-Hexacosanol was detected earlier in oat leaf wax (Tulloch and Hoffmann 1973) and seems to be widely distributed among plants (Karrer et al. 1981). The role of this long- chain alcohol, which we have found to be present not only in the epidermis but also in mesophyll cells, remains to be elucidated.

ACKNOWLEDGEMENTS

We are very grateful to Dr. P. Bigler (Dept. of Organic Chemistry) for the NMR spectra and their discussion and to H. Gfel- let (Dept. of Organic Chemistry) for the mass spectra. We thank Prof. Dr. W. Sucrow (Paderborn) for a sample of synthetic lophe- nol, and the Kentaur AG, Lutzelfl~h, for the generous gift of oat flour.

REFERENCES

Eichenberger W (1982) Plant Cell Reports 1:253-256

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Goad LJ, Goodwin TW (1972) in: Reinhold L, Liwschitz Y (eds.) Progress in Phytochemistry, Vol. III, Interscience Publishers, London, pp 113-198

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Homberg E (1977) J. Chromatogr. 139:77-84

Karrer W, H~rlimann H, Cherbuliez E (1981) Konstitution und Vorkommen der organischen Pflanzenstoffe. Birkh~user, Basel

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229

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