guayano sesquiterpenos de 3 especies de lactarius
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8/9/2019 Guayano Sesquiterpenos de 3 Especies de Lactarius
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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
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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
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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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