Patulin and Penicillic Acid

6 Patulin and Penicillic Acid DAVID M . WILSON

Downloaded by UNIV OF GUELPH LIBRARY on July 25, 2012 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch006

Department of Plant Pathology, University of Georgia, College of Agriculture Experiment Stations, Coastal Plain Station, Tifton, Ga. 31794 The mycotoxins patulin (4-hydroxy-4H-furo[3,2-c]pyran2(6H)-one) and penicillic acid (3-methoxy-5-methyl-4-oxo2,5-hexadienoic acid) are metabolites of several fungi, primarily species of Aspergillus and Penicillium. Their chemical and physical properties and biosynthesis are discussed. Recent work on analytical methods, reports of natural occurrence, and stability in foods and feeds are emphasized. Both patulin and penicillic acid inhibit DNA, RNA, protein synthesis, and some enzymes containing SH groups. The precise mode of toxic action remains uncertain. Since both patulin and penicillic acid are toxic and have been implicated in carcinogenesis and since patulin causes mutations in yeasts, they are potentially dangerous. The biological effects of orally ingested patulin and penicillic acid need further study to assess their potential health hazard. Once upon a time gasoline was a nuisance and the problem was how to get rid of it; once upon a time moulds were a nuisance and the problem was how to get rid of them. Not so today. Since the epoch-making purification of penicillin . . . by Florey and his associates . . . the search for therapeutic agents from moulds has crossed oceans and continents. At the London School of Hygiene and Tropical Medicine in London, England, Professor Harold Raistrick and his associates . . . have assiduously iso lated, purified, and established the structure of a number of therapeutically active compounds from various moulds. Their most recent and most interesting derivative from moulds is "patuline" which is obtained from the mould Penicillium patulum Bainier, and which holds promise of therapeutic activity against the common cold ( 1 ). Patulin was soon found almost useless in curing the common cold (2) and was too toxic for use as an antimicrobial agent. Therefore interest 90 In Mycotoxins and Other Fungal Related Food Problems; Rodricks, J.; Advances in Chemistry; American Chemical Society: Washington, DC, 1976.

6.

Patulin

WILSON

and Penicillic

91

Acid

has n o w s h i f t e d to t h e t o x i c p r o p e r t i e s of p a t u l i n a n d its r e l a t i o n t o t h e p o t e n t i a l c o n t a m i n a t i o n of foods a n d feeds. T h e structure of p a t u l i n

(4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one)

w a s d e t e r m i n e d b y W o o d w a r d a n d S i n g h ( 3 , 4 ) (see


* 0 ^

0

Figure 1.

H

Figure 1).

Patulin

Structure of patulin

has b e e n i s o l a t e d u n d e r v a r i o u s n a m e s : c l a v i c i n , c l a v i t i n , c l a v i f o r m i n , e x p a n s i n , l e u c o p i n , m y c o i n c, p e n i c i d i n , a n d t e r c i n i n ( 5 ) .

I t has b e e n

i s o l a t e d f r o m t h e f o l l o w i n g f u n g i : Pénicillium

[P.

(7)

urticae

a n d p e r h a p s s y n o n y m o u s P . griseo-fulvum

[ P . leucopus

(10)1,

P . claviforme pium

( 1 5 ) , P . lapidosum

(17),

A . giganteus

( 1 4 ) , P . novae-zeelandiae P . equinum

(16),

A . terreus

(18),

patulum

P . expansum

(8)1,

(9)

[ P . diver gens ( I I ) ] , P . Unosum

P. granuhtum

( 1 3 ) , P . melinii

(6)

(6),

P.

(14),

( 1 4 ) , Aspergillus

a n d Byssochlamys

(12), cycloclavatus

nivea

(19)

sp. (20)1 ·

[Gymnoascus

Penicillic acid ( C H i O ) , 8

0

3-methoxy-5-methyl-4-oxo-2,5-hexadienoic

4

a c i d , w a s first i s o l a t e d f r o m P . puberulum

b y A l s b e r g a n d B l a c k (21 ) w h o

n a m e d the c o m p o u n d a n d f o u n d i t t o x i c to m i c e . T h e y also f o u n d t h a t a l t h o u g h P . stoloniferum

produced mycophenolic acid ( C i H o 0 ) , it d i d 7

n o t p r o d u c e p e n i c i l l i c a c i d . B i r k i n s h a w (22)

2

6

showed that the structure

of p e n i c i l l i c a c i d w a s y - k e t o - ^ - m e t h o x y - 8 - m e t h y l e n e - A - h e x a n o i c a c i d o r a

t h e c o r r e s p o n d i n g γ - h y d r o x y l a c t o n e (see

Figure 2).

P e n i c i l l i c a c i d has

Ο OCH II I = C - C - C = C H - C O O H 3

J

CH

*· m

Figure 2.

Structure of penicillic

b e e n i s o l a t e d frorn t h e f o l l o w i n g f u n g i : Pénicillium ( 2 1 ) , P . griseum

berulum (22),

P. thomii

P . fenelliae catum

(29),

lilacinum (33),

(25),

(27), (12),

Aspergillus

[ P . suavolens

P. roqueforti

P. aurantio-virens

P . palitans

(30),

lividum

P . simplicissimum

(23),

(25)1,

( 2 8 ) , P . janthinellum

P . baarnense

P. canescens

(12),

P. chrysogenum

ochraceus

(20),

A . sulphur eus

cyclopium

P. martensii P.

(29),

P . madriti

(31),

(21), (34),

P . pu-

(23), P.

(24),

P.

acid

(32),

(26), viridiP.

olivino-viride

A . Melleus

In Mycotoxins and Other Fungal Related Food Problems; Rodricks, J.; Advances in Chemistry; American Chemical Society: Washington, DC, 1976.

(35)

92

MYCOTOXINS

[ A . quercinus

(34)1,

A. sclerotiorum

a n d Paecilomyces

(36),

Chemical

and

Physical

(36),

ehrlichii

A. alliaceus

A.

(36),

ostianus

(23).

Properties

Patulin. P a t u l i n has a n e m p i r i c a l f o r m u l a of C H 0 , a m o l e c u l a r 7

6

4

w e i g h t of 154, a n d a m e l t i n g p o i n t of 1 1 0 ° - 1 1 2 ° C . P a t u l i n has a n o p t i c a l l y a c t i v e c a r b o n a t o m ; h o w e v e r the r a c e m i c m i x t u r e occurs n a t u r a l l y . T h e crystals are l a r g e m o n o c l i n i c tables ( 0 0 1 ) .

T h e p l a n e of t h e o p t i c


axes is i n c l i n e d at a p p r o x i m a t e l y 2 0 ° to t h e n o r m a l to ( 0 0 1 ) to b).

T h e unit cell dimensions are: a =

(β _

(y is p a r a l l e l

12.42, & = 9.47, c =

7.78A

46.7° ) . T h e d e n s i t y is 1.528 ± .003, a n d the space g r o u p is P 2 t / a ,

Z = 4

(37).

P a t u l i n is s o l u b l e i n w a t e r , a l c o h o l , acetone,

e t h y l acetate,

and

c h l o r o f o r m , s l i g h t l y soluble i n e t h y l ether a n d b e n z e n e , a n d i n s o l u b l e i n p e t r o l e u m ether. I t d e c o m p o s e s s l o w l y i n w a t e r a n d m e t h a n o l , b u t i t is stable i n b e n z e n e , c h l o r o f o r m , a n d m e t h y l e n e c h l o r i d e (38).

P a t u l i n is

n o t stable as a t h i n film f r o m a n e v a p o r a t e d s o l u t i o n ( 3 9 ) . T h e i r s p e c t r u m of p a t u l i n has m a j o r b a n d s at 1768, 1745 a n d 3390 c m " i n n u j o l m u l l (40);

(shoulder),

i n h e x a c h l o r o b u t a d i e n e m u l l there is a

1

b r o a d O H b a n d at 3360 c m " , a n d i n K B r disks there are b a n d s at 1030, 1

1160, 1200, 1740, a n d 1765 c m '

1

e x t i n c t i o n coefficients

P a t u l i n has a single u v a b s o r p

(41, 42).

t i o n m a x i m u m at a b o u t 276 n m ( 4 3 ) .

Scott ( 4 4 ) p r e s e n t e d t h e r e p o r t e d

i n detail. T h e proton N M R spectrum i n C D C 1

3

exhibits c h e m i c a l shifts at δ = 5.97 ( 3 P , c o m p l e x ) , δ 4.73 ( I P , d o u b l e t of d o u b l e t s , A p a r t of A B X system, /

A B

doublets, Β p a r t of A B X system, J

B

7 = 5cps)

(44).

A

= =

1 7 c p s ) , δ 4.40 ( I P , d o u b l e t

of

17 c p s ) , a n d δ 3.46 ( I P , d o u b l e t ,

T h e mass s p e c t r u m of p a t u l i n w a s r e p o r t e d b y Scott

et a l . ( 4 5 ) ; the mass s p e c t r u m of the t r i m e t h y l s i l y l ether was r e p o r t e d b y Scott ( 4 4 ) .

Scott a n d Y a l p a n i (46)

p r o p o s e d structures for seven p r i n

c i p a l f r a g m e n t ions of d e u t e r a t e d p a t u l i n w i t h m/e =

138, 128, 111, 99,

83, 72, a n d 56. P a t u l i n forms a c e t y l , 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e , p h e n y l h y d r a z o n e , s e m i c a r b a z o n e , o x i m e , a n d m e t h y l ether d e r i v a t i v e s . It reduces F e h f i n g s reagent, p o t a s s i u m p e r m a n g a n a t e , a n d a m m o n i a c a l s i l v e r n i t r a t e ( 4 3 , 47, 48).

R e d u c t i o n w i t h s o d i u m b o r o h y d r i d e y i e l d s a s c l a d i o l , a less t o x i c

m y c o t o x i n i s o l a t e d f r o m A . cfovatus

(49).

Hydrogénation gives

p a t u l i n i c a c i d w h i c h has b e e n i s o l a t e d f r o m P . urticae (50).

[P.

P a t u l i n w a s s y n t h e s i z e d b y W o o d w a r d a n d S i n g h (51);

oxime was synthesized starting from acetylenic compounds

(52).

desoxypatuluml patulin For a

d e t a i l e d r e v i e w of p a t u l i n synthesis see K o r z y b y s k i et a l . ( 5 3 ) . Penicillic A c i d . P e n i c i l l i c a c i d , C H i O , has a m o l e c u l a r w e i g h t of 8

0

4

170, a m e l t i n g p o i n t of 8 4 ° - 8 7 ° C ( h y d r a t e d , 5 8 ° - 6 4 ° C ) , a n d a n e u t r a l i -


6.

Patulin and Penicillic

WILSON

z a t i o n e q u i v a l e n t of 169 ( 2 5 ) . s o l u t i o n (54).

93

Acid

The K

a

is 1.26 X 10" at 2 5 ° C i n aqueous 6

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

h o t w a t e r , a l c o h o l , ether, b e n z e n e , c h l o r o f o r m , a n d e t h y l acetate a n d is i n s o l u b l e i n hexane a n d p e t r o l e u m ether. P e n i c i l l i c a c i d absorbs B r , reduces F e h l i n g s s o l u t i o n w h e n h e a t e d , 2

reduces a m m o n i a c a l s i l v e r n i t r a t e , turns y e l l o w i n a l k a l i n e s o l u t i o n a n d d e e p r e d u p o n exposure to a m m o n i a , a n d turns F e C l 55). (56).

b r o w n r e d ( 2 1 , 22,

T h e hydrogenated derivative dihydropenicillic acid was isolated

f r o m a n u n i d e n t i f i e d fungus (57). Downloaded by UNIV OF GUELPH LIBRARY on July 25, 2012 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch006

3

P e n i c i l l i c a c i d has a t e n d e n c y to self-associate s t r o n g l y i n s o l u t i o n T h e a m m o n i a t e d d e r i v a t i v e is

fluores

cent w i t h e x c i t a t i o n m a x i m a at 350 n m a n d e m i s s i o n m a x i m a at 440 n m (58). T h e i n f r a r e d s p e c t r u m has b a n d s at 3270, 1 7 2 8 , 1 6 4 3 , 1352, 1223, 909, a n d 811 c m "

1

i n K B r (57).

K o v a c and Solcaniova (56)

give solution i r

b a n d s for p e n i c i l l i n a c i d i n c a r b o n t e t r a c h l o r i d e a n d c h l o r o f o r m .

The uv

a b s o r p t i o n m a x i m u m is at 227 n m i n H 0 , 225 n m i n 7 6 % e t h a n o l , 224 2

n m i n e t h a n o l , 221 n m i n m e t h a n o l , a n d shifts to 224 n m i n 0 . 0 2 N H C 1 a n d 293 n m i n 0 . 0 2 N N a O H (27, 56, 57, 5 9 ) .

T h e uv absorption m a x i

m u m f o r p e n i c i l l i c a c i d acetate is at 229 n m i n e t h a n o l a n d for m e t h y l p e n i c i l l a t e is at 224 n m i n e t h a n o l

(56).

T h e p r o t o n N M R s p e c t r u m of p e n i c i l l i c a c i d i n d e u t e r a t e d

benzene

exhibits c h e m i c a l shifts for a 3-proton s i g n a l at δ 1.72 a n d δ 3.14 a n d f o r a 1-proton s i g n a l at δ 419, δ 4.91, δ 5.02, a n d δ 5.62 ( 2 7 ) . ( 3 3 ) d e t e r m i n e d the N M R s p e c t r u m i n C D C 1

3

s p e c t r u m c a n b e f o u n d i n C i e g l e r a n d K u r t z m a n (30) (60).

V a n Eijk

(27)

K o b a y a s h i et a l .

a n d D M S O - d . T h e mass 6

a n d S u z u k i et a l .

d i s c u s s e d possible structures f o r f o u r p r i n c i p a l

f r a g m e n t ions i n the mass s p e c t r u m of p e n c i l l i c a c i d b u t c o u l d n o t d i s t i n g u i s h b e t w e e n t h e free a c i d a n d lactone tautomers. P e n i c i l l i c a c i d a n d d i h y d r o p e n i c i l l i c a c i d w e r e s y n t h e s i z e d b y R a p h a e l (55, 64,

65).

T h e t a u t o m e r i s m of p e n i c i l l i c a c i d has b e e n s t u d i e d u s i n g u v a b s o r p t i o n spectroscopy

( 3 1 , 61, 62, 63).

F o r d et a l . (61)

stated that u v

s p e c t r a l measurements h a v e d o u b t f u l v a l u e i n d e t e r m i n i n g t h e c y c l i c o r o p e n c h a i n f o r m a n d c o u l d n o t a d e q u a t e l y define t h e e q u i l i b r i u m b e t w e e n the cyclic and open

c h a i n forms

u s i n g i r spectroscopy.

Using

mass

spectroscopy, v a n E i j k ( 2 7 ) c o u l d n o t d i s t i n g u i s h b e t w e e n the c y c l i c a n d o p e n c h a i n f o r m s o n the basis of possible structures of the i o n f r a g m e n t s . T h e e q u i l i b r i u m a n d existence of the o p e n c h a i n a n d c y c l i c t a u t o m e r s i n v a r i o u s solutions w a r r a n t s f u r t h e r i n v e s t i g a t i o n . Biosynthesis P a t u l i n . B u ' L o c k et a l . (66)

w h o studied conditions favoring p a t u l i n

biosynthesis d e s c r i b e d t w o p h y s i o l o g i c a l phases i n P . urticae:

i n t h e first


94

MYCOTOXINS

p h a s e ( t r o p h o p h a s e ) m y c e l i a l Ν, P , R N A , a n d S H r e a c h m a x i m u m v a l u e s ; i n the s e c o n d p h a s e ( i d i o p h a s e ) these values are r e d u c e d , a n d p a t u l i n a n d other secondary metabolites f r o m 6-methylsalicylic a c i d appear.

Bassett

a n d T a n e n b a u m first p r o p o s e d a s c h e m e for p a t u l i n biosynthesis u s i n g k n o w n m e t a b o l i c p r o d u c t s of P . urticae from

1 4

(67, 68,69).

C - l a b e l e d 6 - m e t h y l s a l i c y l i c a c i d (70).

Patulin was derived

T h e 6-methylsalicyfic a c i d

w a s d e r i v e d f r o m glucose o r acetate w i t h a c e t y l c o e n z y m e A p l a y i n g a n i m p o r t a n t r o l e (71) coenzyme A

as d i d m a l o n a t e , p r e s u m a b l y i n t h e f o r m of m a l o n y l

(72).


B u ' L o c k et a l . (73)

d i s c u s s e d the enzymes t h a t m a y r e g u l a t e p a t u l i n

biosynthesis. T w o of the e n z y m e s r e q u i r e d for the c o n v e r s i o n of 6 - m e t h y l s a l i c y l i c a c i d to p a t u l i n are 6 - m e t h y l s a l i c y l i c a c i d d e c a r b o x y l a s e a n d m-hydroxybenzyl alcohol dehydrogenase L i g h t (76)

(75).

(74)

Also Arihood and

r e p o r t e d t h a t 6 - m e t h y l s a l i c y l i c a c i d synthesis w a s i n h i b i t e d

b y 6 - m e t h y l s a l i c y l i c a c i d a n d some s t r u c t u r a l analogues of this c o m p o u n d . F o r r e s t e r a n d G a u c h e r (77)

described the major p a t h w a y for patulin

biosynthesis s t a r t i n g w i t h a c e t y l C o - A + 3 m a l o n y l C o - A - » 6 - m e t h y l salicylic acid - » m-cresol - » m-hydroxybenzyl alcohol - » m-hydroxybenzaldehyde - » gentisaldehyde - » pre-patulin - » patulin. p r o p o s e d another p a t h w a y l e a d i n g d i r e c t l y f r o m

Scott

et

through an intermediate to patulin ( F i g u r e 3 ) .

OH OH

al.

( 78 )

m-hydroxybenzaldehyde

I

Bioorganic Chemistry

Figure 3. Proposed biosynthetic pathways from 6-methylsalicyclic acid to patulin (78)


6.

Patulin

WILSON

and Penicillic

Penicillic A c i d .

95

Acid

B i r c h et a l . ( 7 9 ) suggested t h a t i n P .

cyclopium

p e n i c i l l i c a c i d w a s f o r m e d w h e n f o u r acetate u n i t s c o n d e n s e d t o f o r m o r s e l l i n i c a c i d f o l l o w e d b y r i n g cleavage a n d d e c a r b o x y l a t i o n t o y i e l d penicillic acid. baarense

M o s b a c h (80)

demonstrated a similar sequence i n P .

as d i d B i r k i n s h a w a n d G o w l a n d ( 3 2 ) i n P . madriti.

a n d K e i l (81,82)

showed that orsellinic acid was formed i n P .

Bentley cyclopium

w h e n o n e a c e t y l c o e n z y m e A u n i t c o n d e n s e d w i t h three m a l o n y l c o e n z y m e A u n i t s w h i c h s u b s e q u e n t l y lost t h r e e m o l e c u l e s o f C 0 f o r m i n g o r s e l l i n i c 2

a c i d a n d w h i c h u n d e r w e n t r i n g cleavage a n d d e c a r b o x y l a t i o n t o y i e l d Downloaded by UNIV OF GUELPH LIBRARY on July 25, 2012 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch006

p e n i c i l l i c a c i d ( F i g u r e 4 ) . C i e g l e r et a l . (83) r e v i e w e d t h e biosynthesis of p e n i c i l l i c a c i d i n m o r e d e t a i l . acetyl CoA + 3 malonyl CoA

I Ο Ο Ο Il II II C H 3 - C - C H - C - C H - C - C H - C 0 S C 0 A I I I COOH COOH COOH J

OH

Journal of Biological Proceedings of the Chemical Society

Figure 4. Intermediates in the biosyn thesis of penicillic acid (81, 82) Toxicity Patulin.

P a t u l i n is a w i d e - s p e c t r u m b i o c i d e .

I t is t o x i c t o m a n y

bacteria, protozoa, fungi, mammals, a n d plants a n d inactivates some viruses.

F o r a d e t a i l e d d i s c u s s i o n o f p a t u l i n t o x i c i t y , see r e v i e w s b y

K o r z y b s k i et a l . ( 5 3 ) , E n o m o t o a n d Satio ( 8 4 ) , Scott ( 4 4 ) f o r t o x i c i t y d e tails, a n d B r o o m et a l . ( 8 5 ) f o r t h e p h a r m a c o l o g y of p a t u l i n . H o w e v e r a b r i e f c o n s i d e r a t i o n o f t h e t o x i c i t y o f p a t u l i n to f a r m a n i m a l s a n d p l a n t s is i m p o r t a n t i n r e l a t i o n to its n a t u r a l occurrence. f r o m soils w h e r e p h y t o t o x i c i t y is o b s e r v e d .

P a t u l i n has b e e n i s o l a t e d

I t c o n t r i b u t e s to t h e p h y t o -

t o x i c i t y of s t u b b l e - m u l c h e d soils (89) a n d to t h e s o i l sickness p r o b l e m i n a p p l e nurseries ( 9 0 ) . T h e r e is o n l y i n d i r e c t e v i d e n c e that p a t u l i n m a y b e associated w i t h a n i m a l diseases c a u s e d b y m y c o t o x i n c o n t a m i n a t e d f e e d .

U k a i et a l .


96

MYCOTOXINS

o b t a i n e d p a t u l i n f r o m a Pénicillium

(86)

sp. t h a t h a d b e e n i s o l a t e d f r o m

a m a l t f e e d associated w i t h the d e a t h of several cattle i n J a p a n . A t o x i c A . davatus

was isolated from a malt causing a feeding p r o b l e m

p a t u l i n m a y h a v e b e e n responsible.

R e c e n t l y , the L D

5 0

(87);

from oral admin

i s t r a t i o n of p a t u l i n for w h i t e L e g h o r n cockrels w a s 170 m g / k g

(88)—

demonstrating moderate toxicity. C h r o n i c a n d sub-acute f e e d i n g s t u d y trials are n e e d e d b e f o r e t h e i m p a c t of p a t u l i n o n f a r m a n i m a l s a n d h u m a n s is k n o w n . T h e effects of p a t u l i n o n the i n t e s t i n a l m i c r o f l o r a n e e d to be e x a m i n e d as w e l l as t h e Downloaded by UNIV OF GUELPH LIBRARY on July 25, 2012 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch006

p o s s i b i l i t y of p a t u l i n a n d other c o m p o u n d s a c t i n g together

to g i v e

a

synergistic toxic reaction. Penicillic A c i d .

A l s b e r g a n d B l a c k (21)

found that penicillic acid

w a s l e t h a l to m i c e at a subcutaneous i n j e c t i o n of ca. 2 0 0 - 3 0 0 m g / k g .

The

a n t i m i c r o b i a l a c t i v i t y was s t u d i e d b y O x f o r d et a l . ; g r a m - n e g a t i v e b a c t e r i a w e r e affected m o r e t h a n g r a m - p o s i t i v e b a c t e r i a b y p e n i c i l l i c a c i d ( 9 1 , 9 2 ) . H o w e v e r , h e a r t b r o t h w a s u s e d i n these experiments, a n d O x f o r d

(93)

l a t e r f o u n d that p e n i c i l l i c a c i d lost bacteriostatic p o w e r i f a b r o t h m e d i u m was used. T h e pharmacology The L D

(94).

5 0

of p e n i c i l l i c a c i d was discussed b y M u r n a g h a n

of p e n i c i l l i c a c i d w a s 110 m g / k g for m i c e b y s u b c u t a n e

ous i n j e c t i o n a n d 250 m g / k g b y intravenous i n j e c t i o n (94).

N o charac

t e r i s t i c features w e r e f o u n d post m o r t e m . F o r r a b b i t s t h e m i n i m a l l e t h a l dose r a n g e d f r o m

100-200 m g / k g

administered subcutaneously

W i t h p e r o v a l a d m i n i s t r a t i o n of p e n i c i l l i c a c i d , u p to 715 m g / k g w e i g h t for 8 days w a s t o l e r a t e d b y w h i t e m i c e w i t h o u t detriment (95).

H a l l et a l . (96)

(95). body

recognizable

s h o w e d that p e n i c i l l i c a c i d h a d a n t i -

p h a g e a c t i v i t y against free b a c t e r i o p h a g e

particles.

T h e possible

anti

t u m o r a n d a n t i v i r a l properties of p e n i c i l l i c a c i d are discussed b y S u z u k i etal.

(60). T h e t o x i c i t y of p e n i c i l l i c a c i d a n d p a t u l i n i n c u l t u r e d cells

s t u d i e d b y N a t o r i et a l . ( 9 7 ) , U m e d a ( 9 8 ) , a n d K a w a s a k i et a l . W i t h H e L a cells, p e n i c i l l i c a c i d at ΙΟμ-g/ml p r o d u c e d

was (99).

a n increase

of

m i t o t i c cells a n d e n l a r g e m e n t of i n t e r p h a s i c cells. E n l a r g e d i n t e r p h a s i c cells c o n t a i n e d large n u c l e i w i t h dotty c h r o m a t i n a n d i r r e g u l a r a n d l a r g e r nucleoli.

Pleomorphism

was

relatively marked

(97).

The

action

of

p e n i c i l l i c a c i d o n H e L a cells was s l o w , a n d the a c c u m u l a t i o n of m e t a p h a s i c cells w a s p r o m i n e n t , a c c o m p a n i e d b y e l o n g a t i o n of the w h o l e c e l l cycle Stability

(99). and Mode of

Action

T h e s t a b i l i t y of p a t u l i n a n d p e n i c i l l i c a c i d at t h e p H of interest a n d t h e r e a c t i o n w i t h c e l l u l a r or m e d i u m constituents are i m p o r t a n t i n c o n -


6.


W I L S O N

97

Acid

s i d e r i n g t h e m o d e of a c t i o n of these m y c o t o x i n s .

Patulin, but not peni

c i l l i c a c i d , loses its b i o l o g i c a l a c t i v i t y i n a l k a l i n e c o n d i t i o n s . B o t h p a t u l i n a n d p e n i c i l l i c a c i d are c a p a b l e of r e a c t i n g w i t h s u l f h y d r y l g r o u p s

or

a m i n o g r o u p s , a n d t h e a d d u c t s seem to h a v e l i t t l e b i o l o g i c a l a c t i v i t y . T h u s i t is necessary to d e t e r m i n e t h e s t a b i l i t y u n d e r a n y set o f e x p e r i m e n t a l c o n d i t i o n s i n o r d e r to evaluate d a t a p r o p e r l y c o n c e r n i n g t h e m o d e of a c t i o n of these m y c o t o x i n s . A l s o i n d e t e r m i n i n g t h e m o d e o f a c t i o n o f patulin a n d penicillic acid, the mycotoxin concentration a n d time of the m e a s u r e d effect is c r i t i c a l to d i s t i n g u i s h b e t w e e n p r i m a r y a n d s e c o n d a r y Downloaded by UNIV OF GUELPH LIBRARY on July 25, 2012 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch006

effects. H e a t l e y a n d P h i l p o t (100)

found that penicillic a c i d was stable

w h e n h e a t e d to 1 0 0 ° C at p H s 2.0 a n d 9.5. T h u s t h e effects of p H o n detoxification o f p e n i c i l l i c a c i d a r e p r o b a b l y m i n i m a l .

Patulin on the

other h a n d w a s stable at p H 2.0 a n d u n s t a b l e at p H 9.5 (100). decomposition

The

of p a t u l i n p r o d u c e d i n t h e c u l t u r e m e d i u m b y p a t u l i n

p r o d u c e r s p r o b a b l y arose f r o m p H changes

(101).

P a t u l i n w a s stable

i n M c l l v a i n e buffer solutions at p H 3.3-6.3; at p H 6.3 s l o w i n a c t i v a t i o n o c c u r r e d (102).

L o v e t t a n d P e e l e r (103) d e t e r m i n e d t h e t h e r m a l d e s t r u c

t i o n k i n e t i c s of p a t u l i n i n M c l l v a i n e s ' buffer of p H 3.5, 4.5, a n d 5.5 h e a t e d to 1 0 5 ° - 1 2 5 ° C . P a t u l i n w a s resistant to t h e r m a l d e s t r u c t i o n a t a l l p H s . However,

the thermal destruction

parameters

increased

as t h e p H

decreased. P a t u l i n is m o d e r a t e l y stable at 22 ° C i n a p p l e a n d g r a p e j u i c e b u t n o t i n o r a n g e juice or flour (104).

H e a t i n g t h e juices to 8 0 ° C f o r s h o r t

p e r i o d s d i d n o t c o m p l e t e l y destroy p a t u l i n . I t w a s stable i n d r y c o r n b u t u n s t a b l e i n w e t c o r n , w h e a t , s o r g h u m , o r aqueous solutions c o n t a i n i n g S0

2

( 3 8 , 1 0 5 ) . T h e s e studies w e r e d o n e at r o o m t e m p r e a t u r e ; f u r t h e r

studies o n t h e p r o d u c t i o n a n d s t a b i l i t y of p a t u l i n a n d p e n i c i l l i c a c i d a r e n e e d e d at different m o i s t u r e a n d t e m p e r a t u r e levels. P e n i c i l l i c a c i d w a s also m o d e r a t e l y stable at 22 ° C i n a p p l e a n d g r a p e j u i c e b u t n o t i n o r a n g e juice, w h o l e w h e a t , or b l e a c h e d

flour

H e a t i n g t h e juices f o r short p e r i o d s to 8 0 ° C d i d n o t c o m p l e t e l y

(104). destroy

the p e n i c i l l i c a c i d . P e n i c i l l i c a c i d w a s m o d e r a t e l y stable i n w e t c o r n a t 0 ° - 1 5 ° C a n d less stable at 2 0 ° C .

I n general, commodities w i t h h i g h p r o

t e i n content ( p e a n u t s , soybeans, a n d c o t t o n s e e d ) either d i d n o t s u p p o r t p e n i c i l l i c a c i d synthesis or p e n i c i l l i c a c i d w a s n o t stable i n t h e m ( 3 0 , 5 9 ) . P a t u l i n w a s stable i n a c i d soils a n d s a n d . I t w a s m o r e stable i n h e a t t r e a t e d s o i l at p H 7.2 t h a n i n u n t r e a t e d s o i l , i n d i c a t i n g t h a t b i o l o g i c a l a c t i v i t y c a u s e d i n a c t i v a t i o n (102).

P a t u l i n w a s m u c h m o r e stable i n s o i l

containing molds that p r o d u c e d patulin than i n garden soil w i t h a n e u t r a l pH

(106). O n e of the first suggested m e c h a n i s m s of t h e a c t i o n of p a t u l i n w a s

t h a t i t r e a c t e d w i t h S H groups of enzymes

a n d e x e r t e d its a n t i b i o t i c


98

MYCOTOXINS

a c t i v i t y i n this w a y . P a t u l i n a n d p e n i c i l l i c a c i d c a n react w i t h free S H g r o u p s as w e l l as N H g r o u p s u n d e r t h e p r o p e r c o n d i t i o n s . O x f o r d 2

(93)

d e m o n s t r a t e d that at p H 7.0 p e n i c i l l i c a c i d s o l u t i o n r e a c t e d w i t h some p r i m a r y a m i n e s a n d a m i n o acids i n a m e d i u m k e p t at 37 ° C for s e v e r a l days. G e i g e r a n d C o n n (107)

f o u n d t h a t cysteine a n d t h i o g l y c o l a t e d i d

n o t g i v e a p o s i t i v e n i t r o p r u s s i d e r e a c t i o n w i t h a n excess of p a t u l i n o r p e n i c i l l i c a c i d . T h e s e r e a c t i o n p r o d u c t s h a d no o b s e r v e d t o x i c p r o p e r ties.

Thiosulfate inactivated patulin only.

Dickens and Cooke

(108)

r e p o r t e d t h a t p a t u l i n a n d p e n i c i l l i c a c i d r e a c t e d r a p i d l y w i t h cysteine. Downloaded by UNIV OF GUELPH LIBRARY on July 25, 2012 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/ba-1976-0149.ch006

G o o d m a n a n d H i a t t ( 1 0 9 ) a n d A n d r a u d et a l . (110)

f o u n d that e n z y m i c

S H groups reacted w i t h patulin. A s h o o r a n d C h u (111 ) f o u n d i n h i b i t i o n constants of a l c o h o l d e h y d r o genase f o r p a t u l i n at 5.0 X 1 0 " M a n d for p e n i c i l l i c a c i d at 1.1 X 1 0 " M 5

4

w h e r e n o n - c o m p e t i t i v e i n h i b i t i o n was o b s e r v e d a n d i n h i b i t i o n constants of l a c t i c d e h y d r o g e n a s e for p a t u l i n at 6.2 X 1 0 " M a n d p e n i c i l l i c a c i d at 6

7.2 X 1 0 " M w h e r e c o m p e t i t i v e i n h i b i t i o n w a s observed. 5

v e r s e d the effect o n l a c t i c d e h y d r o g e n a s e genase.

G o t t l i e b a n d S i n g h (112)

Cysteine re

but not on alcohol dehydro

o b s e r v e d succinate oxidase a n d d e

h y d r o g e n a s e i n h i b i t i o n at h i g h c o n c e n t r a t i o n of p a t u l i n , 5 X 1 0 " M . 2

C i e g l e r et al. ( 2 9 ) f o u n d t h a t a r g i n i n e , h i s t i d i n e , a n d lysine r e a c t e d w i t h p e n i c i l l i c acid. T h e reaction between lysine a n d histidine a n d p e n i c i l l i c a c i d w e n t t o c o m p e t i o n i n n i n e days at p H 7.0. T h e r e a c t i o n b e t w e e n p e n i c i l l i c a c i d a n d g l u t a t h i o n e or cysteine w a s essentially c o m p l e t e i n 7 hrs at p H 5, 6, or 7. T h e r e a c t i o n p r o d u c t s b e t w e e n g l u t a t h i o n e o r c y s t e i n e a n d p e n i c i l l i c a c i d w e r e i d e n t i f i e d as a n a d d i t i o n to the i s o l a t e d d o u b l e b o n d r a t h e r t h a n the c o n j u g a t e d d o u b l e b o n d ( F i g u r e 5 ) .

R Applied Microbiology

Figure 5. Reaction between penicillic acid and RSH when RSH is cysteine or glutathione (29) H o f m a n n et a l . (US)

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

s u l f h y d r y l g r o u p s at p H 7.4 t h a n at p H 5.0.

T h e y postulated that the

a d d i t i o n of R S H c o u l d o c c u r i n s e v e r a l w a y s , b u t t h e r e a c t i o n p r o d u c t s w e r e n o t i s o l a t e d a n d i d e n t i f i e d . I f the a d d i t i o n w e r e at the 3 a n d 7


6.

WILSON

Patulin

and Penicillic

99

Acid

p o s i t i o n w i t h a shift i n t h e d o u b l e b o n d , t h e l a c t o n e r i n g m i g h t b e less stable a n d m o r e l i a b l e to h y d r o l y s i s , e x p l a i n i n g t h e loss of b i o l o g i c a l


activity ( F i g u r e 6 ) .

Figure 6.

One possible reaction patulin and RSH

between

T h e r e a c t i o n of s u l f h y d r y l g r o u p s w i t h β-propiolactone w a s s t u d i e d b y D i c k e n s a n d Jones (114).

T h e y isolated S-2-carboxylethyl-L-cysteine

as t h e m a j o r p r o d u c t w h e n c y s t e i n e a n d β - p r o p i o l a c t o n e r e a c t e d i n n e u t r a l solution.

I n t h e presence o f β - p r o p i o l a c t o n e a n d h e a t - i n a c t i v a t e d l i v e r

supernatant, n o free a c i d w a s p r o d u c e d p r e s u m a b l y b e c a u s e t h e β - p r o p i o l a c t o n e r e a c t e d w i t h t h e d e n a t u r e d proteins

(115).

Compounds

with

α,β-unsaturation m a y r e a c t w i t h S H groups a t t a c h e d to p r i m a r y c a r b o n atoms b u t p r o b a b l y n o t w i t h those a t t a c h e d to t e r t i a r y c a r b o n ( 116).

Jones a n d Y o u n g (117)

(4-hydroxypent-2-enoic

atoms

f o u n d that t h e b i o l o g i c a l l y a c t i v e l a c t o n e

acid lactone)

r e a c t e d w i t h p r i m a r y amines to

y i e l d a n unstable M i c h a e l addition product whereas inactive

lactones

s u c h as 4 - h y d r o x y p e n t - 3 - e n o i c a c i d lactone gave rise to a m i d e d e r i v a t i v e s . F u r t h e r studies b y Jones a n d Y o u n g ( U S ) r e v e a l e d t h a t lactones s u c h as 4 - h y d r o x y p e n t - 2 - e n o i c

carcinogenic

a c i d lactone u n d e r w e n t M i c h a e l

a d d i t i o n w i t h R S H w h i c h t h e n gave rise to t h e S - a l k y l a t e d d e r i v a t i v e . T h e reactions o f p a t u l i n a n d p e n i c i l l i c a c i d w i t h s u l f h y d r y l a n d a m i n o g r o u p s n e e d to b e s t u d i e d i n m o r e d e t a i l , a n d t h e t o x i c i t y o f t h e d e r i v a t i v e s s h o u l d b e d e t e r m i n e d . I n 1961 D i c k e n s a n d Jones (114)

re

ported that patulin a n d penicillic a c i d produced malignant tumors w h e n a d m i n i s t e r e d s u b c u t a n e o u s l y to rats. A l l of t h e c a r c i n o g e n i c a l l y a c t i v e lactones possessed e i t h e r α,β-unsaturation, at p o s i t i o n 4, o r b o t h (119,120,121).

a n external unsaturated b o n d

Because both patulin a n d penicillic

a c i d are q u i t e r e a c t i v e , o r a l f e e d i n g

studies a r e n e e d e d

properly their potential health hazard.

to interpret

S i n c e t h e r e a c t i o n p r o d u c t of

β - p r o p i o l a c t o n e a n d cysteine, S - 2 - c a r b o x y e t h y l - L - c y s t e i n e , h a d w e a k c a r c i n o g e n i c properties, t h e r e a c t i o n p r o d u c t s o f p e n i c i l l i c a c i d a n d p a t u l i n w i t h R S H should b e evaluated for carcinogenic properties. S o m e o f p a t u l i n ' s other effects o n b i o l o g i c a l systems are i n t e r e s t i n g i n r e l a t i o n to d e f i n i n g p r i m a r y a n d s e c o n d a r y effects, b u t t h e list is n o t


100

MYCOTOXINS

i n t e n d e d to b e i n c l u s i v e .

These

i n c l u d e effects o n c a t i o n transfer i n

h u m a n erythrocytes where p a t u l i n i n h i b i t e d K A t h i g h concentrations

transfer at 1 0 " M

+

1 0 " M p a t u l i n i n h i b i t e d succinate

succinate dehydrogenase

(112).

W i t h e r s (123)

3.5 X 1 0 " M i n d u c e d a b n o r m a l metaphases a c i d o n m i t o s i s , G o r i n i et a l . (124)

oxidase a n d

f o u n d t h a t p a t u l i n at

a n d a h i g h percentage

6

p o l y p l o i d cells i n h u m a n c h r o m o s o m e s .

(122).

3

2

of

S t u d y i n g t h e effect o f p e n i c i l l i c

c o n c l u d e d that p e n i c i l l i c a c i d not only

h a d a s t a t m o k i n e t i c a c t i o n b u t also a p a r t i a l i n t e r k i n e t i c b l o c k i n g a c t i o n i n t h e megaloblasts of c h i c k e n e m b r y o s .


1 4

N o t o x i c i t y w a s associated w i t h

C a c t i v i t y i n eggs l a i d b y hens f e d p a t u l i n

1 4

C ( 1 2 5 , 126).

A u s t i n et a l .

(127, J 2 8 ) r e p o r t e d t h a t p a t u l i n i n d u c e d s e r o t y p i c t r a n s f o r m a t i o n s i n Paramecium

aurelia

t h a t m a y i n fact b e m u t a t i o n s .

P e t i t e m u t a n t s o f Saccharomyces

cerevisiae

were induced b y patulin.

E x p o s u r e d u r i n g t h e e x p o n e n t i a l phase p r o d u c e d a h i g h e r m u t a t i o n f r e quency

t h a n d u r i n g t h e s t a t i o n a r y phase

f o u n d t h a t S. cerevisiae

(129).

H a r w i g et a l .

(130)

fermentation of apple juice was not i n h i b i t e d b y

s i m i l a r concentrations of p a t u l i n a n d t h a t t h e p a t u l i n d i s a p p e a r e d d u r i n g t h e f e r m e n t a t i o n process. T h e difference i n the p H of t h e m e d i u m a n d of t h e a p p l e j u i c e m a y b e i m p o r t a n t i n these different

observations.

U s i n g c e l l c u l t u r e systems U m e d a ( 9 8 ) d e m o n s t r a t e d t h a t s i m i l a r concentrations of p a t u l i n o r p e n i c l l i c a c i d d a m a g e d l i v e r , k i d n e y , l u n g , a n d H e L a cells. lin.

In HeLa

H o w e v e r p e n i c i l l i c a c i d w a s less c y t o t o x i c t h a n p a t u

cells p e n i c i l l i c a c i d a c t e d

s l o w l y , a n d a c c u m u l a t i o n of

m e t a p h a s i c cells w a s p r o m i n e n t . P a t u l i n a c t e d r a p i d l y a n d d i r e c t l y , stop p i n g t h e w h o l e c e l l c y c l e ( 9 9 ) . A t 100 f t g / m l o f p e n i c i l l i c a c i d a n d 3.2 / A g / m l of p a t u l i n , D N A synthesis w a s almost e n t i r e l y depressed, R N A a n d p r o t e i n synthesis w a s o n l y p a r t i a l l y depressed. (131)

but

Schaeffer et a l .

f o u n d a depression of R N A synthesis w i t h i n 2 0 m i n a n d a d e p r e s

s i o n o f p r o t e i n synthesis w i t h i n 60 m i n i n C h a n g l i v e r cells t r e a t e d w i t h 2.5 pg/ml o f p a t u l i n .

T h e r R N A species w e r e m o r e i n h i b i t e d t h a n t h e

n o n m e t h y l a t e d species; h o w e v e r

b o t h synthesis a n d m a t u r a t i o n o f t h e

R N A p r e c u r s o r species o c c u r r e d .

T h i s leads t o t h e s p e c u l a t i o n t h a t t h e

i n h i b i t i o n i n R N A biosynthesis m o s t l i k e l y o c c u r r e d at t r a n s c r i p t i o n f r o m D N A r a t h e r t h a n at m a t u r a t i o n f r o m p r e c u r s o r species (131).

Recovery

o c c u r r e d after 6 h r of t r e a t m e n t of H e L a cells w i t h p e n i c i l l i c a c i d , b u t n o r e c o v e r y w a s o b s e r v e d after 1 h r w i t h p a t u l i n ( 9 9 ) . C h a n g l i v e r cells d i d n o t r e c o v e r after a 2 0 m i n t r e a t m e n t w i t h 2.5 f i g / m l o f p a t u l i n P r i o r to these observations H e w i t t et a l . (132)

(131).

observed a decrease i n

R N A content i n p a t u l i n t r e a t e d c a u l i f l o w e r l e a f tissue. P a t u l i n a n d p e n i c i l l i c a c i d at h i g h concentrations i n d u c e d b r e a k s i n H e L a c e l l D N A after a 1-hr i n c u b a t i o n i n b o t h a l k a l i n e a n d n e u t r a l sucrose g r a d i e n t s

(133).

P a t u l i n a n d p e n i c i l l i c a c i d are b o t h capable of interacting w i t h S H g r o u p s o f e n z y m e s a n d a r e m o r e i n h i b i t o r y to s o m e t h i o l e n z y m e s t h a n


6.

W I L S O N

others.


101

Acid

H o w e v e r w i t h the exception of the w o r k b y Ashoor a n d C h u t h e effective c o n c e n t r a t i o n s are p r o b a b l y too h i g h to b e p r i m a r y

(111) effects.

T h e i n h i b i t i o n of p r o t e i n , R N A , a n d D N A syntheses occurs s o o n

after i n t o x i c a t i o n o f c e l l c u l t u r e s , i n d i c a t i n g a p r i m a r y effect. R e s p i r a t i o n seems to b e i n h i b i t e d o n l y at h i g h c o n c e n t r a t i o n s , a n d t h e effects o n r e s p i r a t i o n are p r o b a b l y s e c o n d a r y .

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

n o t c e r t a i n ; i n d e e d p a t u l i n a n d p e n i c i l l i c a c i d m a y h a v e m u l t i p l e effects


i n the cytoplasm.

Analysis P a t u l i n . P a t u l i n has b e e n d e t e c t e d i n s e v e r a l w a y s i n c l u d i n g p a p e r , t h i n l a y e r , gas, a n d l i q u i d c h r o m a t o g r a p h y . P a t u l i n w a s d e t e c t e d o n p a p e r c h r o m a t o g r a m s b y s p r a y i n g w i t h p h e n y l h y d r a z i n e a n d a l k a l i (101).

The

y e l l o w spot v i s i b l e after r e a c t i o n w i t h p h e n y l h y d r a z i n e h a s b e e n u s e d f o r p a t u l i n detection i n thin layer chromatography

(45, 104).

Scott

(44)

d i s c u s s e d i n d e t a i l t h e solvent systems a n d s p r a y reagents t h a t h a v e b e e n u s e d to detect p a t u l i n o n p a p e r a n d t h i n l a y e r c h r o m a t o g r a m s . T h i n l a y e r c h r o m a t o g r a p h y has b e e n u s e d extensively to estimate p a t u l i n i n foods a n d feeds.

Scott a n d Somers

(104)

extracted patulin

w i t h e t h y l acetate, d r i e d the extract w i t h c a l c i u m sulfate, a n d e l u t e d t h e p a t u l i n f r o m a s i l i c a g e l c o l u m n w i t h e t h y l acetate.

T h e patulin was

d e t e c t e d after s p r a y i n g w i t h a m m o n i a a n d p h e n y l h y d r a z i n e h y d r o c h l o r i d e a n d h e a t i n g ; t h e d e t e c t i o n l i m i t w a s 0.02-0.05 fig p a t u l i n (39).

Using

a p - a n i s a l d e h y d e s p r a y reagent c o n t a i n i n g e t h a n o l , acetic a c i d , a n d H S 0 , 2

the d e t c t i o n l i m i t w a s 0.1 μg p a t u l i n (134).

4

Reiss (135, 136) u s e d o - d i -

a n i s i d i n e ( s a t u r a t e d i n g l a c i a l a c e t i c a c i d ) to detect 0.02 μg o f p a t u l i n and N-methylbenzthiazolone-2-hydrazone

(Besthorns hydrazone)

to d e

tect 0.06 μ% p a t u l i n . A c e t o n i t r i l e - h e x a n e ( 4 + 1) w a s u s e d to extract p a t u l i n f r o m c o r n , w h e a t , r y e , oats, a n d s o r g h u m .

T h e a c e t o n i t r i l e phase w a s e v a p o r a t e d ,

and preparative T L C was used for preliminary purification. T h e patulin c o n c e n t r a t i o n w a s e s t i m a t e d u s i n g T L C plates c o n t a i n i n g a 254 n m fluo rescent i n d i c a t o r . T h e l i m i t o f d e t e c t i o n i n c o r n w a s a b o u t 4 0 f t g / k g ( 3 8 ) . A c e t o n i t r i l e - h e x a n e ( 1 0 0 : 4 5 ) w a s u s e d to extract p a t u l i n f r o m m e a t a n d m e a t p r o d u c t s (137). celite 545 column.

T h e acetonitrile phase was passed through a

T h e p a t u l i n content w a s e s t i m a t e d u s i n g d i p h e n y l -

b o r ( i n ) i c a c i d g i v i n g a d e t e c t i o n l i m i t o f 500 jug/kg. A s i m i l a r d e t e c t i o n l i m i t , 4 0 0 - 1 0 0 0 /xg/kg of p a t u l i n i n grains, w a s r e p o r t e d b y Stoloff et a l . (138)

using a multimycotoxin detection method.

E t h y l acetate w a s a n efficient solvent o f p a t u l i n i n a p p l e j u i c e ( 3 9 , 139). acetate

T h e a p p l e j u i c e w a s e x t r a c t e d w i t h e t h y l acetate, t h e d r i e d e t h y l was passed

through a silicagel column

using

benzene-ethyl


102

MYCOTOXINS

acetate ( 7 5 + 2 5 ) as t h e e l u t i n g solvent. P a t u l i n w a s d e t e c t e d b y T L C w i t h 3 - m e t h y l - 2 - b e n z o t h i a z o l i n o n e h y d r o c h l o r i d e as a s p r a y r e a g e n t ; t h e d e t e c t i o n l i m i t w a s 0.01 tig p a t u l i n . P a t u l i n w a s d e t e c t e d i n a p p l e juice u s i n g gas c h r o m a t o g r a p h y after a n i n i t i a l p r e p a r a t i v e T L C p u r i f i c a t i o n ( 4 5 ) . P o h l a n d et a l . (140)

pre

p a r e d t h e s i l y l ether, actate, a n d chloroacetate d e r i v a t i v e o f p a t u l i n a n d u s e d G L C analysis of the chloroacetate d e r i v a t i v e to detect 0.7 f i g / m l of p a t u l i n i n a p p l e juice. S u z u k i et a l . (141), P e r o et a l . (14S)

Pero and Harvan (142), and

d e v e l o p e d m e t h o d s f o r G L C analysis of t h e s i l y l ether


a n d trimethylsilyl derivatives of patulin. O n e l i q u i d c h r o m a t o g r a p h i c m e t h o d to d e t e r m i n e p a t u l i n i n a p p l e j u i c e has b e e n r e p o r t e d ( 1 4 4 ) . T h e i n i t i a l e x t r a c t i o n a n d c o l u m n c l e a n u p w e r e essentially t h e same as that u s e d b y Scott (139).

T h e p a t u l i n eluate

w a s e v a p o r a t e d to dryness a n d i m m e d i a t e l y d i s s o l v e d i n a s m a l l v o l u m e o f e t h y l acetate c o n t a i n i n g ^ - m e t h y l u m b e l l i f e r o n e as a n i n t e r n a l s t a n d a r d . T h e patulin was separated using isooctane/methylene c h l o r i d e / m e t h a n o l ( 8 4 + 15 + 1 ) o n Z o r b a x - s i l s i l i c a w i t h a flow of ca. 0.5 m l / m i n . T h e p e a k w a s c o l l e c t e d f o r T L C c o n f i r m a t i o n or G C - M S

d e t e r m i n a t i o n of t h e

acetate d e r i v a t i v e . T h e m a j o r p r o b l e m w i t h a n y m e t h o d t h a t estimates p a t u l i n o r p e n i c i l l i c a c i d is n o t h o w to extract i t b u t w h e n to extract i t . F o r e x a m p l e i n c a n n e d a p p l e j u i c e a n i n t e r f e r r i n g substance w a s r a p i d l y f o r m e d w h e n e x p o s e d to a i r (139);

i n f r e s h l y pressed, u n c l a r i f i e d j u i c e o n l y 5 0 - 6 0 %

o f t h e p a t u l i n w a s r e c o v e r e d after o v e r n i g h t storage i n a r e f r i g e r a t o r (145).

I n m e a t a n d b r e a d p a t u l i n occurs o n l y t e m p o r a r i l y d u r i n g f e r

m e n t a t i o n or m o l d i n g Penicillic A c i d .

(146,147). P e n i c i l l i c a c i d has b e e n d e t e c t e d u s i n g bioassay,

c o l o r i m e t r i c , t h i n l a y e r c h r o m a t o g r a p h i c , a n d gas c h r o m a t o g r a p h i c m e t h ods.

P e n i c i l l i c a c i d w a s n o n - t o x i c to z e b r a fish l a r v a e at 5 / * g / m l

(148),

a n d b r i n e s h r i m p l a r v a e w e r e m o d e r a t e l y sensitive to 10-20/xg/disc ( 1 4 9 ) . B e t i n a (150)

detected p e n i c i l l i c a c i d on paper chromatograms b y noting

t h e t o x i c i t y to Bacillus

subtillis

o n the chromatogram.

H y d r o x y l a m i n e r e a c t e d w i t h p e n i c i l l i c a c i d to g i v e a r e d c o l o r . A t 530 n m , Beers l a w h e l d t r u e f o r 8 0 - 1 0 0 0 m g / m l o f p e n i c i l l i c a c i d

(151).

A n o t h e r c o l o r i m e t r i c m e t h o d u s e d t h e r e d d i s h - p u r p l e c o m p l e x of a m m o n i a w i t h p e n i c i l l i c a c i d (81). (7:1)

(141),

E t h y l acetate (104),

chloroform-methanol

ethyl acetate-water

(90:10) (59), chloroform-methanol

( 7 0 : 3 0 ) ( 3 6 ) , a n d c h l o r o f o r m ( 2 9 , 152)

have extracted p e n i c i l l i c acid

f r o m v a r i o u s substances. S e v e r a l m e t h o d s h a v e b e e n u s e d f o r p r e l i m i n a r y p u r i f i c a t i o n . Scott a n d Somers (104)

d r i e d t h e e t h y l acetate extract w i t h c a l c i u m sulfate

a n d e l u t e d t h e p e n c i l l i c a c i d w i t h e t h y l acetate b y p a s s i n g i t t h r o u g h a s i l i c a g e l c o l u m n . P e r o et a l . (143)

used preparative thin layer chroma-


6.


W I L S O N

103

Acid

tography for preliminary purification. T h o r p e a n d Johnson (153) parti tioned the penicillic acid w i t h 3 % s o d i u m bicarbonate followed b y acidi fication

a n d e x t r a c t i o n w i t h e t h y l acetate.

T h e penicillic a c i d solution

was passed through a silica g e l c o l u m n u s i n g h e x a n e - e t h y l acetate-formic a c i d ( 7 5 0 + 2 5 0 + 1 ) as t h e e l u t i n g s o l v e n t . P e n i c i l l i c a c i d has b e e n d e t e c t e d o n t h i n l a y e r c h r o m a t o g r a m s b e cause i t

fluoresces

fluorescence

after exposure t o a m m o n i a ( 5 9 ) ; i t gives a y e l l o w

w i t h a m m o n i a a n d p h e n y l h y d r a z i n e (104)

w i t h p-anisaldehyde

a n d a green c o l o r

(134).


T h e t r i m e t h y l s i l y l e t h e r o f p e n c i l l i c a c i d w a s p r e f e r r e d f o r gas c h r o m a t o g r a p h y b y S u z u k i et a l . (141)

o v e r t h e acetate w h i c h f o r m e d

s l o w l y a n d t h e trifluoroacetate w h i c h a l w a y s gave t w o peaks. P e r o et a l . (143) a n d P e r o a n d H a r v a n (142)

d e s c r i b e d c o n d i t i o n s f o r gas c h r o m a t o

g r a p h i c d e t e c t i o n of p e n i c i l l i c a c i d a n d t h e t r i m e t h y l s i l y l ether.

The

t r i m e t h y l s i l y l ether w a s u s e d to d e t e c t p e n i c i l l i c a c i d i n m o l d y t o b a c c o (152).

T h o r p e a n d Johnson (153) successfully obtained t h e trifluoro

acetate of p e n i c i l l i c a c i d f o r gas c h r o m a t o g r a p h i c d e t e c t i o n i n c o r n a n d beans a n d p r e s e n t e d a c o n f i r m a t i o n m e t h o d u s i n g gas c h r o m a t o g r a p h y mass spectrometry. Natural

Occurrence

P a t u l i n . P a t u l i n w a s i m p l i c a t e d i n t h e mass deaths o f o v e r 100 c o w s that w e r e i n t o x i c a t e d b y d r y m a l t f e e d . Pénicillium

urticae

Patulin was obtained

from

that was subsequently isolated from the feed

(86).

P a t u l i n m a y h a v e b e e n e i t h e r t h e cause o f t h e i n t o x i c a t i o n , or i t m a y h a v e b e e n present i n s u b l e t h a l a m o u n t s t h a t a c t e d i n association w i t h other t o x i c substances. M a l e c u l m s i n t o x i c a t i o n o f c a t t l e associated w i t h m o l d y f e e d m a y b e c a u s e d b y p a t u l i n s i n c e t h e disease is associated w i t h A . clavatus

i n v a s i o n of t h e f e e d . A p p a r e n t l y t h e toxic p r i n c i p a l has n o t b e e n

identified

(87).

Pénicillium

expansum

causes a storage r o t of apples, pears, a n d c h e r

ries. B r i a n et a l . ( 154)

i d e n t i f i e d p a t u l i n i n d e c a y e d a p p l e j u i c e b y its

antimicrobial spectrum.

W a l k e r (155)

u s e d p a p e r c h r o m a t o g r a p h y to

detect p a t u l i n i n a p p l e j u i c e b y i t s u l t r a v i o l e t q u e n c h i n g at 254 n m . P a t u l i n was f o u n d i n commercially available apple juice i n C a n a d a a n d the U n i t e d States b y Scott et a l . ( 4 5 ) a n d W i l s o n a n d N u o v o (145).

A

l i m i t e d s u r v e y of c i d e r m i l l s ( 1 4 5 ) r e v e a l e d that p a t u l i n c o n t a m i n a t i o n depended on the proportion of decayed apples used i n m a k i n g fresh apple cider.

B e e r (156)

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

u n s o u n d f r u i t ; p a t u l i n w a s n o t d e t e c t e d i n fresh c i d e r m a d e f r o m u n d e c a y e d apples. A f t e r i n o c u l a t i n g a p p l e s w i t h P . expansum (157)

H a r w i g et al.

f o u n d u p to 250 / x g / m l p a t u l i n p e r d e c a y e d a p p l e i n t h e expressed


104

MYCOTOXINS

j u i c e , a n d W i l s o n a n d N u o v o (145)

f o u n d u p to 146 tig p a t u l i n / m l e x

p r e s s e d juice. L e s s t h a n 1 1 % o f t h e isolates of P . expansum

produced

o v e r 100 j u g / m l of expressed j u i c e i n i n o c u l a t e d apples (145).

Similar

concentrations of p a t u l i n w e r e f o u n d i n lesions of pears a n d stone f r u i t s d e c a y e d b y P . expansum

(158).

P a t u l i n has b e e n r e p o r t e d i n spontaneously m o l d e d b r e a d

(147,159)

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

sausage

T h e s t a b i l i t y of p a t u l i n at different t e m p e r a t u r e s a n d c o n d i t i o n s

(146).

should be studied i n more detail.

T h i s is e v i d e n t w h e n o n e c o m p a r e s


t h e studies o n p e n i c i l l i c a c i d p r o d u c t i o n i n c o r n w i t h t h e f e w studies o n p a t u l i n p r o d u c t i o n i n v a r i o u s c o m m o d i t i e s . P a t u l i n has also b e e n i s o l a t e d f r o m s o i l a n d w h e a t s t r a w residues w h e r e t h e r e is a p h y t o t o x i c p r o b l e m associated w i t h s t u b b l e m u l c h i n g (160).

T h e levels o f p a t u l i n r e c o v e r e d

are h i g h e n o u g h to l e a v e l i t t l e d o u b t t h a t p a t u l i n is a f a c t o r i n this phytotoxicity. Penicillic A c i d .

P e n i c i l l i c a c i d has b e e n f o u n d i n m o l d y t o b a c c o

f r o m c o m m e r c i a l storage

(152)

K u r t z m a n a n d C i e g l e r (59)

a n d i n m o l d y c o r n a n d beans

r e p o r t e d t h a t P . martensii

(153).

molded

high-

m o i s t u r e c o r n at 1 ° C . T h e y i s o l a t e d h i g h levels o f p e n i c i l l i c a c i d f r o m a r t i f i c i a l l y i n o c u l a t e d c o r n i n c u b a t e d at t e m p e r a t u r e s b e t w e e n

1° a n d

15 ° C . T h e p e n i c i l l i c a c i d d i s a p p e a r e d w i t h i n 4 5 days a t h i g h e r t e m p e r a tures. S e v e r a l other species of Pénicillium

that cause b l u e - e y e d

disease

of c o r n w e r e c a p a b l e of p e n i c i l l i c a c i d synthesis o n s e v e r a l c o m m o d i t i e s . P e a n u t s , soybeans, a n d cottonseeds d i d n o t a c c u m u l a t e p e n i c i l l i c a c i d w h e n i n o c u l a t e d w i t h f u n g i c a p a b l e of p e n i c i l l i c a c i d synthesis L i l l e h o j et a l . (161)

(30).

f o u n d t h a t atmospheres e n r i c h e d w i t h 6 0 % C 0

2

r e d u c e d p e n i c i l l i c a c i d a c c u m u l a t i o n b e l o w d e t e c t a b l e levels w h e n h i g h m o i s t u r e c o r n w a s i n o c u l a t e d w i t h P . martensii

a n d w a s s t o r e d at 5 ° C .

A c o m b i n a t i o n of l o w temperatures ( 1 5 ° or 2 2 ° C ) a n d l o w m o i s t u r e favored the production of penicillic a c i d i n autoclaved poultry i n o c u l a t e d w i t h A . ochraceus (163)

(162).

feed

C i e g l e r et a l . ( 2 9 ) a n d F i e l d e r

d i d n o t find p e n i c i l l i c a c i d i n m e a t p r o d u c t s o v e r g r o w n

with

Penicillia. Outlook B o t h p a t u l i n a n d p e n i c i l l i c a c i d s h o u l d b e c o n s i d e r e d as p o t e n t i a l l y d a n g e r o u s m y c o t o x i n s since b o t h are toxic a n d b o t h h a v e b e e n i m p l i c a t e d i n carcinogenesis.

P a t u l i n also causes m u t a t i o n s i n yeast. T h e c a r c i n o

g e n i c p r o p e r t i e s a n d other c h r o n i c effects n e e d f u r t h e r e v a l u a t i o n to assess p r o p e r l y t h e i r i m p o r t a n c e w h e n i n g e s t e d o r a l l y . B o t h mycotoxins are p r o b a b l y prevalent i n the environment a n d are p r o d u c e d b y s e v e r a l f u n g i c a p a b l e of d e c a y i n g f o o d o r f e e d . T h e n a t u r a l


6.

Patulin

WILSON

and Penicillic

Acid

105

o c c u r r e n c e a n d s t a b i l i t y o f either c o m p o u n d i n foods a n d feeds a t differ ent m o i s t u r e levels a n d t e m p e r a t u r e s h a v e n o t b e e n w e l l s t u d i e d ; w e c a n o n l y assume t h e i r presence a n d s t a b i l i t y i n m o l d y foods i f a f u n g u s t h a t p r o d u c e s either m y c o t o x i n is associated w i t h t h e d e c a y . T h e reactions a n d b i o l o g i c a l a c t i v i t y o f a d d u c t s o f p a t u l i n a n d p e n i c i l l i c a c i d w i t h S H groups a n d N H

2

groups n e e d f u r t h e r s t u d y f o r us t o

u n d e r s t a n d h o w d e t o x i f i c a t i o n occurs a n d i f t h e presence o f t h e a d d u c t s i n f o o d a n d f e e d s h o u l d cause c o n c e r n .

T h e question of t h e tautomerism

of p e n i c i l l i c a c i d is i n t e r e s t i n g a n d c o u l d b e better u n d e r s t o o d .

Both


p a t u l i n a n d p e n i c i l l i c a c i d m a y b e u s e f u l as m o d e l s o f a n t i - v i r a l c o m pounds.

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

u n d e r s t a n d o r h e l p us c o n t r o l v i r u s - c a u s e d disorders.

Acknowledgments T h e assistance o f M a r t h a G i r a r d e a u , W i l l i a m T a b o r , a n d T h e o d o s i a F l o w e r s i n the literature retrieval is acknowledged w i t h gratitude. Literature

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Patulin and Penicillic Acid

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