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F d C h e m T o x i c Vol. 27, No. 6, pp. 393-397, 1989 0278-6915/89 3.00 + 0.00

Printed in Great Britain. All rights reserved Copyright 1989 Pergamon Press plc

S S Y S O F T H E B I O L O G I C L C T I V IT I E S O F G U I N E

S E S Q U I T E R P E N O I D S I S O L T E D F R O M T H E F R U I T

B O D I E S O F E D I B L E L C T R I U S S P E C IE S

H. ANKE

Dep artm ent of Biotechnology, University o f Kaiserslautern, D-6750 Kaiserslautern, Fed eral

Republ ic of Germany

a n d

O. BERGENDORFF a n d O . STERNER*

Dep artm ent of Organic Chem istry. Lund Institute of Technology, POB 124, S-221 00 Lund, Sw eden

Received 14 November 1988; revisions received 20 Mar c h 1989)

Ab strac t--T hre e sesquiterpenoids that ar e found in the edible mushrooms

Lactarius deliciosus,

L. deterrimus and L. sanguifluus, have been assayed for biological activity. The compounds tested were

a stearic acid ester of a sesquiterpene I) and a sesquiterpene aldehyde lactaroviolin, II ) and alcoho l

deterrol, III). The assays used were for mutagenic activity in the Ames Salmonella assay, for antim icrobial

activity against bacterial fungi and algae, for cytotoxicity aga inst Ehrlich ascitic tum our cells and L 1210

cells , and for phy totox ic activity again st Lepidium sativum and Seteria italica. All three compounds showed

weak mutagenic activity in the Ames assay. Two compounds II and III) were found to have mode rate

cytotox ic activity and one III) exhibited weak antibacte rial activity. No com poun d revealed phytotoxic,

algicidal or antifungal activity.

INTRODUCTION

F r u i t b o d i e s o f t h e B a s i d i o m y c e t e s L a c t a r i u s d e l i c i o -

s u s F r . , L . d e t e r r i m u s G r 6 g e r a n d L . s angu i f l uus

P a u l e t e x F r . , b e l o n g i n g t o t h e s e c ti o n D a p e t e s o f t h e

g e n u s L a c t a r i u s , a r e g e n e r a l l y c o n s i d e r e d t o b e e d i b l e

a n d d e l ic i o u s, a n d l a r g e a m o u n t s o f th e s e m u s h r o o m s

a r e c o n s u m e d a n n u a l l y . H o w e v e r , a s is o f te n t h e c a s e

f o r e d i b l e m u s h r o o m s , s u r p r i s i n g l y li t t le i s k n o w n

a b o u t t h e b i o l o g i c a l a c ti v i t i e s a n d / o r t o x i c i t y o f

s e c o n d a r y m e t a b o l i t e s p r e s e n t i n th e m . T h e f r u i t

b o d i e s o f t h e t h r e e s p e c i e s m e n t i o n e d a b o v e a r e e a s i ly

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

t h e p r e s e n c e o f a n u m b e r o f a z u l e n e s e s q u i t e r p e n o i d s

w i t h a g u a i a n e s k e l e t o n , f o r i n s t a n c e t h e s t e a r i c a c i d

e s t e r F ig . 1 , I ; Sch mi t t , 1974 ; Vo kac et al . ; 1971),

l a c t a r o v i o l i n F i g . 1, I I ; H e i l b r o n n e r a n d S c h m i d

1953 ; Schm i t t , 1974) and d e te r r o l F ig . 1 , I I I ; B e rgen-

d o r f f a n d S t e r n e r , 1 9 8 8) . T h e n a t u r e o f t h e s e s e s-

q u i t e r p e n e s h a s b e e n s t u d i e d f o r m o r e t h a n 1 0 0 y r

fo r a r ev iew s ee Schmi t t , 1974) , bu t excep t fo r a no te

o n t h e a n t i b a c t e r i a l a c t i v i t y o f l a c t a r o v i o l i n I I )

a g a i n s t t u b e r c l e b a c i l l i W i l l s t a e d t a n d Z e t t e r b e r g ,

1 9 4 6 ) , t o o u r k n o w l e d g e , n o r e p o r t s o n t h e i r b i o -

l o g i c a l a c ti v i t i e s h a v e a p p e a r e d . R e c e n t l y i t w a s

s h o w n t h a t t h e f r u i t b o d i e s o f L . de l i c i o s us a n d

L . d e t e r r i m u s o r i g i n a l l y c o n t a i n o n l y o n e s e s q u i t e r -

p e n e , e x i s ti n g i n t h e m u s h r o o m a s s t e a ri c a c id i .e .

c o m p o u n d I in F i g . 1 ) a n d l i n o l ei c a c i d e st e r s , a n d

t h a t t h e s e e s t e r s a r e e n z y m a t i c a l l y t r a n s f o r m e d i n t o

s e v e r a l s e s q u i t e r p e n e a l c o h o l s a n d a l d e h y d e s , f o r

*To whom correspondence and requests for reprints should

be addressed.

Abbreviation: MIC s = minimal inhibition concentrations.

i n s t a n c e , l a c t a r o v i o l i n F i g . 1 , I I ) a n d d e t e r r o l F i g . 1 ,

I I I ) w h e n t h e f r u it b o d i es a re d a m a g e d B e r g e n d o r f f

and S te rne r , 1988) .

T h e f r u i t b o d i e s o f o t h e r L a c t a r i u s s p e c i es , b e l o n g -

i n g f o r i n s t a n c e t o t h e s e c t i o n A l b a t i h a v e a v e r y

p u n g e n t t a s t e a n d a r e g e n e r a l l y n o t c o n s i d e r e d t o b e

e d i b l e . T h e p u n g e n c y i s c a u s e d b y u n s a t u r a t e d d i a l d e -

h y d e s e s q u i t e r p e n e s w i t h m a r a s m a n e a n d l a c t a r a n e

s k e l e t o n s f o r e x a m p l e F i g . 1 , I V ) , w h i c h a r e f o r m e d

e n z y m a t i c a l l y f r o m a f a t t y - a c i d - e s t e r p r e c u r s o r w h e n

t h es e m u s h r o o m s a r e i n j u r ed S t e r n e r et a l . , 1985).

T h e f o r m a t i o n o f su c h p u n g e n t u n s a t u r a t e d d i a l d e -

h y d e s , w h i c h p o s se s s t r o n g a n t i m i c r o b i a l S t e r n e r

e t aL , 1 98 5) , a n t i f e e d a n t C a m a z i n e et a l . , 1983) and

m u t a g e n i c S t e r n e r et a l . , 1 98 7) a c t i v i ti e s , p r o b a b l y

p r o t e c t s t h e m u s h r o o m s f r o m p a r a s i t e s a n d t h e s e

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

c h e m i c a l d e f e n c e s y s t e m S t e r n e r et a l . , 1985). The

s e s q u i t e r p e n e s i n v e s t i g a t e d h e r e , w h i c h a r e f o u n d i n

t h e f r u i t b o d i e s o f

L . de l i c i o s us , L . de t e r r i mus

a n d

L . s angu i f l uus ,

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

h y d e m o e i t y a n d a r e n o t e s p e c i a ll y p u n g e n t , b u t t h e r e

a r e s t i l l s i m i l a r i t ie s b e t w e e n t h e t w o g r o u p s o f L a c -

t a r i u s s p e c i e s i n t h e c o n v e r s i o n s o f s e s q u i t e r p e n e s i n

i n j u r e d f r u i t b o d i e s . T h e f r u i t b o d i e s o f b o t h g r o u p s

o r i g i n a l l y c o n t a i n f a t t y a c i d e s t e r s o f a s i n g l e

s e s q u i t e rp e n e , w h i c h a r e e n z y m a t i c a l l y c o n v e r t e d t o

s e s q u i t e r p e n e a l d e h y d e s a n d a l c o h o l s i f t h e f r u i t

b o d i e s a r e i n j u r e d .

I n v i e w o f t h e p o t e n t b i o l o g i c a l a c t i v i ti e s o f t h e

s e s q u i t e r p e n e s f o r m e d i n t h e p u n g e n t L a c t a r i u s

s p e c i e s , w e c o n s i d e r e d i t t o b e i m p o r t a n t t o i n v e s t i -

g a t e t h e b i o l o g i c a l a c t iv i t i e s o f t h e g u a i a n e s e s q u i t e r-

p e n e s i s o l a t e d f r o m e d i b l e L a c t a r i u s s p e c ie s . B e c a u se

o f t h e c h e m i c a l i n s t a b i l i t y o f s e v e r a l o f t h e s e

393

8/9/2019 Guayano Sesquiterpenos de 3 Especies de Lactarius

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394

H. ANKE

et al.

o o

rt

~ , . C H O

C H 2 O N ~ ~ ~ CHO

]E[ T~

Fig. 1. The sesquiterpene ester (I), lactaroviolin (II) and

deterrol (II1) are formed in edible Lactarius species, while

isovelleral (IV) is formed in pungent Lactarius species.

sesquiterpenes, the inves tigation was limited to the

reasonably stable compounds (Fig. 1, I, II, and III).

We chose four assays, which together cover a wide

range of biological activities and for which the limited

amounts of the compounds that were available were

sufficient. The assays selected were to determine

mutagenic and antimicrobial activities and cyto- and

phytotoxicity.

MA TERIA LS A ND METH O D S

The three compounds assayed were isolated from

ground fruit bodies of

Lactarius del ic iosus

and

L . de ter r im us

as described earlier (Bergendorff and

Sterner, 1988).

Mutagenic i ty as say .

Mutagenic activity was investi-

gated in the Ames Salmonella/mammalian-micro-

some assay (Ames

et al. ,

1975) with the frameshift

mutant strain TA98 and the base-pair substitution

mutant strain TA100 (the strains were kindly pro-

vided by Professor B. Ames, Universit y of California,

Berkeley, CA, USA). They were carried out both

without and with 3.3 or 10% S-9 mix, which was

prepared from the livers of SPD rats induced with

Arochlor 1254. The assays of the ester (I) and

lactaroviolin (II) were carried out with quintuplicate

plates but in the assay of deterrol (III) the plates were

triplicated because of the limited amounts of this

compound available. The solvent used throughout

was acetone. Before the assay was performed, the

compounds were tested for toxicity towards strain

TAI00 on nutri ent agar plates (about 1000 bac-

teria/plate), and the concent ratio n that caused a 50%

reduction in the n umb er of colonies was taken as the

highest concentration to be tested. As a solvent

control, blanks were run with acetone only. The

average numbers of spontaneous revertants/plate

were as follows: for TA9 8- -53 without S-9 mix, 53

with 3.3% S-9 mix, and 64 with 10% S-9 mix; for

TA100--151 without S-9 mix, 141 with 3.3% S-9

mix, and 151 with 10% S-9 mix. 1-[(Diethyl-

aminoethyl) amino]-4-(methansulphonyloxymethyl)-

thioxanthen-9-one, kindly provided by Winthrop

Ltd, New York, USA was used to determine the

sensitivity of the strains: 1 00 g of this com-

pound /plat e gave 620 colonies for TA98, and 690 for

TAI00 (inclusive of the spontaneous background, in

all cases the average of three plates). As a positive

control of the S-9 activation, 2-amino anthr acene was

tested in strain TAI00:1 pg/plate gave 147 colonies

in the absence of S-9 mix, 1818 colonies in the

presence of 3.3% S-9 mix, and 586 colonies in the

presence of 10% S-9 mix (in all cases the average of

three plates). The analysis by linear regression and

the calcul ation of the correl ation coefficients were

carried out with the software 'Statview' for Apple

Macintosh computers.

Assay o f an t im icrobia l ac t iv iO ,.

Antimicrobial ac-

tivities against two Gram-negative and three Gram-

positive bacteria, three yeasts, four filamentous fungi

and one alga were tested in the plate diffusion assay

(Zfihner and Maas, 1972). Bacteria were grown on

nutri ent agar (Difco, Detroit, USA), yeasts and other

fungi on agar conta inin g (/litre): 4 g yeast extract;

10g malt extract; 4g glucose. The inoculum was

5 l0 s cells or spores /ml. Chlorella vulgaris was

grown under the conditions described by Noll (1987).

The size ofi noc ulu m was 1 1 7 cells/ml. Fil ter discs

(6 mm diameter) bearing the compo unds were placed

on agar plates seeded with the appropriate test organ-

ism, and the plates were incub ated at 27 ~ or 37 C for

24 hr. The minimal inhibi tion concentr ations (MICs)

for the most sensitive organisms were determined

with the broth dilutio n method (Ericsson and Sherris,

1971). The size ofinoculum was 1 6 cells/ml, while the

time of incubation, temperature and media (without

agar) were the same as in the plate diffusion assay.

Assa y o f cy to tox ic ac ti~ ,iO .

For the cytotoxicity

tests Erlich ascitic tumour cells (ECA cells) and L

1210 cells (lymphocytic leukaemia mouse, ATCC

CCL 163) were used (Ekwall, 1983; Leonhard, 1987).

The cells were grown and tested as suspension cul-

tures, as described by Leonhard (1987). The cell

densi ties were 6 l0 s cells (ECA) and 1 106 cells

(L 1210) per ml. Cytotoxic activity was evaluat ed

after 24 and 48 hr.

Assa y o f phy to tox ic ac tiv it y .

Phytotoxic activity

was assayed according to the method of Yopp (1985),

except that

L e p i d i u m s a t i v u m

(dicotyledon) and

Setaria i tal ica

(monocotyle don) were used instead of

Lactuca sat iva.

Filter discs (12-mm diameter) bearing

50 or 100/~g of the com pou nds were placed in plastic

vials (diameter, 13 mm, height 50 ram), and wetted

with 200 pl water. Six seeds of the plants were placed

on each disc, and the vials were incubated in a humid

cham ber at 20~C in the dark for 72 hr, and then for

48 hr in daylight. The germination of the seeds and

the lengths of the seedlings and roots were measured

after 48, 72, 96 and 120 hr, and compared with a

negative control. The assays were carried out in

triplicate.

RESU LTS

The results of the Ames Salmonella assays of the

ester (I), lactaroviol in (II) and deterrol (III) are

shown in Tables 1, 2, and 3, respectively. For all three

compounds the numbers of revertants of at least one

strain increased with the concentration, indicating

mutagenic activity. Assuming linear dose-response

relationships, the slopes and the correlation coeffi-

cients were calculated by linear regression of the

primary data given in Tables 1-3. The ester (I), which

was found to be the least toxic of the three com-

pounds towards the tester strains, gave the strongest

response in strain TAI00 in the absence of S-9 mix

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B i o l o g i c a l a c t i v i t y o f s e s q u i t e r p e n e s

T a b l e I . R e s u l t s o f t e s t i n g t h e se s q u i t e r p e n e e s te r

( I , F i g . 1 )

i n t h e

A m e s S a l m o n e l l a /m a m m a l i a n -m i c r o s o m e m u t a g en i c it y a s sa y

N o . o f r e v e r t a n t s /p l a t e

o m p o u n d

I

c o n c n ~ g / S a l m o n e l l a S -9 i n c lu d i n g

p l a t e ) S t r a i n m i x * s p o n t a n e o u s r e v e r t a n t s )

1 0 0 0 T A 9 8 - - 7 0 , 7 3 , 7 7 , 8 2 , 8 2

T A 9 8 + 3 . 3 6 3 , 6 9 , 7 3 , 7 8 , 8 6

T A 9 8 + 1 0 6 7 , 6 8 , 7 6 , 7 6 , 8 1

T A 1 0 0 - - 3 3 7 , 3 6 6 , 3 7 2 , 3 8 1 , 3 8 3

T A 1 0 0 + 3 . 3 3 3 6 , 3 3 9 , 3 4 9 , 3 5 9 , 3 7 3

T A I 0 0 + 1 0 2 9 0 , 2 9 3 , 3 0 1 , 3 0 6 , 3 3 6

5 0 0 T A 9 8 - - 5 3 , 6 0 , 7 0 , 7 1 , 7 2

T A 9 8 + 3 . 3 5 4 , 6 5 , 7 0 , 7 3 , 7 7

T A 9 8 + 1 0 5 5 , 6 3 , 7 1 , 7 7 , 7 9

T A 1 0 0 - - 2 4 6 , 2 4 9 , 2 7 5 , 2 7 6 , 2 7 8

T A 1 0 0 + 3 . 3 2 4 3 , 2 4 4 , 2 5 1 , 2 6 1 , 2 8 1

T A I 0 0 + 1 0 2 2 9 , 2 3 3 , 2 4 1 , 2 5 3 , 2 6 6

2 5 0 T A 9 8 - - 4 9 , 5 1 , 5 7 , 5 9 , 6 2

T A 9 8 + 3 . 3 4 5 , 5 5 , 5 7 , 6 2 , 7 1

T A 9 8 + 1 0 5 2 , 5 7 , 6 5 , 7 1 , 7 2

T A 1 0 0 - - 1 77 ~ 1 7 8 , 1 9 3 , 2 0 4 , 2 1 5

T A 1 0 0 + 3 , 3 1 7 5 , 1 7 6 , 1 8 4 , 1 8 6 , 1 9 9

1 2 5

T A 1 0 0 + 1 0 1 4 4 , 1 6 5 , 1 6 9 , 1 7 t , 1 8 3

T A 9 8 - - 4 1 , 4 8 , 4 8 , 5 8 , 6 1

T A 9 8 + 3 . 3 5 3 , 5 7 , 6 6 , 6 7 , 7 5

T A 9 8 + 1 0 5 4 , 6 1 , 6 4 , 7 0 , 7 2

T A I 0 0 - - 1 7 2 , 1 7 3 , 1 7 4 , 1 8 3 , 1 9 6

T A 1 0 0 + 3 . 3 1 5 1 , 1 6 4 , 1 7 5 , 1 7 7 , 1 8 9

T A 1 0 0 + 1 0 1 5 5 , 1 5 9 , 1 6 1 , 1 6 3 , 1 8 9

T A 9 8 - - 4 8 , 5 0 , 5 5 , 5 5 , 5 8

T A 9 8 + 3 . 3 4 5 , 5 0 , 5 3 , 5 7 , 5 9

T A 9 8 + 1 0 5 3 , 5 9 , 6 3 , 6 9 , 7 5

T A I 0 0 - - 1 1 8 , 1 4 6 , 1 4 7 , 1 7 1 , 1 7 3

T A I 0 0 + 3 . 3 1 2 5 , 1 3 0 , 1 3 7 , 1 4 5 , 1 4 6

T A I 0 0 + 1 0 1 4 1 , 1 4 5 , 1 4 9 , 1 5 7 , 1 6 1

* P l a te s w e r e m a d e u p w i t h o u t - ) o r w i t h

3 . 3 ( + 3 . 3 ) o r 1 0

( + 1 0 ) S - 9 m i x .

T a b l e 2 . R e s u l t s o f t e s t i n g t h e s es q u i t e r p e n e a l d e h y d e , l a c t a r o v i o l i n

( I I , F i g . 1 ) i n t he A m e s S a l m o n e l l a /m a m m a l i a n -m i c r o s o m e m u t a -

g e n i c i t y a s s a y

L a c t a r o v i o l i n N o . o f r e v e r t a n ts / p la t e

c o n c n g g / S a l m o n e l l a S -9 i n c l u d in g

p l a t e ) s t r a in m i x * s p o n t a n e o u s r e v e r t a n t s )

2 0 0 T A 9 8 - - 4 2 , 4 7 , 5 4 , 5 5 , 6 5

T A 9 8 + 3 . 3 9 2 , 9 7 , 1 0 3 , 1 1 0 , 1 1 9

T A 9 8 + 1 0 9 5 , t 1 7 , 1 2 2 , 1 2 9 , 1 3 0

T A I 0 0 - - 1 5 1 , 1 7 0 , 1 7 6 , 1 8 8 , 1 9 2

T A 1 0 0 + 3 . 3 2 0 1 , 2 0 2 , 2 1 6 , 2 2 0 , 2 3 1

T A 1 0 0 + 1 0 2 8 9 , 2 9 9 , 3 0 6 , 3 2 0 , 3 4 3

1 0 0 T A 9 8 - - 4 5 , 5 2 , 5 5 , 5 5 , 6 1

T A 9 8 + 3 . 3 1 0 1 , 1 0 3 , 1 0 6 , 1 1 2 , 1 2 9

T A 9 8 + 1 0 9 5 , 9 7 , 9 9 , 9 9 , 1 1 0

T A I 0 0 - - 1 6 2 , 1 7 5 , 1 7 7 , 1 8 3 , 2 0 3

T A I 0 0 + 3 . 3 1 6 5 , 1 8 8 , 1 8 9 , 1 9 3 , 2 0 1

T A 1 0 0 + 1 0 1 7 9 , 1 8 5 , 2 0 3 , 2 1 2 , 2 1 7

5 0 T A 9 8 - - 3 7 , 3 9 , 4 7 , 5 1 , 6 0

T A 9 8 + 3 . 3 8 3 , 8 9 , 9 6 , 1 0 1 , 1 0 7

T A 9 8 + 1 0 7 7 , 7 9 , 8 2 , 9 0 , 9 1

T A I 0 0 - - 1 5 1 , 1 5 7 , 1 6 6 , 1 7 0 , 1 7 9

T A I 0 0 + 3 . 3 1 5 5 , 1 7 7 , 1 8 3 , 1 8 9 , 1 9 0

T A 1 0 0 + 1 0 1 6 9 , 1 8 3 , 1 8 7 , 1 9 3 , 2 0 1

2 5 T A 9 8 - - 4 9 , 5 1 , 5 6 , 5 9 , 6 2

T A 9 8 + 3 . 3 6 0 , 6 3 , 7 0 , 7 6 , 8 0

T A 9 8 + 1 0 6 2 , 7 2 , 7 9 , 8 3 , 9 0

T A I 0 0 - - 1 4 3 , 1 5 8 , 1 6 2 , 1 7 4 , 1 7 6

T A 1 0 0 + 3 . 3 1 3 6 , 1 4 6 , 1 5 2 , t 6 0 , 1 6 6

T A I 0 0 + 1 0 1 2 4 , 1 3 9 , 1 4 5 , 1 5 0 , 1 5 5

0 T A 9 8 - - 4 8 , 5 0 , 5 5 , 5 5 , 5 8

T A 9 8 + 3 . 3 4 5 , 5 0 , 5 3 , 5 7 , 5 9

T A 9 8 + 1 0 5 3 , 5 9 , 6 3 , 6 9 , 7 5

T A I 0 0 - - 1 1 8 , 1 4 6 , 1 4 7 , 1 7 1 , 1 7 3

T A 1 0 0 + 3 . 3 1 2 5 , 1 3 0 , 1 3 7 , 1 4 5 , 1 4 6

T A I 0 0 + 1 0 1 4 1 , 1 4 5 , 1 4 9 , 1 5 7 , 1 6 1

* P l a te s w e r e m a d e u p w i t h o u t - ) o r w i t h

3 . 3 ( + 3. 3 ) o r 1 0

( + l O ) S - 9 m i x .

3 9 5

T a b l e 3 . R e s u l t s o f t e s t i n g t h e s e s q u it e r p e n e a l c o h o l , d e t e r r o l

( I I I ,

F i g . I )

i n t h e A m e s S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i ty

a s s a y

D e t e r r o l N o . o f r e v e r t a n t s / p l a t e

c o n c n g g / S a l m o n e l l a S -9 i n c lu d i n g

p l a t e ) s t r a in m i x * s p o n t a n e o u s r e v e r t a n t s )

5 0 T A 9 8 - - 8 6 , 1 0 6 , 1 2 0

T A 9 8 + 3 . 3 1 6 2 , 1 6 8 , 2 0 4

T A 9 8 + 1 0 1 4 2 , 1 5 4 , 1 6 0

T A 1 0 0 - - 1 6 4 , 1 6 4 , 1 6 5

T A 1 0 0 + 3 . 3 1 7 5 , 1 7 9 , 2 0 0

T A 1 0 0 + 1 0 2 4 9 , 2 7 6 , 2 8 3

2 5 T A 9 8 - - 8 3 , 8 4 , 9 6

T A 9 8 + 3 . 3 8 2 , 9 2 , 9 7

T A 9 8 + 1 0 9 6 , 1 0 3 , 1 2 5

T A 1 0 0 - - 1 4 8 , 1 5 1 , 1 5 7

T A 1 0 0 + 3 . 3 1 5 4 , 1 5 9 , 1 7 9

T A 1 0 0 + 1 0 1 8 7 , 1 9 6 , 2 0 9

1 2 .5 T A 9 8 - - 6 7 , 6 9 , 9 0

T A 9 8 + 3 . 3 8 1 , 8 5 , 8 8

T A 9 8 + 1 0 7 5 , 9 5 , 1 1 0

T A I 0 0 - - 1 4 2 , 1 6 0 , 1 7 7

T A I 0 0 + 3 . 3 1 7 6 , 1 8 3 , 2 0 7

T A I 0 0 + 1 0 1 7 5 , 1 8 8 , 1 9 3

0 T A 9 8 - - 4 8 , 5 0 , 5 5 , 5 5 , 5 8

T A 9 8 + 3 . 3 4 5 , 5 0 , 5 3 , 5 7 , 5 9

T A 9 8 + 1 0 5 3 , 5 9 , 6 3 , 6 9 , 7 5

T A I 0 0 - - 1 1 8 , 1 4 6 , 1 4 7 , 1 7 1 , 1 7 3

T A I 0 0 + 3 . 3 1 2 5 , 1 3 0 , 1 3 7 , 1 4 5 , 1 4 6

T A I 0 0 + 1 0 1 4 1 , 1 4 5 , 1 4 9 , 1 5 7 , 1 6 1

* P l a te s w e r e m a d e u p w i th o u t - ) o r w i t h

3 . 3 ( + 3 . 3 ) o r 1 0

( + 1 0 ) S - 9 m i x .

( s l o p e = 0 . 2 2 r e v e r t a n t s / g / p l a t e , c o r r e l a t io n c o e f f i-

c i e n t - - 0 . 9 5 ) . T h e r e s p o n s e w a s n o t s i g n i fi c a n t ly a l -

t ered by t he pres ence o f 3 .3% S- 9 mi x , w hi l e 10% S - 9

mi x reduced i t s ubs t ant i a l l y . L ac t arov i o l i n ( I I ) i n -

creas ed t he num ber o f r ever t ant s on l y i n t he pres ence

o f S - 9 m i x . T h e s t r o n g e s t r e s p o n s e w a s o b t a i n e d i n

s t ra i n T A 1 0 0 i n th e p r e s en c e o f 1 0% S - 9 m i x

( s l o p e = 0 . 8 3 r e v e r ta n t s // ~ g /p l a te , c o r r e la t i o n c o e f-

f i c ient = 0 .90 ) , w hi l e t he re s pon s es i n t he s ame s t ra in

i n t he pres ence o f 3 .3% S- 9 mi x , a s w e l l a s i n s t ra i n

T A 98 i n t he pres ence o f 3 .3 or 10% S- 9 mi x , w ere

cons i derabl y l ow er . D e t erro l ( I I I ) i s t he mos t t ox i c

c o m p o u n d t o w a r d s t h e S a l m o n e l l a t e st e r s tr a in s ,

5 0 / ~ g / p l a t e w a s f o u n d t o b e t h e h i g h e s t n o n - t o x i c

concent ra t i on . D e t erro l ( l l I ) gave t he s t ronges t r e -

s p o n s e i n T A 9 8 i n t h e p r e s e n c e o f 3 . 3 % S - 9 m i x

( s l o p e = 2 . 4 r e v e r t a n t s /~ g / p l a t e , c o r r e la t i o n c o e f -

f i c ient = 0 .91 ) , a nd i n T A 10 0 i n t he pres ence o f 10%

S- 9 mi x ( s l ope = 2 .3 rever t an t s / ~g / p l a t e , corre l a t i on

coef f ic ient = 0 .94) .

A s s h o w n i n T a b l e 4, n o a n t i f u n g a l a c t i v it y c o u l d

be de t ec t ed f or e i t her compound ( up t o 50 / ~g / d i s c ) .

N o r w a s

Ch l o r e l l a v u l g a r i s

af f ec t ed ( a t 50 g / d i s c ) ,

a n d n o p h y t o t o x i c a c t iv i t y to w a r d s

L e p i d i um sa t i v um

a n d

Se t a r i a i t a l i c a

w as de t ec t ed ( t he h i ghes t concen-

t ra t i on t e s t ed w as 500 g / ml ) . D e t erro l ( I I I ) i s t he

o n l y c o m p o u n d t o e x h i b i t w e a k a n t i b a c t er i a l a c t i v it y ,

a n d t h e m o s t s e n s i t i v e o r g a n i s m w a s f o u n d t o b e

c i n e t o bae t e r c a l c oa c e t i c u s

f o r w h i c h t h e M I C w a s

5 / ~g / m l i n the brot h d i l u t i on t e st . T he M I C f or

M i c r o c o c e u s l u t eu s

a n d

P ro t eu s v u l g a r i s

w a s

30 / ~g / ml . D e t erro l ( I I I ) a l s o s how s moderat e cy t o -

t o x i c a c t i v i t y t o w a r d s E C A c e l l s a n d w e a k t o x i c i t y

t ow ards L 1210 ce ll s (T abl e 5 ) . L ac t a rov i o l i n ( I I )

exh i b i t s a w eak i nh i b i t ory e f fec t t ow ards E C A ce l l s ,

w hi l e t he e s t er ( I ) d i d not i mpai r e i t her ce l l l i ne a t

50/~ g/m l

8/9/2019 Guayano Sesquiterpenos de 3 Especies de Lactarius

4/5

3 9 6 H . A N K E

et al.

Table 4. Antimicrobial activity of three sesquiterpene compounds in the plate diffusion assay.

Inhibition zone (mm) after treatment with*:

Incubation I II III

temperature

Compound conch . . . .

Test organism (C)

( g/disc)...

10 50 10 50 10 50

Bacteria

Acinetobacter calcoaceticus

27 0 0 0 0 12 15

Bacillus brevis

37 0 0 0 0 0 0

B. subtilis 37 0 0 0 0 I) 0

Micrococcus luteus

37 0 0 0 0 0 0

Proteus vulgaris

37 0 0 0 0 0 12

Yeas ts and fungi

Fusarium oxysporum 27 NT 0 NT 0 NT 0

Mucor miehei

37 NT 0 NT 0 NT 0

Nematospora coo'li

27 NT 0 NT 0 NT 0

Paecilomyces variotii

37 NT 0 NT 0 NT 0

Penicillium notatum

27 NT 0 NT 0 NT 0

Saccharomyces cerevisiae

iS1 27 NT 0 NT 0 NT 0

Saccharomyces cerevisiae S 288 27 NT 0 NT 0 NT 0

Algae

Chlorella t'ulgaris

NT 0 NT 0 NT 0

NT = not tes ted

*The three com pou nds tested are shown in Fig. I, and were a sesquiterpene ester (I) lactaroviolin (1I) and deterrol (Ill) .

DISC USSION

T h e r e w e r e l a r g e d i f fe r e n c e s b e t w e e n t h e b i o l o g ic a l

a c t i v i ti e s o f t h e s e s q u i t e r p e n e s i s o l a t e d f r o m t h e f r u i t

b o d i e s o f e d i b l e L a c t a r i u s s p e c i e s ( t h i s i n v e s t i g a t i o n )

a n d t h o s e is o l a te d f r o m t h e p u n g e n t L a c t a r i u s

s p e c i e s . T h e u n s a t u r a t e d d i a l d e h y d e s , s u c h a s i s o -

v e l l er a l ( IV ) , t h a t a r e p r e s e n t i n t h e p u n g e n t s p e c i e s ,

a r e r e a c ti v e c o m p o u n d s a n d g i ve t h e ir e ff e c ts

p r o m p t l y . T h i s i s d e m o n s t r a t e d b y t h e i r i n t e n s e p u n -

g e n t t a s t e , t h e i r m u t a g e n i c i t y , a n d t h e i r s t r o n g a n t i -

b i o t i c a c ti v i t y . F o r c o m p a r i s o n , i s o v e l l e r a l ( I V ) h a s

b e e n s h o w n t o i n d u c e 1 55 re v e r t a n t s # g / p l a t e i n

s t r a in T A I 0 0 , i n t h e a b s e n c e o f S - 9 m i x ( S t e r n e r

et al.,

1 9 87 ), a n d i t s M I C s a g a i n s t Acinetobaeter calcoaceti-

cus, Micrococcus luteus

a n d

Proteus vulgaris

a r e t e n

t i m e s l o w e r t h a n t h o s e r e p o r t e d h e r e f o r d e t e r ro l ( I II ;

A n k e

et al.,

1 9 89 ). F u r t h e r m o r e , i s o v e l l e r a l ( I V ) h a s

b e e n s h o w n t o s u p p r e s s c o m p l e t e l y g e r m i n a t i o n o f

Lepidium sat ivum

a n d

Setaria italica

a t 5 0 / ~ g / m l

( A n k e

et al.,

1 98 9) , a n d t o i n h ib i t t h e g r o w t h o f E C A

a n d L 1 2 1 0 c e l ls a t 1 - 2 p g / m l ( A n k e

et al.,

1 9 8 9 ) . T h e

g u a i a n e s e s q u i t e r p e n e s a s s a y e d h e r e a p p e a r t o b e le ss

p o t e n t , a n d a r e a c ti v a t e d r a t h e r t h a n i n a c t i v a t e d b y

r a t -l i v er m i c r o s o m a l e n z y m e s ( S -9 ) in t h e A m e s

S a l m o n e l l a a s s a y . I t w a s u n e x p e c t e d t h a t d e t e r r o l

( I I I ) is m o r e t o x i c t o w a r d s b o t h t h e A m e s t e s t e r

s t r a i n s a n d t h e E C A a n d L 1 2 1 0 c e l ls t h a n l a c t a r o -

v i o l i n (I I ) w a s , s i n c e a n a l d e h y d e f u n c t i o n a l i t y i s

Table 5. Cytotoxic act ivi ty of three sesqui terpene co mp ound s agains t

ECA and L 1210 cel ls

Concentration (pg/ml) needed for

growth inhibitions of

C el l l i n e C ompound* 10-20% 50% 100%

ECA I > 50 > 50 > 50

II 10 20 50

III 5 10 20

L 1210 1 > 50 > 50 > 50

1I 50 > 50 > 50

III 10 50 > 50

*The three compounds tes ted are shown in Fig. I and were a

sesquiterpene ester (I), lactaroviolin (II) and deterrol (III).

+Con centrations (,ug/ml) needed for reduction in ce ll numb ers in

comp arison w ith untreated control c ell cultures.

n o r m a l l y m o r e r e a c ti v e a n d t h e r e b y m o r e t o x i c t h a n

t h e c o r r e s p o n d i n g a l c o h o l f u n c t i o n a l i t y , a n d t h is

s u g g e s t s t h a t t h e a z u le n e p o r t i o n o f th e s e c o m p o u n d s

c o n t r i b u t e s s i g n i f i c a n t ly t o t h e b i o l o g i c a l a c t i v i t i e s

a s s a y e d h e r e .

E v e n i f t h e o b s e r v e d a c t i v i t i e s a r e l o w , t h e w e a k

r e s p o n s e s i n t h e A m e s S a l m o n e l l a a s s a y o f a ll t h r e e

c o m p o u n d s i n v e s t ig a t e d h e r e , a s w e l l a s th e c y t o -

t o x i c i t y o f d e t e r r o l ( I I I) t o w a r d s E C A c e ll s s h o u l d b e

o f s o m e c o n c e r n . H o w e v e r , t h e s e c o m p o u n d s a r e

r a t h e r s e n s i t i v e , a n d i f l e ft a t r o o m t e m p e r a t u r e f o r

d a y s o r h e a t e d t o 5 0 C f o r h o u r s ( f o r in s t a n c e d u r i n g

e v a p o r a t i o n o f s o lv e n t s ) t h ey a r e l a r g e ly d e s t r o y e d .

W e t h e r e f o r e b e l ie v e , a l t h o u g h w e h a v e n o t i n v e s t i-

g a t e d t h i s, t h a t t h e s e c o m p o u n d s w il l d i s a p p e a r i f t h e

m u s h r o o m s a r e th o r o u g h l y c o o k e d p r i o r to in g e s t io n .

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