Plant Cell Reports

Plant Cell Reports (1982) 1:253-256

© Springer-Verlag1982

Incorporation of [4-14C] Cholesterol into Steryl Derivatives and Saponins of Oat (Avena sativa L.) Plants Waldemar Eichenberger Department of Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland Received June 23, 1982/August 2, 1982

ABSTRACT

MATERIALS

Sterols from both green and e t i o l a t e d oat plants (Avena sativa) c o n t a i n sitosterol, stigmasterol, cholesterol, ~ 7 - s t i g m a s t e n o l , ~ 7 - c h o l e s t e n o l and campesterol. In the samonin fraction a v e n a c o s i d e s A and B and 26-desglucoa v e n a c o s i d e s A and B were detected. E t i o l a t e d plants i n c o r p o r a t e d [ 4 - 1 4 C ] c h o l e s t e r o l into steryl d e r i v a t i v e s (esters, g l y c o s i d e s and a c y l a t e d glycosides) and also into all of the 4 saponins. [4-14C]sitosterol, however, is i n c o r p o r a t e d only into steryl derivatives, but not in saponins. F r o m this it is concluded that cholesterol, but not s i t o s t e r o l is %he in vivo p r e c u r s o r of oat sanonins. A b b r e v i a t i o n s : GLC, gas liquid chromatography; I.D., inner diameter; TMS, trimethylsilyl; dpm, d e c o m p o s i t i o n s per minute.

Oat seeds (Vatter were soaked for 1 day in tap w a t e r and g e r m i n a t e d for six days on V e r m i c u l i t e at 23 ° C, under 5000 ix fluorescent light (for green plants) and in the dark for e t i o l a t e d Dlants.

INTRODUCTION Steroid saponins of oat (Avena sativa) have been k n o w n for several years (Tschesche and S c h m i d t 1966). They have been named avenacoside A (Tschesche e t a l . 1969) and avenacoside B (Tschesche and L a u v e n 1971). Two other saponins, i d e n t i f i e d as 2 6 - d e s g l u c o a v e n a c o s i d e s A and B, were later found in h o m o g e n a t e s of oat p l a n t s (Tschesche and W i e m a n n 1977, K e s s e l m e i e r and Strack 1981). All of the four c o m p o u n d s c o n t a i n n u a t i g e n i n as an a g l y c o n , a n d rhamnose and glucose in their c a r b o h y d r a t e residues. Oat saponins have been r e p o r t e d to be c o n s t i t u e n t s of i s o l a t e d m r o l a m e l l a r bodies of e t i o l a t e d oat leaves, where they are obv i o u s l y i n v o l v e d in the f o r m a t i o n of tubular s t r u c t u r e s (Kesselmeier and B u d z i k i e w i c z 1979; K e s s e l m e i e r and Ruppel 1979). First c o n s i d e ~ ed to be a v e n a c o s i d e s A and B, these compounds later turned out to be 2 6 - d e s g l u c o a v e n a c o s i d e s A and B (Kesselmeier 1982). The w o r k p r e s e n t e d in this paper is conc e r n e d with the b i o g e n e s i s of avenacosides, w h i c h has not been i n v e s t i g a t e d so far. In order to i d e n t i f y the p o s s i b l e in vivo precursors, the sterols of oat plants were analysed. Two m a j o r c o m p o n e n t s were fed to the plant as labelled precursors, and the incorp o r a t i o n of r a d i o a c t i v i t y into saponins and steryl d e r i v a t i v e s was measured.

AND METHODS

Extraction

and F r a c t i o n a t i o n

of Lipids

Leaves were frozen and p o w d e r e d in liquid N~ and e x t r a c t e d e x h a u s t i v e l y with m e t h a n o l aid ethyl ether. For the n r e p a r a t i o n of saponins, 3.3 g of lipids were d i s s o l v e d ~ chlorof o r m - m e t h a n o l 95:5 (v/v) and loaded on a column of 30 g S i l i c a g e l 60 (0.067 - 0.2 mm, Merck) s u s p e n d e d in the same solvent. E l u t i o n was o b t a i n e d with 150 ml c h l o r o f o r m - m e t h a n o l 9 5 : 5 (v/v) (fraction I), 150 ml c h l o r o f o r m m e t h a n o l 90:10 (v/v) (fraction II), 400 ml c h l o r o f o r m - m e t h a n o l - H g O 70:30:4 (by vol.) (fraction III), iOO mI c h l o r o f o r m - m e t h a n o l 3:2 (v/v) (fraction IV), iO0 ml c h l o r o f o r m m e t h a n o l i:i (v/v) (fraction V) and iOO ml m e t h a n o l (fraction VI). Isolation

and A n a l y s i s

of Sterols

Sterols were isolated from c o l u m n fractions I and II by a l k a l i n e s a p o n i f i c a t i o n and p r e c i p i t a t i o n w i t h d i g i t o n i n (Eichenberger and Menke 1966). S i l y l a t i o n was c a r r i e d out in i00 ~i p y r i d i n and 20 N1 each of hexam e t h y l d i s i l a z a n e and t r i m e t h y l c h l o r o s i l a n e . For analysis of sterol-TMS, a glass column (1.8 m length, 3 m m I.D.) m a c k e d with 2 % OV-IOI on C h r o m o s o r b W at 180 ° C and a flow of 30 ml N g / m i n was used. Mass s~ectra were o b t a i n e d oN line after s e p a r a t i o n of sterols on a SE-52 c a D i l l a r v column (14 m length) at 220 - 270 ° C on a V a r i a n MAT 44 S. Isolation

and A n a l y s i s

of SaDonins

Saponins a p p e a r e d in fractions I I I - V and were further p u r i f i e d on S i l i c a g e l 60 (Merck) plates with c h l o r o f o r m - m e t h a n o l - H g O 70:30:5.5 (Kesselmeier and B u d z i k i e w i c z 1979) as a solvent. For the i s o l a t i o n of aglyca, saponins were h y d r o l y z e d in m e t h a n o l i c HCl (Tschesche et al. 1961). S a p o g e n i n s were chromatographed on S i l i c a g e l 60 (Merck) plates with chlorof o r m - e t h a n o l 95:5 (v/v) (Kesselmeier and B u d z i k i e w i c z 1979), e l u t e d and used for mass spectrometry.

0721-7714/82/0001/0253/$ 01.00

254 F o r the i d e n t i f i c a t i o n of sugars, s a p o n ins w e r e h y d r o l y z e d in K i l i a n i s o l u t i o n (Kopp et al. 1981) or in 0.5 M H^SO. in d i o x a n e - H g O 1:3 (v/v) ( G e s t e t n 4 r et ~ 1 . 9 1 9 6 6 ) . S u g a r = T M S p r e p a r e d in the same w a y as s t e r o l TMS w e r e s e p a r a t e d by GLC on a g l a s s c o l u m n (1.8 m length, 3 m m I.D.) p a c k e d w i t h 2.5 % s i l i c o n e E - 3 O I on C h r o m o s o r b W at 176 ° C and a flow of 31 ml Ng/min. L ( + ) - r h a m n o s e and D(+)-glucose (Merdk) w e r e u s e d as r e f e r e n c e s . Incubation with precursors: [4-14C]choi e s t e r o l (Amersham) 2.16 G B q / m m o l or [4-14C] sitosterol (Amersham), 2.15 G B q / m m o l w e r e s o l u b i l i z e d in a 2% T w e e n 80 s o l u t i o n . 1 D1 c o n t a i n i n g 105 d p m (0.78 nmol) w a s i n j e c t e d into the p r i m a r y leaf of e a c h plant.

b)

I d e n t i f i c a t i o n of S a n o n i n s and I n c o r p o r a t i o n of L a b e l l e d S t e r o l s into S t e r y l D e r i v a t i v e s and S a p o n i n s

In o r d e r to e x a m i n e the p o s s i b l e in L vlvo p r e c u r s o r s o [ 4 s a m o n i n s , 6 - d a y - o l d p l a n t s w S r e f e d w i t h [4C]cholesterol (105 dpm, O1.78 nmol), as d e s c r i b e d in M e t h o d s . A f t e r 3 days of i n c u b a t i o n , the lipids w e r e extracted, c h r o m a t o g r a p h e d on t h i n layer p l a t e s and autoradiographed. In the a u t o r a d i o g r a m , 8spots w e r e o b s e r v e d as i l l u s t r a t e d in Fig. 2. All of the r a d i o a c t i v e spots c o i n c i d e d w i t h redd i s h c o l o u r e d spots on the c h r o m a t o g r a m obt a i n e d by s p r a y i n g w i t h 25% D e r c h l o r i c acid.

RESULTS a)

Sterol

Composition

W h e n the s t e r o l s f r o m g r e e n and from e t i o l a t e d oat l e a v e s w e r e a n a l y s e d by GLC, a m i x t u r e of 6 c o m p o n e n t s was f o u n d in e a c h case (Fig. i). The m a j o r c o m p o n e n t s w e r e s i t o s t e r o l , s t i g m a s t e r o l , and c h o l e s t e r o l , all i d e n t i f i e d by r e f e r e n c e c o m p o u n d s , ret e n t i o n d a t a and m a s s spectra. I n _ a d d i t i o n , A7-cholestenol, c a m p e s t e r o l and A l - s t i g m a s t e n o l w e r e p r e s e n t as m i n o r c o m p o n e n t s . The two A 7 - s t e r o l s w e r e i d e n t i f i e d by m a s s spectra and r e l a t i v e r e t e n t i o n data: (Homberg 1977). The r e l a t i v e amounts, w h i c h are also g i v e n in Fig. i, w e r e c a l c u l a t e d f r o m the p e a k areas. The s t e r o l p a t t e r n s w e r e the same

15 rain

10

Front

5

1

I

GREEN J

;

~

Fig. 1 - S e p a r a t i o n g r e e n and e t i o l a t e d

AREA

PERCENT

Mw GREENETIOLATED

CHOLESTEROL 386 LA7- CHOLESTENOL 386 CAMPESTEROL 400 STIGMASTEROL 412 SITOSTEROL 414 AT- STIGMASTENOL 414

Rf

1

0.95

2 3

0.87 0.82

4

0.70

5

0.39

6 7

0.31 0.23

8

0.15

Start

ETIOL~ L] L

Spot

9.7 3.6 3.5 33.& 41,7

14.3 5.3 4.0 26.3 46.0

7.5

6.0

by GLC of T M S - s t e r o l s oat leaves.

of

for b o t h g r e e n and e t i o l a t e d plants. However, the r e l a t i v e a m o u n t of c h o l e s t e r o l was h i g h er in e t i o l a t e d t h a n in g r e e n leaves. The s t e r o l p a t t e r n o b t a i n e d here for oat l e a v e s is s i m i l a r to that p r e v i o u s l y r e p o r t e d for oat seeds (Knights 1965; K n i g h t s and L a u r i e 1967), e x o e p t that the l a t t e r a u t h o r s also d e t e c t e d two m o r e a v e n a s t e r o l s (24-ethylidene~ 5 - c h ~ e s t e n o l and 24-ethylidene-~choles~nol).

Fig. 2 - A u t o r a d i o g r a m of leaf l i p i d s from oat p l a n t s i n c u b a t e d w i t h [ 4 - 1 4 C ] c h o l e s t e r o l for 3 days.

By c o m p a r i s o n to r e f e r e n c e c o m p o u n d s , spots 1 - 4 c o u l d be i d e n t i f i e d as s t e r y l e s t e r s (i), s t e r o l s (2), a c y l a t e d s t e r y l glyc o s i d e s (3), a n d s t e r y l g l y c o s i d e s (4). The s l o w e r m o v i n g spots 5 - 8 w e r e s u p p o s e d to be s a p o n i n s ; to v e r i f y t h e i r i d e n t i t i e s , they w e r e c o m p a r e d to s a p o n i n s w h i c h w e r e p r e p a r e d in pure f o r m f r o m u n l a b e l l e d lipids, by a c o m b i n a t i o n of c o l u m n and t h i n layer c h r o m a t o g r a p h y . The a g l y c a w e r e o b t a i n e d by m e t h a n o lysis a n d s h o w e d the same p r o p e r t i e s in all four cases: a) On S i l i c a g e l G p l a t e s w i t h c h l o r o f o r m e t h a n o l 95:5 (v/v) as a s o l v e n t and perc h l o r i c a c i d as a spray reagent, a r e d d i s h spot w i t h rf 0.37 was o b t a i n e d . b) M a s s s p e c t r a c o n t a i n e d s i g n a l s (M-), 399, 342, 324, 300, 282, and 155.

at m / e 430 271, 253

h e m a s s s p e c t r a and the b e h a v i o u r on thin layer c h r o m a t o g r a p h y are b o t h t y p i c a l of the

255 sapogenin nuatigenin k i e w i c z 1979). T h e s e cate that nuatigenin c o m p o u n d s 5 - 8.

(Kesselmeier and Budziresults strongly indiis the a g l y c o n of the

steryl esters, glycosides and acylated glycosides. M o s t of the r a d i o a c t i v i t y a p p e a r e d in the s t e r o l e s t e r f r a c t i o n , c h o l e s t e r o l b e i n g esterified even more rapidly than sitosterol. Cholesterol was incorporated into glycosides a n d a c y l a t e d g l y c o s i d e s to the e x t e n t of 5 % each, w h e r e a s a b o u t t w i c e as m u c h s i t o s t e r o l was incorporated into these derivatives. How-

The sugar moieties obtained by hydrolysis of t h e c o m p o u n d s 5 - 8 w e r e i d e n t i f i e d a n d q u a n t i f i e d as T M S d e r i v a t i v e s b y GLC. B y c o m p a r i s o n w i t h r e f e r e n c e h e x o s e s all of the

5 RHAMNOSE

/ MIN 10

5

0

10

5

0

10

5

0

10

5

0

MOLAR RATIO GLUCOSE/RHAMNOSE

Fig.

2.03

2.93

3.13

4.06

+0.48

+0.5

+0.54

+0.43

3 - Separation

b y GLC o f T M S - s u g a r s

four compounds contain glucose and rhamnose as t h e o n l y m a j o r c o m p o n e n t s , as s h o w n in Fig. 3. T h e m o l a r r a t i o s of g l u c o s e / r h a m n o s e as c a l c u l a t e d f r o m the p e a k a r e a s in 4 - 8 exp e r i m e n t s are a l s o g i v e n in Fig. 3. B a s e d o n t h e s e r e s u l t s , t h e r e is g o o d e v i d e n c e t h a t c o m p o u n d 5 is i d e n t i c a l w i t h 2 6 - d e s g l u c o a v e n a c o s i d e A ( g l c / r h a 2:1) a n d c o m p o u n d 6 with 26-desglucoavenacoside B ( g l c / r h a 3:1) ( T s c h e s c h e a n d W i e m a n n 1977). C o m p o u n d s 7 a n d 8 t h e r e f o r e c o r r e s p o n d to a v e n a c o s i d e A (glc/ r h a 3:1) ( T s c h e s c h e e t al. 1969) a n d a v e n a c o s i d e B ( g l c / r h a 4:1) ( T s c h e s c h e a n d L a u v e n 1971), r e s p e c t i v e l y . T h e p h e n o m e n o n of deg l u c o s y l a t i o n of a v e n a c o s i d e s i n t o 2 6 - d e s glucoavenacosides has been reported previously ( T s c h e s c h e a n d W i e m a n n 1977; K e s s e l m e i e r 1982). In a s e p a r a t e e x p e r i m e n t , the s a p o n i n patterns of extracts from fresh or frozen p l a n t s w e r e c o m p a r e d to t h o s e f r o m e x t r a c t s of s m a s h e d p l a n t s . In e x t r a c t s of f r o z e n o r fresh plants, mainly compounds 7 and 8 could be detected whereas extracts from smashed p l a n t s c o n t a i n e d m a i n l y c o m p o u n d s 5 a n d 6. T h e s e r e s u l t s f u r t h e r c o r r o b o r a t e the i d e n t i f i c a t i o n o f c o m p o u n d s 7 a n d 8 as a v e n a c o s i d e s a n d 5 a n d 6 as 2 6 - d e s g l u c o a v e n a c o s i d e s . In o r d e r to i n v e s t i g a t e the i n c o r p o r a tion of different precursors, 6-day-old etiol a t e d p l a n t s w e r e i n c u b a t e d for 5 d a y s w i t h either- [4-14C]cholesterol or [4-14C]sito s t e r o l . T w o to t h r e e p l a n t s w e r e e x t r a c t e d at a t i m e a n d the l i p i d s s e p a r a t e d o n T L C plates. After visualization with perchloric acid, the s p o t s w e r e s c r a p e d o f f a n d c o u n t e d by liquid scintillation spectrometry. and

As s h o w n in Fig. 4, b o t h c h o l e s t e r o l s i t o s t e r o l w e r e r a o i d l y i n c o r p o r a t e d into

from compounds

5 - 8.

]

15

PRECURSOR : 14C-CHOLESTEROL []

10

D~

/ []

D

-0,5

o

t3_

o1¢ Z

p 1

2

/PREc0

3

4

5

INCUBATION

s0R

1

s TOSTE OL

2

3

4

5

DAYS

Fig. 4 ~ 4 1 n c o r m o r a t i o n of [ 4 - 1 4 C ] c h o l e s t e r o l a n d [4C]sitosterol into steryl derivatives and saoonins by etiolated oat leaves. Steryl e s t e r s (D), s t e r y l g l y c o s i d e s ([]) , a c y l a t e d steryl glycosides (I), d e s g l u c o a v e n a c o s i d e A (O), d e s g l u c o a v e n a c o s i d e B (@), a v e n a c o s i d e A (~), a v e n a c o s i d e B (Q) . V a l u e s m l o t t e d are the m e a n of 2 - 3 d e t e r m i n a t i o n s .

256 ever, analysis of the saponin fractions rev e a l e d that 3 - 8 % of c h o l e s t e r o l administered to the plant was i n c o r p o r a t e d into each of the four saponins. M e t h a n o l y s i s of the saponins was carried out to show that radioa c t i v i t y was l o c a l i z e d e x c l u s i v e l y in the n u a t i g e n i n moiety. Sitosterol, on the other hand, is i n c o r p o r a t e d only in trace amounts, s u g g e s t i n g that this C 2 9 - s t e r o l is not a precursor of avenacosides.

DISCUSSION N u a t i g e n i n as an a g l y c o n of saponins has b e e n found in S o l a n u m s i s y m b r i i f o l i u m (Tschesche and R i c h e r t 1964) and Avena sativa (Tschesche and Schmidt 1966). I s o n u a t i g e n ~ was recently d e t e c t e d in S o l a n u m a b u t i l o i d e s (Evans et al. 1981), but the b i o g e n e s i s of these two compounds has not been i n v e s t i g a t e d so far. The results d e s c r i b e d in this paper show that labelled cholesterol, but not sitosterol, is i n c o r p o r a t e d into n u a t i g e n i n by oat leaves. Since c h o l e s t e r o l accounts for i0 - 14 % of the total sterols, it is likely that this sterol is the in vivo p r e c u r s o r of nuatigenin, at least in oat plants. The function of c h o l e s t e r o l as a precursor of other sapogenins has been reported in D i o s c o r e a s D i c u l i f l o r a (Bennett and H e f t m a n n 1965), L y c o p e r s i c o n p i m p i n e l l i f o l i u m (Bennett et al. 1967), and D i g i t a l i s lanata (Tschesche and Hulpke 1966; Varma et al. 1969). Tissue cultures of D i o s c o r e a d e l t o i d e a also used sitosterol as a p r e c u r s o r of sapogenins (Stohs et al. 1974). In the sequence of reactions leading from the sterol to the sapogenin, h y d r o x y l a tion at C-26 is g e n e r a l l y c o n s i d e r e d to be the first step (Takeda 1972). With respect to the f o r m a t i o n of the c a r b o h y d r a t e moiety, a stepwise g l y c o s y l a t i o n at C-3 m a y be assumed, as shown in the case of c a r d e n o l i d e s of C o n v a l l a r i a m a j a l i s (L~ffelhardt and Kopp 1981). W h e t h e r a similar m e c h a n i s m is also w o r k i n g in oat plants, remains to be clarified. T s c h e s c h e and H u l p k e (1966) reported that c h o l e s t e r o l from c h o l e s t e r y l g l u c o s i d e was t r a n s f o r m e d into sapogenins by D i g i t a l i s lanata. It is, however, not clear w h e t h e r the hexose part was also i n c o r p o r a t e d into the saponin. Since c h o l e s t e r o l is also rapidly i n c o r p o r a t e d into steryl g l y c o s i d e s and acylated g l y c o s i d e s by oat leaves, the role of these d e r i v a t i v e s in the further transformation of the sterol part has to be elucidated. Based on the present results, a role of 2 6 - d e s g l u c o a v e n a c o s i d e s as i n t e r m e d i a t e s in the synthesis of a v e n a c o s i d e s seems unlikely. Since the latter are very rapidly deglucosylated to 2 6 - d e s g l u c o a v e n a c o s i d e s by endogenous enzymes, as o b s e r v e d also by Kesselmeier (1982), the label found in 26-desglucoa v e n a c o s i d e s o r i g i n a t e s m o r e likely from secondary d e g r a d a t i o n of avenacosides. This h y d r o l y s i s could be due to tissue lesions either caused by the i n j e c t i o n of substrate or by the extraction.

ACKNOWLEDGEMENTS This work has been s u p p o r t e d by the Swiss N a t i o n a l Science Foundation. I thank A n d r e a Koster and Renate Kr~uchi for technical assistance, and Dr. Leticia M e n d i o l a - M o r g e n thaler for critical r e a d i n g of the manuscript. Mass spectra were o b t a i n e d by H. Gfeller, Dep a r t m e n t of O r g a n i c Chemistry.

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RD, H e f t m a n n

E

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W, Menke W

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BA

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Knights 407-416

BA, Laurie W

139:

H

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77-84

(1979)

Z Pflan-

Z Naturforsch

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