1996-Effect of Acetylsalicylic Acid on Secondary Metabolism of Catharanthus Roscus Tumor Suspension Cultres

7/24/2019 1996-Effect of Acetylsalicylic Acid on Secondary Metabolism of Catharanthus Roscus Tumor Suspension Cultres

1/4

P l a n t C e l l R e p o r t s ( 1 9 9 7 ) 1 6 : 2 8 7 - 2 9 0

Plant ell

R e p o r t s

Springer-Verlag 1997

f f ec t o f a ce t y l s a l i cy l i c a c i d o n s eco n d a ry m et a b o l i s m

o f

a t h a r a n t h u s r o s eu s

tumor suspens ion cu l tures

G r e g o r i o G o d o y H e r n ~ n d e z a n d V i c t o r M . L o y o l a V a r g a s

D e p a r t a m e n t o d e B i o l o g i a E x p e r i m e n t a l , D i v i s i 6 n d e B i o l o g ia V e g e ta l , C e n t r o d e I n v e s t i g a c i d n C i e n ti f i c a d e Y u c a t /m ,

A p a r t a d o P o s t a l 8 7 , C o r d e m e x , 9 7 3 1 0 , Y u c a t f i n , M ~ x i c o

R e c e i v ed 1 7 M a y 1 9 9 6 / R e v is e d v e r s i o n r ec e i ve d 2 3 J u l y 1 9 9 6 - C o m m u n i c a t e d b y F . C o n s t a b e l

Sum ma ry . Add i t ion o f var ious concen t ra t ions (0 .5 -20

raM ) o f ace ty lsa l icy l ie ac id (ASA ) to tum or l ines o f

C a t h a r a n t h u s r o s e u s cu l t iva ted in v i t ro and requ i r ing

c o rn s t a r c h a s c a rb o n s o u rc e , p ro d u c e d r e m a rk a b l e

e f f e c t s o n s e c o n d a ry m e t a b o l i t e p ro d u c t i o n . An

increase o f 505 to ta l a lka lo ids per cu l tu re (ce il s p lus

l iqu id med ium), 1587 to ta l pheno l ics ( l iqu id

med ium), 612 to ta l fu ranoc oum arins ( l iqu id

med ium)~ and 1476 to ta l an thocyan ins (l iquid

m e d i u m ) wa s d e te c t e d . 1 m M A S A i n c o m b i n a t i o n

wi th o ther e l i c i to rs , such as homogenates o f A s p e r g i l l u s

f u m i g a t u s or t rans -c innamic ac id , d id no t fu r ther

increase the metabo l i t e con ten t subs tan t ia l ly . The

resu l t s sugges t tha t AS A cou ld ac t as a new b io t ic

e l i c i t o r o f m e t a b o l i t e p ro d u c t i o n i n C . r o s e u s cel l

suspens ion cu l tu re .

Ke y w o rd s : C a t h a r a n t h u s r o s e u s Acetylsal icyl ic acid -

Asp i r in - Alka lo ids - suspens ion cu l tu res - tumo r

Introduct ion

Applicat ion of sal icyl ic acid (SA) or acetylsal icyl ic acid

(ASA) to h igher p lan t s , a lgae and cu l tu red p lan t ce l l s

h a s b e e n s h o wn t o i n f l u e n c e d i v e r se p la n t d e v e l o p m e n t

p rocesses such as germina t ion (Reyno lds 1978) , g rowth

of mesoco ty l s , co leop t i l es , and roo ts (Kumar and

Nanda 1981) , f lower ing induct ion (Kumar and Nanda

1981 ; Klmrana and Maheshwar i 1983 ; Kaihara e t a l .

1 98 1; Kh u ra n a a n d M a h e s h w a r i 1 9 7 8 ; W a t a n a b e a n d

Tak imoto 1979) , shoo t induct ion in

N i c o t i a n a t a b a c u m

t i s sue (Lee and Skoog 1996) , vege ta t ive rep roduct ion

in F u c u s s p i r a l i s (Fr ies 1984) , inh ib i t ion o f tum or t i s sue

g ro wt h i n N . t a b a c u r n (Saint-Pierre et al . 1984).

L i k e wi s e a f f e c t e d h a v e b e e n a w i d e v a r ie t y o f m e t a b o l i c

p rocesses . Fo r a rev iew see (Rask in 1992 ; Kless ig and

Malamy 1994; Lee et al . 1995).

Correspondence to: V . M . L o y o l a - V a r g a s

At p resen t no in fo rmat ion i s ava i lab le regard ing the

e f f e c t o f AS A o n s e c o n d a ry m e t a b o l i t e p ro d u c t i o n .

T h e a im o f t h e p r e s e n t c o m m u n i c a t i o n i s t o e x a m i n e

t h e e f f e c t o f AS A o n s e c o n d a ry m e t a b o l i sm i n a

C a t h a r a n t h u s r o s e u s tumor ce l l l ine cu l t iva ted in v i t ro

i n t h e a b s e n c e o f g ro wt h r e g u l a t o r s a n d w i t h c o rn

s ta rch as a carbo n source .

M a t e r i a ls a n d m e t h o d s

C e l l l i n e LB A1 C u l t u re s . Catharanthus roseu~fL~G D o n - t r a n s f o r m e d

t u mo r c u l t u re s we re o b t a i n e d f ro m s t e ms o f p l a n t s g ro wn a t t h e

g re e n h o u se , a n d wh i c h h a d b e e n i n fe c t e d wi t h t h e PC 2 5 0 0 s t ra i n o f

Agrobacterium tumefaciens ( J . Sc h e l l -Ge n t s tr a i n ) . Tu m o rs fo rm e d

we re d i s se c t e d i n t o p i e c e s o f 0 .5 c m a n d t ra n s fe r re d t o PC me d i u m

(Phi l l ips and Col l ins 1979) wi thout g rowth regula tors for ca l lus

fo rma t i o n . C a l l u s wa s su b c u l tu re d i n t h e sa m e m e d i u m e v e ry 1 5 d a y s

fo r t wo y e a rs . Th e re a f t e r 1 g c a l l u s wa s t r a n s fe r re d t o 5 0 ml o f PC

me d i u m i n 2 5 0 ml E r l e n me y e r f l a sk s fo r t h e o b t e n t i o n o f c e l l

su sp e n s i o n c u l tu re s . On e y e a r l a t e r c e l l s we re t r a n s fe r re d t o 5 0 m l o f

PC me d i u m wi t h a su p p l e me n t o f 1 0 g / L o f c o rn s t a rc h , wi t h o u t

su c ro se , i n 2 5 0 Er l e n me y e r f l a sk s a n d ro u t i n e l y su b c u l t u re d u n d e r

c o n t i n u o s l i g h t, t e mp e ra t u re o f 2 8 C a n d 1 0 0 rp m. F i n a l l y , ce l l

su sp e n s i o n s (3 g f re sh we i g h t ) we re su b c u l t u re d i n 1 0 0 ml o f PC

me d i u m wi t h c o rn s t a rc h (1 0 g / L ) i n 5 0 0 ml E r l e n me y e r f l a sk s a n d

ro u t i n e l y su b c u l t u re d u n d e r t h e a b o v e c o n d i t i n s . Al l e x p e r i me n t s

we re p e r fo rme d wi t h 1 0 d a y -o l d c e l l su sp e n s i o n s (1 2 / 1 0 0 ml ) , i n

t h re e re p l ic a t e s . Vi a b i l it y wa s m e a su re d u s i n g f l u o re sc e i n d i a c e t a t e .

Ex p e r i me n t s we re re p e a t e d t wi c e in t r i p l ic a t e .

Acetylsalicylic

acid preparation

A S A ( S i g m a ) w a s p r e p a r e d a t

d i f fe re n t c o n c e n t ra t i o n s (0 .5 - 2 0 mM ) i n d i s ti l le d wa t e r a n d t h e p H

wa s a d j u s t e d t o 5 .8 wi t h KOH. Pre p a ra t i o n s we re s t e r i l i z e d b y

f i l t r a t i o n (M i l l i p o re me mb ra n e 4 5 / ~ m) p r i o r t o t h e i r a d d i t i o n t o t h e

1 0 od a y-o ld su sp e n s i o n c u l t u re s . Th e c u l t u re s we re sa mp l e d a f t e r 7 2

h o u rs o f i n c u b a t i o n .

Fungal hom ogenate preparation Aspergil lus fumigatus (ATCC-10787)

h o m o g e n a t e s we re a d d e d t o t h e 1 0 ~d a y-o ld c u l t u re s t o re n d e r a f i n a l


2/4

288

c o n c e n t ra t i o n o f 1 5 mg / ml o f t o t a l su g a r (g l u c o se ) . Th e c u l t u re

c o n d i t i o n s a n d h o mo g e n a t e p re p a ra t i o n h a v e b e e n p re v i o u s l y

d e sc r i b e d (G o d o y -H e rn f i n d e z a n d Lo y o l a -V a rg a s 1 9 91 ) ,

e t e r m i n a t i o n o f t o t a l s u g a r s

(g lucose) . T he me thod for

d e t e rm i n a t i o n o f t o t a l su g a rs w a s a c c o rd i n g t o D u b o i s e t a l.

(1956).

e t e r m i n a t i o n of to ta l a lkalo id . Tota l a lkalo id concent ra t ion , in ce ll s

a n d l i q u i d me d i u m, w a s d e t e rmi n a t e d a c c o rd i n g t o

G o d o y -H e rn ~ n d e z a n d Lo y o l a -V a rg a s (1 99 1 ).

D e t e r mi n a t i o n o f t o t a l p h e n o l ie s . To t a l p h e n o l c o n c e n t ra t i o n w a s

d e t e rmi n a t e d a c c o rd i n g t o Se i t z e t a l . (1 9 8 9 ) . Th e c u l t u re me d i u m

w a s se p a ra t e d f ro m c e l ls b y f i l t r a t io n (Wh a t ma n N o . 1 ) u n d e r

su c t i o n . F i l t e re d me d i u m (0 .5 ml ) w a s d i l u t e d i n 6 ml o f d i s t i l l e d

w a t e r a n d 0 .5 ml o f Fo l i n re a g e n t . A f t e r i n c u b a t i o n fo r 3 mi n , 1 ml o f

sa t u ra t e d so d i u m c a rb o n a t e so l u t i o n w a s a d d e d . Th e mi x t u re w a s

d i l u t e d t o a f i n a l v o l u me o f 1 0 ml w i t h d i s ti l le d w a t e r. T h e so l u t i o n

was a l lowed to s tan d for 1 h and a bsor banc e (OD) (~,=~x = 725 nm)

w a s d e t e rmi n e d . A c a l i b ra t i o n c u rv e w a s c o n s t ru c t e d w i t h c a f fe ic

a c i d a s s t a n d a rd .

D e t e r m i n a t i o n o f t o t a l f u r a n o c o n m a r i n s . T h e m e t h o d t o

d e t e rm i n a t e t o t a l fu ra n o e o u ma r i n c o n c e n t ra t i o n w as s i mi l a r t o t h e

o n e re p o r t e d b y D a v i s a n d H a h l b ro c k (1 9 8 7 ) , w i t h s l i g h t

mo d i f i c a t i o n s . C u l t u re me d i u m (2 0 ml ) w a s e x t ra c t e d t h re e t i me s

w i t h 2 0 ml c h lo ro fo rm. T h e e x t ra c t s w e re c o m b i n e d a n d e v a p o ra t e d

t o d ry n e ss . Th e re s i d u e w a s re su sp e n d e d i n 1 .0 ml o f a n a l y ti c a l g ra d e

me t h a n o l a n d a 1 :1 0 (v/ v) d i l u t i o n i n t o me t h a n o l w a s p re p a re d a n d

a b s o r b a n c e a t 3 2 0 n m w a s d e t e r m i n e d . M o l a r f u r a n o c o u m a r i n

c o n t e n t w a s c a l c u l a t e d u s i n g a n e x t i n c t io n c o e f f ic i e n t o f 1 2 ,00 0 L / mo l

x c m. A l l me a su re me n t s w e re d o n e i n t r ip l i c at e .

e t e r m i n a t i o n o f t o t a l a n t h o e y a n i n s . Th e me t h o d o f t o t a l

a n t h o c y a n i n c o n c e n t ra t i o n w a s s imi l a r t o t h a t r e p o r t e d b y D o a n d

C o rm i e r (1 9 9 0) , w i t h so me mo d i f i c a t io n s . To t a l a n t h o c y a n i n s w e re

e x t ra c t e d f ro m f re e z e -d r i e d me d i a w i t h e t h a n o l -H C 1 1

(85:15 v /v) . Opt ica l densi ty of ex t rac ts was measured a t 535 nm.

M o l a r a n t h o c y a n i n c o n t e n t w a s c a l c u l a t e d u s i n g a n e x t i n c t i o n

c o e f f i c i e n t o f 9 8 .2 l / mo l x c m. A l l me a su re me n t s w e re d o n e i n

t r ip l ica te .

e s u l t s a n d d i s c u s s i o n

AS A p ro d u c e d a g e n e ra l i n c r e a s e i n s e c o n d a ry

m e t a b o l i t e c o n c e n t r a t i o n i n C roseus ce l l suspens ion

cu l tu res. To ta l a lka lo ids increased a t each o f the AS A

conce n t ra t ions use d (F ig . 1 ) . Add i t ion o f 1 mM AS A

for 72 h increased the a lka lo ids 160 in the cu l tu re

med ium , wh i le in the ce ll s the increase was 345 over

the co n t ro l . Th is e f fec t was h igher than the o ne

ob ta ine d wi th 1 mM t rans -c innamic ac id

(Godoy-Hern f indez and Loyo la-Vargas 1991) . In

genera l , there was a 2 to 2 .5 - fo ld increase in the to ta l

alkaloids pe r f lask (Fig . 1).

3OO 300

~3oo ~ o

1 5 0 '~ 1 5 0

i =

0 0 ~ 1 5 0

5 0 ~ 5 0

3 0 0

I I t I

5 1 0 1 5 2 0

A C E T Y L S A L I C Y L I C A C I D ( r aM )

2 5 0

2 0 0 o

o

z

1 5 0 i

1 0 0

3

n

Fi g . 1. To t a l a l k a l o i d c o n t e n t i n c e l ls a n d l i q u i d me d i u m a s a

funct ion of acety lsa l icy l ic ac id concent ra t ion in C ros eus

t r a n s fo rme d c e l l su sp e n s i o n c u l t u re s (1 0 -d a y s o l d ) a f t e r 7 2 h o f

t r e a t m e n t .

AS A a l s o a f f e c t e d o t h e r s e c o n d a ry m e t a b o l i t e

pa thways . Pheno l ics , fu ranocumarins and an thocyan ins

were increased in a dose-response manner (F ig . 2A -

2C .

A t 2 0 m M o f A S A there was a 1587 increase in

pheno l ics wi th respec t to the con t ro l (F ig . 2A) . Whi le

fu ra n o c o u m a r i n c o n t e n t i n c re a se d 6 1 2 a t 1 0 m M

ASA (Fig . 2B) . Th is accumula t ion o f pheno l ics and

fu ranocoumarins i s co rre la ted wi th the induct ion o f

enzymes invo lved in genera l pheny lp ropano id

metabo l i sm, e .g . pheny la lan ine ammonia lyase (PAL)

a n d 4 -c o u m a ra t e : C o A l i g a s e (4 C L ) (Ko m b r i n k a n d

Ha h l b ro c k 1 9 8 6 ) a n d i n t h e fu r a n o c o u m a r i n

b iosyn the t ic pa thway , eg . S-adenosy l -L-meth ion ine :

a n t h o t o x o l O-m e t h y l t r a n s f e r a s e (XM T ) (Da v i s a n d

Ha h lb ro ck 1987).

3 5

2 5

s

15O0

~ o

|

=

C

o 5 1o 15 2o

ACEl lYL AUCYBC CID mb l)

Fi g u re 2 . To t a l p h e n o l i c s (2 A ) , fu ra n o c o u ma r i n s (2 B ) a n d

anthocyanins (2C) in l iqu id medium as a funct ion of acety lsa l icy l ie

a c i d c o n c e n t ra t i o n in C roseus t r a n s fo rm e d c e l l su sp e n s i o n c u l t u re s

(10-days o ld) af ter 72 h of t rea tme nt .

No t o n l y t h e s y n t h e s i s o f p h e n o l i c c o m p o u n d s wa s

induced , bu t an thocyan ins a l so (F ig . 2C) . At 20 mM

ASA, there was an a lmos t f i f t een -fo ld increase


3/4

(1476 ) in the con ten t o f these p igmen ts . Th is

i n c re a s e i n a n t h o c y a n i n s w i t h t h e AS A t r e a t m e n t wa s

1250 h igher than tha t ob ta ined wi th osmo t ic s t ress

(sucrose o r mann i to l ) by Do and C ormier (1990) in

cel ls of Vitis vinifera; 1180 h igher than tha t ob ta ined

wi t h h i g h a m m o n i u m c o n c e n t r a t i o n s (Do a n d C o rm i e r

1991) and 1050 h igher than the t rea tm en t wi th low

n i t r a te a n d h i g h s u g a r c o n c e n t r a t i o n (D o a n d C o rm i e r

1991) wi th the same p lan t ce l l suspens ion . However , a

1 0 0 0 i n c re a s e i n a n t h o c y a n in p ro d u c t io n h a s b e e n

o b t a i n e d b y m e d i u m w i t h lo w n i t ra t e a n d h i g h s u c ro s e

c o n c e n t r a t i o n i n a Vitis hybr id ce l l cu l tu re (Hi rasuna e t

a l . 1991) . Rais ing the sucrose concen t ra t ion f rom 2 to

11 in C roseus ce l l suspens ion caused an increase in

a n t h o c y a n i n c o n c e n t r a t i o n o f m o re t h a n 3 0 0 i n a n

exper im en t by Kno b loch e t a l. (1982) , whereas

s t a rv a ti o n o f p h o s p h a t e a n d n i t ro g e n i n M S m e d i u m

c a u s e d a n i n c re a s e o f 1 2 0 0 .

250

225

200

175

150

125

100

75

5O

25

0

C

T

AF AF ASAASAASA TC TC

CULTURE CONDITIONS

Fig . 3 . T o ta l a lka lo id c on te n t in c e l l s 1~) a n d l iqu id m e d ium r - --7 )

d u e t o t h e e f f e c t o f d i f f e r e n t c u l t u r e c o n d i t i o n s i n C rose us

t r a ns fo rm e d c e l l s us pe ns ion c u l tu re s 10 -days o ld ) a f t e r 72 h o f

t r e a t m e n t . C ) : c o n t r o l a t d a y 1 3 ; A F ) A s p e r g i l l u s f u m i g a tu s

h o m o g e n a t e 1 5 m g / m l g l u c o s e) ; A F + A S A ) A s p e r g i l l u s f u m i g a t u s

hom oge na te 15 m g/m l g luc os e ) p lus 1 .0 m M a c e ty l s a li c y l i c a c id ;

A S A ) 1 .0 m M a e e ty l s a li c y l ic a c id ; A S A + T C ) 1 .0 m M

a c e ty l s a l i c y l i c a c id p lus 1 .0 m M t r a ns -c inna m ic ac id; T C) 1 .0 m M

tra ns -c inna m ic a c id .

T h e r e s p o n s e i n t h e a l k a l o i d c o n t e n t t o s o m e

c o m p o u n d s a n d t r e a t m e n t s c a n b e a d d i t i v e o r

synerg i s t i c (Godoy-Hern f indez and Loyo la-Vargas

1 99 1) . T o t e s t i f t h is p h e n o m e n o n h a d b e e n h a p p e n i n g

i n t h e p r e s e n c e o f AS A , t h e c o m b i n a t i o n o f

h o m o g e n a t e s of Aspergillusfumigatus or t rans -c innamic

a c i d a n d AS A wa s t e s t e d .

T h e a d d i t i o n o fA . fumigatus h o m o g e n a t e s d i d n o t a l t e r

the a lka lo id o f the ce l l s (F ig , 3 ) . When the

h o m o g e n a t e s w e re u s e d i n c o m b i n a t i o n w i t h AS A, t h e y

re v e r t e d t h e i n c re m e n t i n d u c e d b y t h is c o m p o u n d . On

2 8 9

the o ther hand , the use o f i m M o f t rans -c innamic ac id

prod uce d an increase o f 282 in the a lka lo id con ten t

(F ig . 3 ) . S imi la r resu l t s have been ob ta in ed in C roseus

ce l l suspens ion cu l tu re ( l ine BAP-2) g rown wi th

s u c ro s e a s a c a rb o n s o u rc e (Go d o y -He rn f i n d e z a n d

Loyo la-Vargas 1991) . When the t rans -c innamic ac id

was used in the p resenc e o f ASA , the in it i a l e f fec t was

reverted (Fig . 3).

T h e a d d i t i o n o f

A fumigatus

h o m o g e n a t e s d i d n o t

m o d i fy t h e p h e n o l c o n t e n t o f t h e c u l t u re s , b u t

d e c re a s e d t h e e f f e c t o f AS A. W h i l e t h e p r e s e n c e o f

t rans -c innamic ac id d id no t mod i fy the AS A increase in

the pheno l con ten t (F ig . 4A) .

The add i t ion o fA.

f umiga tus

homogenates decreased

the leve l o f fu ranocoumarins compa red to con t ro l and

AS A t rea tmen t . T rans -c innamic ac id d id no t enhance

the A SA effec t on fu ranocoumarin p roduc t ion (F ig . 4B) .

T h e a d d i t i o n o f A furnigatus h o m o g e n a t e s i n c re a s e d

the an thocyan ins m ore than 3 -fo ld wi th respec t to the

c o n t ro l . Ho w e v e r , wh e n i t wa s a d d e d i n c o m b i n a t i o n

wi t h AS A t h e e f f e c t d i m i n is h e d b y a l m o s t 5 0 . T h e

add i t ion o f t rans-c innamic ac id a l so increased the

an thocyan in con ten t and d id no t a f fec t the increase

p ro d u c e d b y AS A (Fi g. 4 C ) .

1500

I A

250

1

i

A F A F A SA A SA A SA TC TC

CULTUREONDITIONS

4 A ) , f u r a n o c o u m a r i n sig . 4 . T o ta l phe no l ic s 4B) a nd

a n t h o c y an i n s 4 C ) i n l iq u i d m e d i u m d u e t o t h e e f f e ct o f d i ff e r e n t

c u l t u r e c o n d i t i o n s

in C roseus

t r a ns fo rm e d c e l l s us pe ns ion c u l tu re s

10-days o ld ) a f t e r 72 h o f t r e a tm e n t . C) : c on t ro l a t da y 13 ; AF )

A s p o g i l l u s f u m i g a t u s h o m o g e n a t e 1 5 m g / m l g l u c o s e ); A F + A S A )

A s p e r g i l l u s f u m i g a t us h o m o g e n a t e 1 5 m g / m l g l u c o s e ) p l u s 1 . 0 m M

a c e ty l sa l ic y l ic a c id ; AS A) 1 .0 m M a c e ty l s a l i cy l i c a c id ; AS A + T C)

1 .0 m M a c e ty l s a l ic y l i c a c id p lus 1 .0 m M t r a ns -c inna m ic a cid ; T C)

1 .0 m M t r a ns -c inna m ic a c id .

The ro le o f AS A in p lan t ce l ls is s ti ll unkno wn and i s

under inves t iga t ion . I t has been sugges ted to ac t as

che la to r (Watanabe and Tak imoto 1979) to inh ib i t

e thy lene syn thes i s in cu l tu red p lan t ce l ls by b lock ing

the ac t ion o f the e thy lene fo rming enzym e (Les l ie and


4/4

2 9 0

Romani 1986) . Other au thors have hypo the th ized tha t

AS A c o u l d f a v o r t h e e n z y m e p ro d u c t i o n t h a t d e g ra d e s

o r inac t iva tes endogenous aux ins o r behaves l ike a

horm one , and m ay t r igger o ther p rocesses in s ide

plants .

R e s u l t s s u g g e s t t h a t AS A m a y a c t a s m o d u l a t o r o f

pheny lp ropano id metabo l i sm, because i t s e f fec t on the

p ro d u c t i o n o f d i f f e r e n t s e c o n d a ry m e t a b o l i t e s wa s

noted, e . g . alkaloids (505 ) , phenolics (1587 ) ,

fu ranocoumarins (612 ) and an thocyan ins (1476 )

(F ig . 1 and 2A - 2C) . ASA i s a more powerfu l e l i c i to r

t h a n

A s p e r g i l l u s f u m i g a t u s

h o m o g e n a t e o r

t rans -c innamic ac id (TCA) fo r s t imula t ion o f

second ary metab o l i sm (F ig . 3 and 4A - 4C) .

T h e ro l e o f AS A o n a n t h o c ya n i n p ro d u c t i o n i n li n e

L B A 1 o f

C r o s e u s

t rans fo rmed ce l l suspens ion cu l tu res

i s u n k n o wn , o n e m a y h y p o t h e s i z e t h a t AS A c o u l d b e

ac t iva t ing the key enzyme (Chalcone syn thase) in the

an thoeyan in b iosyn the t ic pa thway .

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