Plant Cell Reports
Plant Cell Reports (1988) 7:508-511
© Springer-Verlag 1988
Examination of triterpenoids produced by callus and cell suspension cultures of Glycyrrhiza glabra H. Hayashi, H. Fukui, and M. Tabata Faculty of Pharmaceutical Sciences, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, Japan Received July 13, 1988/Revised version received October 10, 1988 - Communicated by F. Constabel
ABSTRACT
C a l l u s and c e l l s u s p e n s i o n c u l t u r e s of Glycyrrhiza glabra failed to produce d e t e c t a b l e amounts of g l y c y r r h i z i n , the major oleanane-type triterpene glycoside of the thickening root, or of its 1 I deoxoderivative. However, b e t u l i n i c acid, a l u p a n e - t y p e t r i t e r p e n e , w h i c h w a s f o u n d in the root bark, and a small amount of Bamyrin, a p o s s i b l e p r e c u r s o r of o l e a n a n e - t y p e triterpenes, were detected in cel 1 suspension c u l t u r e s in a d d i t i o n to lupeol, a f u n d a m e n t a l form of l u p a n e - t y p e triterpenes. T h e s e f i n d i n g s s u g g e s t t h a t the a b s e n c e of glycyrrhizin in u n d i f f e r e n t i a t e d cultured c e l l s m a y be p a r t l y due to i n t e r r u p t i o n of the later reactions leading to the synthesis of glycyrrhizin from a triterpenoid intermediate. INTRODUCTION
Glycyrrhizin, the main triterpene s a p o n i n c o n t a i n e d in the r o o t of G__~.g l a b r a , is u s e d in l a r g e q u a n t i t i e s as a n a t u r a l sweetener and a l s o for medical treatments of chronic hepatitis in Japan. It was reported that g l y c y r r h i z i n was produced in s u b s t a n t i a l amounts by c a l l u s and cell suspension c u l t u r e s of G__~.g l a b r a ( T a m a k i et al. 1972; Yoo and Kim 1976), but its chemical i d e n t i f i c a t i o n remained ambiguous. The present study has been u n d e r t a k e n in an attempt to c l a r i f y whether g l y c y r r h i z i n is accumulated by c a l l u s or c e l l suspension c u l t u r e s d e r i v e d f r o m v a r i o u s o r g a n s of _G. g l a b r a seedlings, and to find out what kinds of t r i t e r p e n o i d s are produced by the c u l t u r e d cells. MATERIALS
AND
METHODS
Chemicals Glycyrrhizin and 11-deoxoglycyrrhizin, s y n t h e t i c a l l y d e r i v e d from g l y c y r r h i z i n , were supplied by M a r u z e n K a s e i Co. Ltd., Japan. T~ latter compound was i d e n t i f i e d by IH- and - N M R in c o m p a r i s o n s w i t h an a u t h e n t i c s a m p l e of 1 1 - d e o x o g l y c y r r h i z i n i s o l a t e d from licorice r o o t by K i t a g a w a et al.(1 987).
Offprint requests to." H. Hayashi
A u t h e n t i c s a m p l e s of b e t u l i n i c acid, B-amyrin and l u p e o l were o b t a i n e d from Dr. W. Kamisako of M u k o g a w a Women's U n i v e r s i t y , Japan. P l a n t m a t e r i a l and c u l t u r e m e t h o d The seeds, l e a v e s , stems, a n d r o o t s of G__~. g l a b r a L. w e r e c o l l e c t e d from fruitbearing plants (plant height: c a 90 cm) g r o w i n g w i l d by the r o a d s i d e in the s u b u r b of Mu~, Turkey in August, 1986. Callus cultures were derived from various p a r t s (root, h y p o c o t y l , stem, a n d leaf) of 4 - w e e k - o l d seedlings on n u t r i e n t agar medium (Linsmaier and Skoog 1 965) c o n t a i n i n g 100 ~M 1 - n a p h t h a l e n e a c e t i c acid (NAA) a n d I ~M 6 - b e n z y l a d e n i n e (BA), a n d s u b c u l t u r e d on the same m e d i u m at i n t e r v a l s of 5 w e e k s for 18 m o n t h s in the d a r k at 25°C. Suspension cultures (RNS-strain) initiated from the root c a l l u s were s u b c u l t u r e d in the a b o v e m e d i u m (I00 ml) w i t h o u t a g a r in 3 0 0 m l E r l e n m e y e r flasks a g i t a t e d on a r e c i p r o c a l shaker (100 strokes/min) at i n t e r v a l s of 3 to 4 weeks for a period of 1 5 months. Suspension cultures (RDS-strain) were also e s t a b l i s h e d in LS m e d i u m c o n t a i n i n g I ~M 2,4dichlorophenoxyacetic a c i d (2,4-D) and I ~M kinetin. For s t u d y i n g the t i m e c o u r s e of g r o w t h a n d t r i t e r p e n e p r o d u c t i o n , c e l l s (I g) w e r e inoculated in e a c h 100 ml flask (30 m l medium) and incubated under the same c o n d i t i o n s as i n d i c a t e d a b o v e . Cells were h a r v e s t e d from three flasks e v e r y three days d u r i n g a c u l t u r e p e r i o d of 24 d a y s to m e a s u r e the c e l l growth and the contents of b e t u l i n i c acid and phytosterols. To p r o v i d e a l a r g e s a m p l e of c e l l s for c h e m i c a l a n a l y s i s , c__aa.120 g of c e l l s in I 1 of s p e n t m e d i u m w e r e u s e d to i n o c u l a t e a 4 ljar fermentor c o n t a i n i n g 3 1 of fresh medium, and cultured at 2 5 ° C in t h e d a r k . The m e d i u m w a s a g i t a t e d by a s c r e w - t y p e s t i r r e r (60 rpm) a n d a e r a t e d by b u b b l i n g (200 ml air/min). Cells were harvested four weeks a f t e r i n o c u l a t i o n a n d a i r - d r i e d at 6 5 ° C for one week. Isolation and identification of triterDenes and p h y t o s t e r o l s Dried c e l l s (73 g) m e n t i o n e d a b o v e were e x t r a c t e d with M e O H (I 1 x 3) by reflux, then the M e O H was e v a p o r a t e d in v a c u o at 40 °C.
509 The residue (c__{a. 10g) was s u s p e n d e d in 300 ml of H20, a n d the pH was a d j u s t e d to 4 w i t h 0.1N HCI. T h i s s o l u t i o n was e x t r a c t e d w i t h EtOAc ( 3 0 0 m l x3); the EtOAc layer was e v a p o r a t e d in v a c u o to g i v e an e x t r a c t (2.4 g), which was s u b j e c t e d to s i l i c a gel c o l u m n c h r o m a t o g r a p h y (80 g W a k o g e l C-I00 in 3 cm x 30 cm column) using a d m i x t u r e s of C H C I 3 - E t O A c (20:0, 19:1, 18:2, 16:4, 10:10, 500 ml each) and of E t O A c - M e O H (20:0, 19:1, 18:1, 16:4, 10:10, 0:20, 500 ml each) in t h a t o r d e r as solvents. A f r a c t i o n (0.38 g) e l u t e d w i t h CHCI3-EtOAc (18:2) was r e c r y s t a l l i z e d from M e O H to y i e l d c o l o r l e s s n e e d l e s (92 mg), m.p. 283-286°C. This compound was identified as b e t u l i n i c acid, 3 - h y d r o x y - l u p - 2 0 ( 2 9 ) e n - 2 8 - o i c acid,1 by TLC, MS, G C - M S (as m e t h y l ester), IR, H-NMR, and high resolution MS in c o m p a r i s o n with an a u t h e n t i c sample. From a fraction (0.45 g) e l u t e d w i t h CHCI 3 - E t O A c (I 9:1), a mixture of triterpenes were o b t a i n e d by p r e p a r a t i v e TLC. This sample was a n a l y z e d by c a p i l l a r y GC-MS: c a p i l l a r y c o l u m n (Shimadzu C B P 5 - M 2 5 - 0 2 5 ) , gas flow: He {0.5 k g / c m 2, s p l i t 40 m l / m i n , purge 2 m l / m i n ) , c o l u m n temp.: 2 0 0 - 3 0 0 ° C (10°C/rain). Lupeol and B-amyrin were identified by comparison with authentic samples. Phytosterols (sitosterol and stigmasterol) contained in the above fraction were identified by GC-MS. Q u a n t i t a t i v e a n a l y s i s of b e t u l i n i c acid and phytosterols A p o w d e r e d s a m p l e (100 rag) of c u l t u r e d c e l l s was e x t r a c t e d w i t h C H C l 3 (4 ml x2) by reflux. After evaporation of C H C I 3 , the r e s i d u e was d i s s o l v e d in E t 2 0 a n d t r e a t e d with d i a z o m e t h a n e to m e t h y l a t e the c a r b o x y l group of b e t u l i n i c acid. C h o l e s t e r o l (0.5mg) was a d d e d to the s o l u t i o n as an i n t e r n a l s t a n d a r d . An a l i q u o t ( 2 0 pl) of t h i s s a m p l e (I ml) was a n a l y z e d by GC: g l a s s c o l u m n (3 mm x I m) p a c k e d w i t h S i l i c o n e O V - 1 7 (1%} on C h r o m o s o r b W ( A W - D M C S ) 8 0 - 1 0 0 mesh, c a r r i e r gas: N? (30 ml/min), c o l u m n temperature: 100280°C [5°C/min), detector: FID. The contents of betulinic acid and phytosterols (sitosterol, stigmasterol) were calculated f r o m t h e r a t i o of t h e p e a k a r e a of e a c h c o m p o u n d to t h a t of the i n t e r n a l s t a n d a r d . The m i n i m u m quantity detectable by t h i s method was 0.001% w/w of the sample. D e t e c t i o n of b e t u l i n i c acid in intact p l a n t s A p o w d e r e d s a m p l e (100 mg) of l i c o r i c e was e x t r a c t e d w i t h C H C I 3 and m e t h y l a t e d as m e n t i o n e d above. The sample was a n a l y z e d by GC-MS. B e t u l i n i c a c i d was d e t e c t e d by the f r a g m e n t ion p e a k of m/z 189, w h i c h is the base peak of b e t u l i n i c acid methyl ester, as w e l l as by other typical ions. Q u a n t i t a t i v e a n a l y s i s o~f saponins in intact p l a n t s and c u l t u r e d c e l l s A powdered s a m p l e (100 mg) was e x t r a c t e d with hot 70% M e O H (5 ml x2). The extract was a d j u s t e d to 10 ml by M e O H , a n d s u b j e c t e d to r e v e r s e p h a s e ion p a i r H P L C a c c o r d i n g to a m o d i f i e d method of Sagara et ai.(1985), which is s u i t a b l e for s e p a r a t i n g g l y c y r r h i z i n from minute amounts of c o - e x i s t i n g phenolic substances. The c o n d i t i o n s of H P L C w e r e s t a i n l e s s c o l u m n (4.6 mm x 150 mm) p a c k e d w i t h T S K g e l ODS 1 2 0 A (Toyo Soda), s o l v e n t system: M e O H - H20 (60 : 40) c o n t a i n i n g 20 mM
t e t r a - ~ - b u t y l a m m o n i u m bromide a d j u s t e d to pH 6.0 w i t h 0.1 N NaOH, f l o w rate: 1.5 m l / m i n , column temperature: 50°C, detection: UV d e t e c t o r (254 nm). The m i n i m u m q u a n t i t y of g l y c y r r h i z i n d e t e c t a b l e was 0.01% w/w of the sample. A n a l y s i s of 1 1 - d e o x o g l y c y r r h i z i n was made by HPLC under the same conditions as used for g l y c y r r h i z i n except for: s o l v e n t system: MeCN - H20 c o n t a i n i n g 0.1% v / v H 3 P O 4 (45 : 55), f l o w rate: I ml/min, column temperature: 40°C, d e t e c t o r : U V d e t e c t o r (205 nm). The d e t e c t i o n l i m i t for t h i s c o m p o u n d was 0.05% w/w of the sample.
RESULTS
F o r m a t i o n of b e t u l i n i c acid B e t u l i n i c a c i d was i s o l a t e d f r o m the cells cultured in a j a r f e r m e n t o r and identified by various a n a l y t i c a l methods. GC and G C - M S a n a l y s e s r e v e a l e d the p r e s e n c e of stigmasterol ( 0 . 0 4 - 0 . 0 7 % of c e l l d r y wt), sitosterol (0.06-0.12%) and s m a l l amounts of ~-amyrin and lupeol. Neither g l y c y r r h i z i n nor 11-deoxoglycyrrhizin c o u l d be d e t e c t e d by H P L C in the 70% M e O H e x t r a c t s of a n y c a l l u s and cell suspension cultures examined. Similarly, no g l y c y r r h e t i n i c acid was d e t e c t e d in b o t h c u l t u r e s by GC a n d G C - M S . Furthermore, g l y c y r r h i z i n was u n d e t e c t a b l e by H P L C in t h e a q u e o u s layer and all the f r a c t i o n s of the E t O A c l a y e r o b t a i n e d f r o m the MeOH extract of c e l l s u s p e n s i o n c u l t u r e s (see experimental). By contrast, b e t u l i n i c a c i d was d e t e c t e d by GC in a l l the c a l l u s cultures derived from various organs ( h y p o c o t y l , root, stem, a n d l e a f ) of o n e m o n t h - o l d G. g l a b r a seedlings, a l t h o u g h its c o n t e n t v a r i e d f r o m 0.01% to 0.17% a m o n g c u l t u r e strains (Table I).
T a b l e 1. Content of b e t u l i n i c acid in c a l l u s and s u s p e n s i o n c u l t u r e s of G l y c y r r h i z a g l a b r a
Culture strain
Origin of cultures
Callus cultures I) LC-I Leaf LC-2 Leaf LC-3 Leaf SC-I Stem SC-2 Stem HC-I Hypocotyl HC-2 Hypocotyl RC-I Root RC-2 Root RC-3 Root Suspension cultures RNS-12) Root RNS-22) Root RDS-I 3) Root
Content (% of dry wt) of betulinic acid
0.05 0.14 0.05 0.01 0.13 0.01 0.06 0.12 0.17 0.01 0.15 0.05 0.04
i) c a l l u s cultures o n LS a g a r medium containing 100 ~ M N A A a n d I p M B A f o r 5 w e e k s , 2) s u s p e n s i o n c u l t u r e s in LS m e d i u m containing 100 p M N A A a n d I ~ M B A f o r 3 w e e k s , 3) s u s p e n s i o n c u l t u r e s in LS m e d i u m c o n t a i n i n g I ~M 2 , 4 - D a n d I uM k i n e t i n for 4 w e e k s , g l y c y r r h i z i n was not d e t e c t e d in a l l c u l t u r e s a b o v e (~
.z:
5o~
~o 50 o
o I
r
I
I
Betulinic acid 0
3£
I
I
.
0
I
1
.
I
, L
E v 20 o x~
10
o
E 0
I
I
I
I
I
F
I
3
6
9
12
15
18
21
I
24 (days)
Fig I. T i m e c o u r s e of c e l l growth and p r o d u c t i o n of b e t u l i n i c acid and p h y t o s t e r o l s in G. g l a b r a c e l l s u s p e n s i o n c u l t u r e s . B a r s r e p r e s e n t s.e. (n=3).
Table 2. D i s t r i b u t i o n of g l y c y r r h i z i n b e t u l i n i c acid in G__ t. g l a b r a p l a n t s
Organ Leaf I) Stem I) Root I) (% 32 mm) Xylem Bark Root 2) Thickening Root (~ 6 mm) Radicle (~ < I mm)
and
Content (% of dry wt) of glycyrrhizin betulinic acid n.d. 3) n.d. 3) 3.78 0.06
n.d. 4) n.d. 4) n.d. 4) >0.1
0.28
0.01-0.1
n.d. 3)
0.01-0.1
i) c o l l e c t e d in Turkey, 2) c u l t i v a t e d for 6 months in K y o t o , J a p a n , 3) n o t d e t e c t e d (
Plant Cell Reports (1988) 7:508-511
© Springer-Verlag 1988
Examination of triterpenoids produced by callus and cell suspension cultures of Glycyrrhiza glabra H. Hayashi, H. Fukui, and M. Tabata Faculty of Pharmaceutical Sciences, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, Japan Received July 13, 1988/Revised version received October 10, 1988 - Communicated by F. Constabel
ABSTRACT
C a l l u s and c e l l s u s p e n s i o n c u l t u r e s of Glycyrrhiza glabra failed to produce d e t e c t a b l e amounts of g l y c y r r h i z i n , the major oleanane-type triterpene glycoside of the thickening root, or of its 1 I deoxoderivative. However, b e t u l i n i c acid, a l u p a n e - t y p e t r i t e r p e n e , w h i c h w a s f o u n d in the root bark, and a small amount of Bamyrin, a p o s s i b l e p r e c u r s o r of o l e a n a n e - t y p e triterpenes, were detected in cel 1 suspension c u l t u r e s in a d d i t i o n to lupeol, a f u n d a m e n t a l form of l u p a n e - t y p e triterpenes. T h e s e f i n d i n g s s u g g e s t t h a t the a b s e n c e of glycyrrhizin in u n d i f f e r e n t i a t e d cultured c e l l s m a y be p a r t l y due to i n t e r r u p t i o n of the later reactions leading to the synthesis of glycyrrhizin from a triterpenoid intermediate. INTRODUCTION
Glycyrrhizin, the main triterpene s a p o n i n c o n t a i n e d in the r o o t of G__~.g l a b r a , is u s e d in l a r g e q u a n t i t i e s as a n a t u r a l sweetener and a l s o for medical treatments of chronic hepatitis in Japan. It was reported that g l y c y r r h i z i n was produced in s u b s t a n t i a l amounts by c a l l u s and cell suspension c u l t u r e s of G__~.g l a b r a ( T a m a k i et al. 1972; Yoo and Kim 1976), but its chemical i d e n t i f i c a t i o n remained ambiguous. The present study has been u n d e r t a k e n in an attempt to c l a r i f y whether g l y c y r r h i z i n is accumulated by c a l l u s or c e l l suspension c u l t u r e s d e r i v e d f r o m v a r i o u s o r g a n s of _G. g l a b r a seedlings, and to find out what kinds of t r i t e r p e n o i d s are produced by the c u l t u r e d cells. MATERIALS
AND
METHODS
Chemicals Glycyrrhizin and 11-deoxoglycyrrhizin, s y n t h e t i c a l l y d e r i v e d from g l y c y r r h i z i n , were supplied by M a r u z e n K a s e i Co. Ltd., Japan. T~ latter compound was i d e n t i f i e d by IH- and - N M R in c o m p a r i s o n s w i t h an a u t h e n t i c s a m p l e of 1 1 - d e o x o g l y c y r r h i z i n i s o l a t e d from licorice r o o t by K i t a g a w a et al.(1 987).
Offprint requests to." H. Hayashi
A u t h e n t i c s a m p l e s of b e t u l i n i c acid, B-amyrin and l u p e o l were o b t a i n e d from Dr. W. Kamisako of M u k o g a w a Women's U n i v e r s i t y , Japan. P l a n t m a t e r i a l and c u l t u r e m e t h o d The seeds, l e a v e s , stems, a n d r o o t s of G__~. g l a b r a L. w e r e c o l l e c t e d from fruitbearing plants (plant height: c a 90 cm) g r o w i n g w i l d by the r o a d s i d e in the s u b u r b of Mu~, Turkey in August, 1986. Callus cultures were derived from various p a r t s (root, h y p o c o t y l , stem, a n d leaf) of 4 - w e e k - o l d seedlings on n u t r i e n t agar medium (Linsmaier and Skoog 1 965) c o n t a i n i n g 100 ~M 1 - n a p h t h a l e n e a c e t i c acid (NAA) a n d I ~M 6 - b e n z y l a d e n i n e (BA), a n d s u b c u l t u r e d on the same m e d i u m at i n t e r v a l s of 5 w e e k s for 18 m o n t h s in the d a r k at 25°C. Suspension cultures (RNS-strain) initiated from the root c a l l u s were s u b c u l t u r e d in the a b o v e m e d i u m (I00 ml) w i t h o u t a g a r in 3 0 0 m l E r l e n m e y e r flasks a g i t a t e d on a r e c i p r o c a l shaker (100 strokes/min) at i n t e r v a l s of 3 to 4 weeks for a period of 1 5 months. Suspension cultures (RDS-strain) were also e s t a b l i s h e d in LS m e d i u m c o n t a i n i n g I ~M 2,4dichlorophenoxyacetic a c i d (2,4-D) and I ~M kinetin. For s t u d y i n g the t i m e c o u r s e of g r o w t h a n d t r i t e r p e n e p r o d u c t i o n , c e l l s (I g) w e r e inoculated in e a c h 100 ml flask (30 m l medium) and incubated under the same c o n d i t i o n s as i n d i c a t e d a b o v e . Cells were h a r v e s t e d from three flasks e v e r y three days d u r i n g a c u l t u r e p e r i o d of 24 d a y s to m e a s u r e the c e l l growth and the contents of b e t u l i n i c acid and phytosterols. To p r o v i d e a l a r g e s a m p l e of c e l l s for c h e m i c a l a n a l y s i s , c__aa.120 g of c e l l s in I 1 of s p e n t m e d i u m w e r e u s e d to i n o c u l a t e a 4 ljar fermentor c o n t a i n i n g 3 1 of fresh medium, and cultured at 2 5 ° C in t h e d a r k . The m e d i u m w a s a g i t a t e d by a s c r e w - t y p e s t i r r e r (60 rpm) a n d a e r a t e d by b u b b l i n g (200 ml air/min). Cells were harvested four weeks a f t e r i n o c u l a t i o n a n d a i r - d r i e d at 6 5 ° C for one week. Isolation and identification of triterDenes and p h y t o s t e r o l s Dried c e l l s (73 g) m e n t i o n e d a b o v e were e x t r a c t e d with M e O H (I 1 x 3) by reflux, then the M e O H was e v a p o r a t e d in v a c u o at 40 °C.
509 The residue (c__{a. 10g) was s u s p e n d e d in 300 ml of H20, a n d the pH was a d j u s t e d to 4 w i t h 0.1N HCI. T h i s s o l u t i o n was e x t r a c t e d w i t h EtOAc ( 3 0 0 m l x3); the EtOAc layer was e v a p o r a t e d in v a c u o to g i v e an e x t r a c t (2.4 g), which was s u b j e c t e d to s i l i c a gel c o l u m n c h r o m a t o g r a p h y (80 g W a k o g e l C-I00 in 3 cm x 30 cm column) using a d m i x t u r e s of C H C I 3 - E t O A c (20:0, 19:1, 18:2, 16:4, 10:10, 500 ml each) and of E t O A c - M e O H (20:0, 19:1, 18:1, 16:4, 10:10, 0:20, 500 ml each) in t h a t o r d e r as solvents. A f r a c t i o n (0.38 g) e l u t e d w i t h CHCI3-EtOAc (18:2) was r e c r y s t a l l i z e d from M e O H to y i e l d c o l o r l e s s n e e d l e s (92 mg), m.p. 283-286°C. This compound was identified as b e t u l i n i c acid, 3 - h y d r o x y - l u p - 2 0 ( 2 9 ) e n - 2 8 - o i c acid,1 by TLC, MS, G C - M S (as m e t h y l ester), IR, H-NMR, and high resolution MS in c o m p a r i s o n with an a u t h e n t i c sample. From a fraction (0.45 g) e l u t e d w i t h CHCI 3 - E t O A c (I 9:1), a mixture of triterpenes were o b t a i n e d by p r e p a r a t i v e TLC. This sample was a n a l y z e d by c a p i l l a r y GC-MS: c a p i l l a r y c o l u m n (Shimadzu C B P 5 - M 2 5 - 0 2 5 ) , gas flow: He {0.5 k g / c m 2, s p l i t 40 m l / m i n , purge 2 m l / m i n ) , c o l u m n temp.: 2 0 0 - 3 0 0 ° C (10°C/rain). Lupeol and B-amyrin were identified by comparison with authentic samples. Phytosterols (sitosterol and stigmasterol) contained in the above fraction were identified by GC-MS. Q u a n t i t a t i v e a n a l y s i s of b e t u l i n i c acid and phytosterols A p o w d e r e d s a m p l e (100 rag) of c u l t u r e d c e l l s was e x t r a c t e d w i t h C H C l 3 (4 ml x2) by reflux. After evaporation of C H C I 3 , the r e s i d u e was d i s s o l v e d in E t 2 0 a n d t r e a t e d with d i a z o m e t h a n e to m e t h y l a t e the c a r b o x y l group of b e t u l i n i c acid. C h o l e s t e r o l (0.5mg) was a d d e d to the s o l u t i o n as an i n t e r n a l s t a n d a r d . An a l i q u o t ( 2 0 pl) of t h i s s a m p l e (I ml) was a n a l y z e d by GC: g l a s s c o l u m n (3 mm x I m) p a c k e d w i t h S i l i c o n e O V - 1 7 (1%} on C h r o m o s o r b W ( A W - D M C S ) 8 0 - 1 0 0 mesh, c a r r i e r gas: N? (30 ml/min), c o l u m n temperature: 100280°C [5°C/min), detector: FID. The contents of betulinic acid and phytosterols (sitosterol, stigmasterol) were calculated f r o m t h e r a t i o of t h e p e a k a r e a of e a c h c o m p o u n d to t h a t of the i n t e r n a l s t a n d a r d . The m i n i m u m quantity detectable by t h i s method was 0.001% w/w of the sample. D e t e c t i o n of b e t u l i n i c acid in intact p l a n t s A p o w d e r e d s a m p l e (100 mg) of l i c o r i c e was e x t r a c t e d w i t h C H C I 3 and m e t h y l a t e d as m e n t i o n e d above. The sample was a n a l y z e d by GC-MS. B e t u l i n i c a c i d was d e t e c t e d by the f r a g m e n t ion p e a k of m/z 189, w h i c h is the base peak of b e t u l i n i c acid methyl ester, as w e l l as by other typical ions. Q u a n t i t a t i v e a n a l y s i s o~f saponins in intact p l a n t s and c u l t u r e d c e l l s A powdered s a m p l e (100 mg) was e x t r a c t e d with hot 70% M e O H (5 ml x2). The extract was a d j u s t e d to 10 ml by M e O H , a n d s u b j e c t e d to r e v e r s e p h a s e ion p a i r H P L C a c c o r d i n g to a m o d i f i e d method of Sagara et ai.(1985), which is s u i t a b l e for s e p a r a t i n g g l y c y r r h i z i n from minute amounts of c o - e x i s t i n g phenolic substances. The c o n d i t i o n s of H P L C w e r e s t a i n l e s s c o l u m n (4.6 mm x 150 mm) p a c k e d w i t h T S K g e l ODS 1 2 0 A (Toyo Soda), s o l v e n t system: M e O H - H20 (60 : 40) c o n t a i n i n g 20 mM
t e t r a - ~ - b u t y l a m m o n i u m bromide a d j u s t e d to pH 6.0 w i t h 0.1 N NaOH, f l o w rate: 1.5 m l / m i n , column temperature: 50°C, detection: UV d e t e c t o r (254 nm). The m i n i m u m q u a n t i t y of g l y c y r r h i z i n d e t e c t a b l e was 0.01% w/w of the sample. A n a l y s i s of 1 1 - d e o x o g l y c y r r h i z i n was made by HPLC under the same conditions as used for g l y c y r r h i z i n except for: s o l v e n t system: MeCN - H20 c o n t a i n i n g 0.1% v / v H 3 P O 4 (45 : 55), f l o w rate: I ml/min, column temperature: 40°C, d e t e c t o r : U V d e t e c t o r (205 nm). The d e t e c t i o n l i m i t for t h i s c o m p o u n d was 0.05% w/w of the sample.
RESULTS
F o r m a t i o n of b e t u l i n i c acid B e t u l i n i c a c i d was i s o l a t e d f r o m the cells cultured in a j a r f e r m e n t o r and identified by various a n a l y t i c a l methods. GC and G C - M S a n a l y s e s r e v e a l e d the p r e s e n c e of stigmasterol ( 0 . 0 4 - 0 . 0 7 % of c e l l d r y wt), sitosterol (0.06-0.12%) and s m a l l amounts of ~-amyrin and lupeol. Neither g l y c y r r h i z i n nor 11-deoxoglycyrrhizin c o u l d be d e t e c t e d by H P L C in the 70% M e O H e x t r a c t s of a n y c a l l u s and cell suspension cultures examined. Similarly, no g l y c y r r h e t i n i c acid was d e t e c t e d in b o t h c u l t u r e s by GC a n d G C - M S . Furthermore, g l y c y r r h i z i n was u n d e t e c t a b l e by H P L C in t h e a q u e o u s layer and all the f r a c t i o n s of the E t O A c l a y e r o b t a i n e d f r o m the MeOH extract of c e l l s u s p e n s i o n c u l t u r e s (see experimental). By contrast, b e t u l i n i c a c i d was d e t e c t e d by GC in a l l the c a l l u s cultures derived from various organs ( h y p o c o t y l , root, stem, a n d l e a f ) of o n e m o n t h - o l d G. g l a b r a seedlings, a l t h o u g h its c o n t e n t v a r i e d f r o m 0.01% to 0.17% a m o n g c u l t u r e strains (Table I).
T a b l e 1. Content of b e t u l i n i c acid in c a l l u s and s u s p e n s i o n c u l t u r e s of G l y c y r r h i z a g l a b r a
Culture strain
Origin of cultures
Callus cultures I) LC-I Leaf LC-2 Leaf LC-3 Leaf SC-I Stem SC-2 Stem HC-I Hypocotyl HC-2 Hypocotyl RC-I Root RC-2 Root RC-3 Root Suspension cultures RNS-12) Root RNS-22) Root RDS-I 3) Root
Content (% of dry wt) of betulinic acid
0.05 0.14 0.05 0.01 0.13 0.01 0.06 0.12 0.17 0.01 0.15 0.05 0.04
i) c a l l u s cultures o n LS a g a r medium containing 100 ~ M N A A a n d I p M B A f o r 5 w e e k s , 2) s u s p e n s i o n c u l t u r e s in LS m e d i u m containing 100 p M N A A a n d I ~ M B A f o r 3 w e e k s , 3) s u s p e n s i o n c u l t u r e s in LS m e d i u m c o n t a i n i n g I ~M 2 , 4 - D a n d I uM k i n e t i n for 4 w e e k s , g l y c y r r h i z i n was not d e t e c t e d in a l l c u l t u r e s a b o v e (~
.z:
5o~
~o 50 o
o I
r
I
I
Betulinic acid 0
3£
I
I
.
0
I
1
.
I
, L
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10
o
E 0
I
I
I
I
I
F
I
3
6
9
12
15
18
21
I
24 (days)
Fig I. T i m e c o u r s e of c e l l growth and p r o d u c t i o n of b e t u l i n i c acid and p h y t o s t e r o l s in G. g l a b r a c e l l s u s p e n s i o n c u l t u r e s . B a r s r e p r e s e n t s.e. (n=3).
Table 2. D i s t r i b u t i o n of g l y c y r r h i z i n b e t u l i n i c acid in G__ t. g l a b r a p l a n t s
Organ Leaf I) Stem I) Root I) (% 32 mm) Xylem Bark Root 2) Thickening Root (~ 6 mm) Radicle (~ < I mm)
and
Content (% of dry wt) of glycyrrhizin betulinic acid n.d. 3) n.d. 3) 3.78 0.06
n.d. 4) n.d. 4) n.d. 4) >0.1
0.28
0.01-0.1
n.d. 3)
0.01-0.1
i) c o l l e c t e d in Turkey, 2) c u l t i v a t e d for 6 months in K y o t o , J a p a n , 3) n o t d e t e c t e d (
