Plant Cell Reports

Plant Cell Reports (1984) 3:226-229

© 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 s e e d s and l e a v e s of oats ( A v e n a sativa L.) 12 d i f f e r e n t s t e r o l s ( c h o l e s t e r o l , c h o l e stanol, ~ -cholestenol, campesterol, campestanol stigmas~erol lophenol _sitosterol stlgm~stanol, ~ -avenasterol, A-avenasterol and ~ - - s t i g m a s t e n o l ) h a v e b e e n i d e n t i f i e d . The s t e r o l p a t t e r n is q u a l i t a t i v e l y the same, b u t the r e l a t i v e c o m p o s i t i o n is d i f f e r e n t in l e a v e s and in seeds. L e a v e s c o n t a i n m a i n l y s i t o s t e r o l , s t i g m a s t e r o l , c h o l e s t e r o l and c a m p e s t e r o l , b u t o n l y m i n o r p o r t i o n ~ of aven~s t e r o l s . S e e d s c o n t a i n s i t o s t e r o l , A - - a n d ~'avenasterol, campesterol, but only minor a m o u n t s of s t i g m a s t e r o l and c h o l e s t e r o l . In leaf l i p i d s l-hexaecsanol (2.35 wt % of t o t a l lipid) has a l s o b e e n i d e n t i f i e d . INTRODUCTION The s t e r o l c o m p o s i t i o n of oat ( A r e n a sativa L.) s e e d s has p r e v i o u s l y b e e n i n v e s t i g a t ed ( K n i g h t s 1965; K n i g h t s 1967a, K ~ i g h t s 1968, Y o u n g s etTal. 1977). S i t o s t e r o l , A - a v e n a s t e rol and A - - a v e n a s t e r o l w e r e f o u n d to b e the m a j o r c o m p o n e n t s . In a d d i t i o n , c h o l e s t e r o l , b~assicasterol, campesterol, stigmasterol, -•s t i g m a s t e n o l _,/ c h o l e s t a n o l , campestanol,_ stlgmastanol, ~-cholestenol, 24-methyl-~/c h o l e s t e n o l _,! 2 4 - m e t h y l e n e c h o l e s t e r o l and 24methylene-~-cholenstenol w e r e d e t e c t e d as m i n o r c o m p o u n d s . T h e r e f o r e , oat s e e d s c o n t a i n the C _ _ C . ^ and C~^ s t e r o l s e r i e s . F r o m z/' ZU Z

t%ese, the saturate~ c o m p o u ~ d ~ , ~

-

A'~mgg99~gs as w e l l as the A ....... - and ' " - d i e n e s are p r e s e n t .

and

the

In g r e e n leaves, in c o n t r a s t , c h o l e s t e rol, s i t o s t e r o l and s t i g m a s t e r o l , b u t no a v e n a s t e r o l s w e r e f o u n d as m a j o r c o m p o n e n t s ( E i c h e n b e r g e r 1982). In o r d e r to c l a r i f y the s t e r o l c o m p o s i t i o n of oat leaves, w h i c h s e e m s to b e d i f f e r e n t f r o m that of seeds, we ext e n d e d our i n v e s t i g a t i o n s a l s o to the m i n o r c o m p o n e n t s u s i n g c a p i l l a r y gas c h r o m a t o g r a p h y and m a s s s p e c t r o m e t r y as a n a l y t i c a l tools. MATERIALS

AND M E T H O D S

Oat p l a n t s w e r e c u l t i v a t e d as d e s c r i b e d p r e v i o u s l y ( E i c h e n b e r g e r 1982). F r o z e n l e a v e s were extracted with methanol. The extract was s a p o n i f i e d by a l k a l i and the s t e r o l s w e r e p r e c i p i t a t e d by d i g i t o n i n ( E i c h e n b e r g e r and

M e n k e 1966). S e e d s t e r o l s w e r e o b t a i n e d by e x t r a c t i o n of e i t h e r oat s e e d s or of c o m m e r c i a l l y a v a i l a b l e oat flour. 4 - M e t h y l s t e r o l s w e r e s e p a r a t e d f r o m 4 - d e m e t h y l s t e r o l s on S i l i c a g e l 60 (Merck) p l a t e s u s i n g c h l o r o f o r m e t h a n o l 1 0 0 : 2 or h e x a n e - a c e t o n e 8 5 : 1 5 (Licht e n t h a l e r et al. 1982) as s o l v e n t s . S t e r o l s w e r e s i l y l a t e d ( E i c h e n b e r g e r 1982) and the TMS-derivatives s e p a r a t e d on a c a p i l l a r y col u m n (0.25 mm I.D., i0 m of length) c o a t e d w i t h S E - 5 2 as a s t a t i o n a r y phase. T h e temp e r a t u r e w a s 2 5 0 ° C a n d the f l o w 1 ml H 2 per min. M a s s s p e c t r a w e r e o b t a i n e d on line on a Varian M A T 445 m a s s s p e c t r o m e t e r . RESULTS

AND

DISCUSSION

S t e r o l s , i s o l a t e d f r o m g r e e n l e a v e s of o a t s by d i g i t o n i n p r e c i p i t a t i o n and TLC p u r i f i c a t i o n , y i e l d e d 19 p e a k s w h e n s e p a r a t e d by GLC. 12 of them c o u l d be i d e n t i f i e d by t h e i r r e t e n t i o n time and b y m a s s s p e c t r o m e t r y . T h e d a t a are g i v e n in T a b l e i. Cholesterol, campesterol, stigmasterol and s i t o s t e r e l : T h e i d e n t i t y of t h e s e f o u r c o m p o n e n t s has b e e n r e p o r t e d p r e v i o u s l y ( E i c h e n b e r g e r 1982) and is d e m o n s t r a t e d by t h e i r m o l e c u l a r ions m/e 458, 472, 484 and 486, r e s p e c t i v e l y . T h e y all p r o d u c e the p r o m inent s i g n a l s d u e to the f r a g m e n t a t i o n s 2, 4 and 9, w h i c h are t y p i c a l of A unsaturation. T h e s p e c t r a w e r e i d e n t i c a l to t h o s e o b t a i n e d from reference sterols. A7-Cholestenol: T h e m o l e c u l a r ion m/e 458 s u g g e s t s a C.., m o n o e n e . F r a g m e n t s ii a n d 14 s h o w that t h e Z ~ o s i t i o n of the d o u b l e b o n d is n u c l e a r . T h e A ~ u n s a t u r a t i o n is s t r o n g l y s u g g e s t e d by the fact that s i g n a l s ii and 14 are of h i g h e r i n t e n s i t y , s i g n a l s 2, 4, 9, i0 and 13 of l o w e r i n t e n s i t y than the c o r r e s p o n d ing f r a g m e n t s of A sterols.

The Lophenol (4~-methyl-~7-cho!estenol): m o l e c u l a r ion m/e 472 s u g g e s t s a C 2 R m o n o e n e . F r a g m e n t s ii and 16 s h o w that a d o u D l e b o n d and an a d d i t i o n a l m e t h y l g r o u p are p r e s e n t in the n u c l e u s . T h e A - u n s a t u r a t i o n is s t r o n g ly s u g g e s t e d by the p r o m i n e n t s i g n a l s ii and 14 and the low i n t e n s i t y ~ o f s i g n a l s 2, 4, 9, i0 and 13, c o m p a r e d to ~ sterols. The spectra w e r e i d e n t i c a l to t h o s e o b t a i n e d f r o m the r e f e r e n c e c o m p o u n d . S i n c e l o p h e n o l d o e s not s e p a r a t e f r o m s t i g m a s t e r o l in GLC u n d e r the

227 Table

i.

Identification of s t e r o l s from oat leaves. ( R e l a t i v e i n t e n s i t y of s i g n a l s in b r a c k e t s ; R = SiMe3; SC = side chain; RRT = r e t e n t i o n time r e l a t i v e to c h o l e s t e r o l TMS) . The s i g n a l s w e r e a t t r i b u t e d to f r a g m e n t s a c c o r d i n g to K n i g h t s (1967b) and M e r c e r and B a r t l e t t (1974).

Fragmentation

o ,-~

¢

,-4

O

O -e

r"--

0

O~

I

0

O~

E n~

-,d 4J

~=

D., O

0

-.~ -H

O~

,,-I 4J

~ o

o ~

o

®

-~

@

~ kO

I

I

M+

458(17)

458(32)

472(30)

484(26)

472(45)

486(26)

488(5)

443(9)

443(14)

457(13)

469(7)

457(11)

471(10)

473(10) 469(3)

471(6)

469(3)

2 M-ROH

368(62)

368(10)

382(69)

394(50)

382(14)

396(74)

398(6)

396(4)

394(2)

383(11)

379(5)

4

M-[Cl-C3+RO]

5 M-SC

353(47)

353(21)

367(55)

379(18)

367(25)

381(44)

329(100)

329(5)

343(100)

355(21)

343(5)

357(100)

345(2)

345(8)

345(5)

379(6)

381(10)

355(5)

357(4)

345(5)

343(4)

345(2)

484(2)

345(6)

6 M-[SC+2H]

343(100)

351(22)

7 M-[C25-C27+ROH] 8 M-[CI5-C17+SC]

303(2)

9 M-[C3-C7+ROH]

301(9)

303(5)

303(3) 315(13)

329(3)

317(4)

303(12)

305(13)

329(10)

i0 M-[CI-C7+ROH]

275(16)

275(4)

289(16)

301(3)

ii M-[SC+ROH]

255(55)

255(100)

255(72)

255(100)

269(100)

255(71)

257(6)

301(5)

13 M-[Cl-C7,CI0,1018+ROH]

247(50)

247(9)

261(59)

273(7)

247(11)

275(37)

275(3)

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

229(15)

229(50)

229(25)

229(15)

243(35)

229(17)

12 M-[SC+ROH+2H]

255(31) 255(98)

255(19)

253(33)

253(75)

229(47) 257(38)

15 M-[C23-C29+H+CI-C3+RO] 16 M-[CIs-CI7+SC+ROH]

394(9)

486(19)

~< r--

i M-Me

3 M-[Me+ROH]

484(4)

~o

I r'--.~

213(62)

213(94)

213(72)

213(63)

227(88)

213(62)

215(100)1213(45) 213(100)

213(55)

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

211(67)

18 M-[Cm3-C29+H]

386(45)

386(30)

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

371(14)

371(8)

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

296(100)

296(13)

281(76)

281(21)

281(13)

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

RRT sample reference

1.00

1.14

1.28

1.39

1.40

1.60

1.63

1.66

1.82

1.89

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 the l a t t e r

used, it had by TLC.

to be

separated

from

Stigmastanol: M + 488 i n d i c a t e s a satur a t e d C ^ ^ - s t e r o l and f r a g m e n t ii the n u c l e u s z~ of a d e m e t h y l s t e r o l . However, f r a g m e n t ii is of lower i n t e n s i t y ~han the c o r r e s p o n d i n g f r a g m e n t of ~ or ~ monoenes. ~5-Avenasterol: M + 484 i n d i c a t e s the C29 diene. F r a g m e n t s ii, 12, 16 and 17 suggest one n u c l e a r d o u b l ~ b o n d and f r a g m e n t 15 strongly s u g g e s t s its ~ ~ p o s i t i o n . F r a g m e n t s 15 and 18-21 are t y p i c a l of the 2 4 - e t h y l i d e n e s t r u c ture. A7Avenasterol:

M + 484

indicates

the C29

d i e n e and f r a g m e n t s ll, 1 2 7 a n d 16 s u g g e s t one n u c l e a r d o u b l e bond. The A - s t r u c t u r e is shown by the p r o m i n e n t f r a g m e n t 6 a~d by the a b s e n c e of f r a g m e n t 15. As in the A zsomer, the f r a g m e n t s 15 and 18-21, w h i c h are typical of the 2 4 - e t h y l i d e n e s t r u c t u r e , are p r e s e n t . A7-Stigmastenol: M + 486 s u g g e s t s a C_^ monoen-e7 ~ i-i and 12 show that th~ 9 d o u b l e b o n d is nuclear. The ½ n t e n s e f r a g m e n t s ii, 12 and 14 i n d i c a t e the A- u n s a t u r a t i o n . The s p e c t r a were i d e n t i c a l to those o b t a i n e d from the r e f e r e n c e c o m p o u n d i s o l a t e d from s p i n a c h ( E i c h e n b e r g e r and M e n k e 1966). •

gave

All the s t e r o l s i s o l a t e d from oat leaves the same r e t e n t i o n time as the c o r t e s -

228 p o n d i n g r e f e r e n c e c o m p o u n d s , as s h o w n in T a b l e 1. C h o l e s t a n o l (RRT 1.02) and c a m p e s t a n o l (RRT 1.30) w e r e i d e n t i f i e d by t h e i r r e t e n t i o n d a t a only. T h e s t e r o l p a t t e r n of s e e d s or s e e d f l o u r is q u a l i t a t i v e l y the s a m e as in leaves, as s h o w n in T a b l e 2. H o w e v e r , the r e l a t i v e

Table

2.

8terols

of s e e d s weight

leaves

and

% of

leaves

total

seeds*

of oat

sterols 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

Stigmasterol

28.4

Lophenol

trace

3.9

4.6

1.8

Sitosterol

42.6

Stigmastanol

0.6 42.9

30.6

0.8

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

1.8

7.3

17.2

identified

* A sample from Dr.

l - H e x a c o s a n o l was d e t e c t e d e a r l i e r in oat leaf wax ( T u l l o c h and H o f f m a n n 1973) and s e e m s to be w i d e l y d i s t r i b u t e d a m o n g p l a n t s ( K a r r e r et al. 1981). T h e role of this l o n g c h a i n a l c o h o l , w h i c h we have f o u n d to b e p r e s e n t not o n l y in the e p i d e r m i s b u t also in m e s o p h y l l cells, r e m a i n s to be e l u c i d a t e d .

trace

of oat s e e d s t e r o l s w a s J. K e s s e l m e i e r , C o l o g n e

ACKNOWLEDGEMENTS

obtained (Germany).

a m o u n t s are d i f f e r e n t . L e a v e s c o n t a i n m u c h l a r g e r a m o u n t s of b o t h c h o l e s t e r o l and s t i g m a s t e r o l , b u t l o w e r p r o p o r t i o n s of a v e n a s t e r o l s than s e e d s or s e e d flour. A p o s s i b l e r e a s o n for s u c h d i f f e r e n c e s mgy be the f a c t that a v e n a s t e r o l s as w e l l as A - s t i g m a s t e n o l are c o n s i d e r e d to be p r e c u r sors of the c o m m o n C 9 . s t e r o l s ( s i t o s t e r o l and s t i g m a s t e r o l ) i n ~ [ h e g e n e r a l b i o s y n t h e t i c p a t h w a y of p l a n t s t e r o l s (Goad and G o o d w i n 1972). T h e r e f o r e , the a c c u m u l a t i o n of a v e n a s t e r o l s in s e e d s c o u l d be d u e to the i n h i b i tion of one or s e v e r a l s t e p s l e a d i n g f r o m t h e s e p r e c u r s o r s to s i t o s t e r o l and s t i g m a s t e r o l . F u r t h e r m o r e , the p r e s e n c e of l o p h e n o l , w h i c h has not b e e n d e t e c t e d b e f o r e in oat p l a n t s , is in a g r e e m e n t w i t h the g e n e r a l v i e w that this s t e r o l is an i n t e r m e d i a t e in the b i o s y n t h e s i s of c h o l e s t e r o l ( G r u n w a l d 1975). H o w e v e r , l o p h e n o l , in c o n t r a s t to avenasterols, does not a c c u m u l a t e in oat seeds. In o a t $1our, the p e r c e n t a g e of s i t o s t e r o l and ~ - - a v e n a s t e r o l is lower, b u t the a m o u n t of u n i d e n t i f i e d s t e r o l s is c o n s i d e r ably h i g h e r than in seeds. T h i s d i f f e r e n c e c o u l d be e x p l a i n e d by a l t e r a t i o n s of the s t e r o l p a t t e r n at e l e v a t e d t e m p e r a t u r e s d u r ing the p r o c e s s i n g of s e e d s to flour. B e s i d e s the free s t e r o l s , oat l e a v e s also c o n t a i n s t e r y l g l y c o s i d e s and a c y l a t e d glycosides. Their quantity and sterol compos i t i o n as w e l l as the d i s t r i b u t i o n of s t e r o l d e r i v a t i v e s in the leaf are p r e s e n t l y u n d e r investigation. It is of

interest

to n o t e

that

leaf

p i d s also c o n t a i n c o n s i d e r a b l e a m o u n t s of a v e r y l o n g c h a i n a l c o h o l w h i c h c o u l d be i d e n t i f i e d as l - h e x a c o s a n o l . It a c c o u n t s for 2.35 wt % of e t h e r - s o l u b l e leaf l i p i d s comp a r e d to 0.53 % t o t a l free s t e r o l s . On S i l i ca gel G (Merck) w i t h c h l o r o f o r m - e t h a n o l 1 0 0 : 2 as a s o l v e n t , l - h e x a c o s a n o l g a v e a Rf v a l u e of 0.38 c o m p a r e d to 0.29 for the 4 - d e m e t h y l s t e r o l s . T h e TMS d e r i v a t i v e g a v e a r e t e n t i o n time of 0.63 ( r e l a t i v e to c h o l e sterol-TMS) . Mass spectra showed a very weak s i g n a l at m/e 454 (M+), b u t an i n t e n s e signal at m/e 439 (M-Me). T h e f r e e a l c o h o l g a v e a w e a k s i g n a l at m/e 382 (M+) , but i n t e n s e s i g n a l s at m/e 364 and 336, w h i c h c o u l d be a t t r i b u t e d to M - H ? O and M - [ H g O + CgHa], resp e c t i v e l y . T h e e x a c t mass o f - t h e m T e - 3 6 4 f r a g m e n t is 3 6 4 . 4 0 ~ and c o r r e s p o n d s to the ~ o r m u l a of C 2 6 H ~ 2 . - ~ C - N M R (25.2 MHz CDCI~ H - b r o a d b a n d d e c o u p l i n g ) g a v e s l g n a l s at ~ 6 3 . 1 (C-l), ~ 3 2 . 9 (C-2), d 2 5 . 8 (C-B), ~ 2 9 . 7 (C-4 to C-23], ~ 3 1 . 9 (C-24), W 2 5 . 8 (C-25) and @ 1 4 . 0 (C-26). l - H e x a c o s a n o l (Fluka) u s e d as r ~ e r e n c e g a v e i d e n t i c a l d a t a in GC, MS and ~C-NMR.

li-

We are v e r y g r a t e f u l to Dr. P. B i g l e r (Dept. of O r g a n i c C h e m i s t r y ) for the N M R s p e c t r a and t h e i r d i s c u s s i o n and to H. G f e l let (Dept. of O r g a n i c C h e m i s t r y ) for the m a s s s p e c t r a . We t h a n k Prof. Dr. W. S u c r o w ( P a d e r b o r n ) for a s a m p l e of s y n t h e t i c l o p h e nol, and the K e n t a u r AG, L u t z e l f l ~ h , for the g e n e r o u s g i f t of oat flour. REFERENCES Eichenberger 1:253-256

W

(1982)

E i c h e n b e r g e r W, M e n k e schung 21b:859-867

Plant W

Cell

(1966)

Reports

Z. N a t u r f o r -

G o a d LJ, G o o d w i n TW (1972) in: R e i n h o l d L, L i w s c h i t z Y (eds.) P r o g r e s s in Phytochemistry, Vol. III, I n t e r s c i e n c e P u b l i s h e r s , L o n d o n , pp 1 1 3 - 1 9 8 Grunwald C 26:209-236 Homberg

(1975)

E (1977)

Ann.

Rev.

Plant

J. C h r o m a t o g r .

Physiol.

139:77-84

K a r r e r W, H ~ r l i m a n n H, C h e r b u l i e z E (1981) Konstitution und Vorkommen der organischen Pflanzenstoffe. Birkh~user, Basel Knights

BA

(1965)

Knights Knights 282

BA BA

(1967a) (1967 b)

Knights

BA

(1968)

Phytochemistry

4:857-862

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