Photodynamic Action of Hypericin - American Chemical Society

C h a p t e r 19 Photodynamic

Action

of

Hypericin

1

J.PaulKnox ,Richard I. Samuels, and Alan D. Dodge

Downloaded by CORNELL UNIV on September 1, 2016 | http://pubs.acs.org Publication Date: May 7, 1987 | doi: 10.1021/bk-1987-0339.ch019

School of Biological Sciences, University of Bath, Bath, Avon BA2 7AY, United Kingdom

Hypericin, a photodynamic quinone, occurs in certain species of the genus Hypericum (the St. John's worts). Aspects of its photobiology and photochemistry, especially in relation to its ability to generate singlet molecular oxygen,have been investigated. Its phototoxicity, including that towards Manduca sexta larvae, is also discussed.

The quinones p r o v i d e many examples o f n a t u r a l l y o c c u r i n g photodynamic compounds, and h y p e r i c i n , found p r e d o m i n a n t l y i n t h e H y p e r i c a c e a e i s h i s t o r i c a l l y t h e most i m p o r t a n t o f t h e s e (1#2). The p h o t o s e n s i t i z a t i o n o f g r a z i n g a n i m a l s f o l l o w i n g t h e i n g e s t i o n o f c e r t a i n Hypericum s p e c i e s (the St. John's worts) i s due t o t h e p r e s e n c e o f h y p e r i c i n (3,4). This condition, hypericism, m a n i f e s t i n s k i n i r r i t a t i o n and i n f l a m m a t i o n , i s most commonly caused by t h e i n g e s t i o n o f Hypericum p e r f o r a t u m and has been most p r e v a l e n t i n N o r t h A m e r i c a and A u s t r a l i a (1,5). Chemistry The s t r u c t u r e o f h y p e r i c i n and i t s b i o s y n t h e t i c pathway were e l u c i d a t e d by Brockmann (7,8). H y p e r i c i n (4,5,7,4',5',7'hexahydroxy-2,2'-dimethylnaphthodianthrone) i s a h i g h l y condensed quinone and o f t e n o c c u r s i n t h e presence o f c l o s e l y r e l a t e d photodynamic compounds, most. coir>pionly p s e u d o h y p e r i c i n (9).

'Current address: John Innes Institute, Colney Lane, Norwich NR4 7 U H , United Kingdom

0097-6156/87/0339-0265$06.00/0 © 1987 American Chemical Society

Heitz and Downum; Light-Activated Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LIGHT-ACTIVATED PESTICIDES

266

0

OH

OH

()

HO'

R

Hypericin

H

HO.

-R

0

2 1

2

R =R =CH

\ OH

1

OH

3

Pseudohypericin 1

2

R =CH OH R =CH


2

3

In e t h a n o l , h y p e r i c i n and p s e u d o h y p e r i c i n display identical and d i s t i n c t i v e a b s o r p t i o n s p e c t r a , w i t h a s e r i e s o f a b s o r p t i o n maxima between 500 and 600nm, and marked red f l u o r e s c e n c e (10,11). They can be d i s t i n g u i s h e d by t h e i r s p e c t r a i n a c i d i c e t h a n o l and aqueous a l k a l i (10). Hypericin i s photostable i n both aqueous and o r g a n i c s o l v e n t s . Distribution The l a r g e s t s u r v e y o f t h e d i s t r i b u t i o n o f h y p e r i c i n w i t h i n t h e H y p e r i c a c e a e r e v e a l e d t h a t approx. 60% o f t h e 200 s p e c i e s i n v e s t i g a t e d contained hypericins. These s p e c i e s were c o n c e n t r a t e d i n t h e s e c t i o n s Euhypericum and Campylosporus (12). T h i s study u t i l i s e d a l e a f p r i n t t e c h n i q u e t h a t was unable t o d i s c r i m i n a t e between h y p e r i c i n and p s e u d o h y p e r i c i n . These compounds do d i f f e r i n t h e i r d i s t r i b u t i o n between s p e c i e s . H. p e r f o r a t u m c o n t a i n s both, IL h i r s u t u m o n l y h y p e r i c i n and H. montanum and fL c r i s p u m o n l y p s e u d o h y p e r i c i n (8). Their d i s t r i b u t i o n w i t h i n p l a n t t i s s u e a l s o d i f f e r s w i d e l y among species. In H^ p e r f o r a t u m t h e l e a v e s , stem and f l o w e r s c o n t a i n the h y p e r i c i n s , whereas i n IL h i r s u t u m h y p e r i c i n o c c u r s o n l y i n the m u l t i c e l l u l a r t r i c l u o i e s o f t h e c a l y x (10). In a l l cases the h y p e r i c i n s a r e r e s t r i c t e d t o d i s c r e t e glands. I n t e r e s t i n g l y , t h e h y p e r i c i n m o l e c u l e appears t o have o t h e r d i v e r s e o c c u r r e n c e s i n nature. The most n o t a b l e examples a r e as the chromophore o f t h e p h o t o r e c e p t o r o f S t e n t o r c o e r u l e u s (a b l u e - g r e e n c i l i a t e ) (13) and i n t h e integument o f an A u s t r a l i a n i n s e c t (Nipaecoccus a u r i l a n a t u s ) (14). I n a d d i t i o n , buckwheat (Fagopyrum esculentum) c o n t a i n s f a g o p y r i n , a d e r i v a t i v e o f h y p e r i c i n (9), t h e mould P e n i c i l l i o p s i s c l a v a r i a e f o r m i s c o n t a i n s p e n c i l l i o p s i n which can be o x i d i s e d and i r r a d i a t e d t o form h y p e r i c i n (9) and t h e c i l i a t e B l e p h a r i s i n a c o n t a i n s a pigment which i s a p o s s i b l e polymer o f h y p e r i c i n (5).


19.

KNOX ET AL.

Hypericin

Photodynamic Action of Hypericin

267

and s i n g l e t m o l e c u l a r oxygen


E a r l y s t u d i e s on t h e e f f e c t o f h y p e r i c i n on mammals demonstrated that i t s p h o t o s e n s i t i z i n g a c t i o n r e q u i r e d v i s i b l e l i g h t and oxygen, i.e. was photodynamic. The p r o d u c t i o n o f s i n g l e t m o l e c u l a r oxygen by c e r t a i n photodynamic r e a c t i o n s and i t s r o l e as t h e t o x i c s p e c i e s i n p h o t o o x i d a t i v e damage has s i n c e been d e m o n s t r a t e d (15). We have r e c e n t l y i s o l a t e d h y p e r i c i n from JL h i r s u t u m and i n v e s t i g a t e d i t s p o t e n t i a l t o p h o t o g e n e r a t e s i n g l e t oxygen (10). P u r i f i e d h y p e r i c i n was o b s e r v e d t o promote oxygen consumption from aqueous s o l u t i o n s when i r r a d i a t e d i n t h e presence o f i m i d a z o l e (capable o f r e a c t i n g w i t h s i n g l e t oxygen). T h i s p h o t o o x i d a t i o n was promoted i n t h e p r e s e n c e o f d e u t e r i u m o x i d e and d i m i n i s h e d by the a d d i t i o n o f a z i d e i o n s , s u g g e s t i v e o f s i n g l e t oxygen i n v o l v e m e n t . In a f u r t h e r model system, t h e i r r a d i a t i o n o f h y p e r i c i n was o b s e r v e d t o promote t h e p e r o x i d a t i o n o f methyl l i n o l e n a t e , as measured by t h e appearance o f ethane and m a l o n d i a l d e h y d e . L i n o l e n a t e o x i d a t i o n was reduced when c r o c i n (a water s o l u b l e c a r o t e n o i d c a p a b l e o f the e f f i c i e n t quenching o f s i n g l e t oxygen) was added t o t h e r e a c t i o n m i x t u r e . In t h i s system a c o n c e n t r a t i o n o f h y p e r i c i n g r e a t e r than lOuM was o b s e r v e d t o reduce l i p i d p e r o x i d a t i o n r e l a t i v e t o c o n t r o l s w i t h o u t h y p e r i c i n (unpublished o b s e r v a t i o n ) . T h i s may r e f l e c t the d i r e c t s c a v e n g i n g o f l i p i d r a d i c a l s by h y p e r i c i n . In both o f the above systems t h e use o f f i l t e r s i n d i c a t e d t h a t t h e e f f e c t i v e i r r a d i a t i o n was 500-600nm. These o b s e r v a t i o n s c l e a r l y demonstrate t h e a b i l i t y o f h y p e r i c i n t o promote type I I photodynamic r e a c t i o n s . Hypericin i s thus p o t e n t i a l l y d i s r u p t i v e o f b i o l o g i c a l s y s t e m s i n w h i c h i t i s i r r a d i a t e d i n p r o x i m i t y t o v u l n e r a b l e c e l l u l a r components such as t h e u n s a t u r a t e d l i p i d s o f membranes (2,15). In a d d i t i o n , e v i d e n c e f o r t h e p h o t o g e n e r a t i o n o f s u p e r o x i d e a n i o n s by t h e i r r a d i a t i o n o f h y p e r i c i n i n a r e d u c i n g environment (in t h e p r e s e n c e o f methionine) has been o b t a i n e d i n a system i n v o l v i n g the r e d u c t i o n o f n i t r o b l u e t e t r a z o l i u m (unpublished observations). The e x t e n t t o which type I photodynamic r e a c t i o n s ( i n c l u d i n g t h e g e n e r a t i o n o f s u p e r o x i d e anions) a r e a component o f t h e photodynamic damage s e n s i t i z e d by h y p e r i c i n i s unknown. Phototoxic

action of hypericin

Photodynamic r e a c t i o n s a r e g e n e r a l l y not s p e c i e s s p e c i f i c . A l t h o u g h h y p e r i c i n p o t e n t i a l l y has a wide t o x i c i t y , i t s a c t i o n w i l l be g r e a t l y modulated by v a r i a t i o n s i n i t s s e q u e s t r a t i o n and m e t a b o l i s m among s p e c i e s and w i t h i n t i s s u e s . As y e t t h e p h o t o t o x i c i t y o f h y p e r i c i n has been i n v e s t i g a t e d i n o n l y a few systems. As a l r e a d y s t a t e d the e a r l y i n v e s t i g a t i o n s upon t h e t o x i c i t y o f h y p e r i c i n were conducted due t o t h e p r e v a l e n c e o f h y p e r i c i s m (5). H y p e r i c i n must be i n g e s t e d by mammals t o r e s u l t i n hypericism and, u n l i k e the f u r a n o c o u m a r i n s , does n o t appear t o be




268

absorbed t h r o u g h the o u t e r l a y e r s of the e p i d e r m i s (5). After i n g e s t i o n a n i m a l s r e m a i n s e n s i t i v e t o s u n l i g h t f o r a week o r more. S k i n i r r i t a t i o n and i n f l a m m a t i o n i s most s e v e r e i n r e g i o n s of unpigmented s k i n d e v o i d of h a i r i.e. the mouth, nose and e a r s (5) . As a l r e a d y p o i n t e d out h y p e r i c i n o c c u r s as a component o f the p h o t o r e c e p t o r of c o e r u l e u s and p r e d i s p o s e s t h i s o r g a n i s m t o l e t h a l photodynamic i n j u r y (16). Exogenous h y p e r i c i n promotes t h i s i n j u r y and the use of quenchers i n t h i s system p r o v i d e s e v i d e n c e o f the i n v o l v e m e n t of s i n g l e t oxygen. The p r e c i s e r o l e of h y p e r i c i n i n Hypericum s p e c i e s i s u n c l e a r , a l t h o u g h i t would appear t o be a d e f e n s i v e one. A l t h o u g h t h i s compound can be a s o u r c e o f i r r i t a t i o n t o mammals, i t i s r a r e l y f a t a l and does not appear t o d e t e r g r a z i n g a n i m a l s (5). I t has been suggested t h a t h y p e r i c i n may a c t as a d e t e r r e n t t o phytophagous i n s e c t s (5), a f r e q u e n t l y proposed r o l e f o r p h o t o s e n s i t i z i n g p l a n t secondary m e t a b o l i t e s (2,17). Hypericin i s r e p o r t e d t o be p h o t o t o x i c t o mosquito (Aedes a t r o p a l p u s ) l a r v a e (17). We have u t i l i s e d t h i r d i n s t a r l a r v a e o f the t o b a c c o hawkmoth (Manduca s e x t a , L e p i d o p t e r a : Sphingidae) as a model i n s e c t h e r b i v o r e f o r the i n v e s t i g a t i o n of the p h o t o t o x i c i t y of h y p e r i c i n t o w a r d s i n s e c t s . The normal h o s t range o f s e x t a does not i n c l u d e any s p e c i e s o f the H y p e r i c a c e a e . H y p e r i c i n , i s o l a t e d as d e s c r i b e d p r e v i o u s l y (10), was o b s e r v e d t o be p h o t o t o x i c t o M. s e x t a l a r v a e . At the moderate r a d i a n c e l e v e l used i n t h i s study (22 Wm~ , p r o v i d e d by w h i t e f l u o r e s c e n t tubes) the L D was found t o be 16pg/g l a r v a l i n i t i a l fr.wt., w h i c h r e p r e s e n t s approx. a l u g dose t o a t h i r d i n s t a r l a r v a (Table I ) . In t h e s e e x p e r i m e n t s , the h y p e r i c i n was a d m i n i s t e r e d t o l a r v a e on t o b a c c o l e a f d i s c s (7mm diameter) a f t e r l h o f s t a r v a t i o n from an a r t i f i c i a l d i e t , and o b s e r v a t i o n s were made d u r i n g the subsequent c o n t i n u o u s i r r a d i a t i o n f o r up t o 48h. No m o r t a l i t y or any e f f e c t s upon w e i g h t g a i n were o b s e r v e d i n the h y p e r i c i n t r e a t e d but d a r k - m a i n t a i n e d c o n t r o l l a r v a e . Reduced i r r a d i a n c e r e s u l t e d i n d e c r e a s e d m o r t a l i t y , a l t h o u g h a f t e r 48h the s u r v i v i n g l a r v a e a t the l o w e r l i g h t l e v e l s d i s p l a y e d reduced w e i g h t g a i n r e l a t i v e t o dark c o n t r o l s . The m o d u l a t i o n of l i g h t q u a l i t y by a cut o f f f i l t e r ( a l l o w i n g i r r a d i a t i o n o n l y w i t h wavelengths g r e a t e r than 500nm) reduced the m o r t a l i t y r a t e by o n l y 20%, c o n f i r m i n g t h a t a c t i v e wavelengths i n h y p e r i c i n t o x i c i t y a r e g r e a t e r than 500nm (data not shown). I f a f t e r consumption of the h y p e r i c i n t r e a t e d l e a f d i s c s the l a r v a e were m a i n t a i n e d i n darkness on an a r t i f i c i a l d i e t , t h e p h o t o t o x i c e f f e c t upon subsequent i r r a d i a t i o n was r a p i d l y l o s t . M o r t a l i t y was reduced t o 6% i f i r r a d i a t i o n was d e l a y e d f o r 8h a f t e r t r e a t m e n t (Table I ) . I f the l a r v a e were not s u p p l i e d w i t h a r t i f i c i a l d i e t d u r i n g t h i s p e r i o d o f darkness, the p o t e n t i a l f o r a h i g h m o r t a l i t y r a t e upon subsequent i r r a d i a t i o n o f the l a r v a e s u p p l i e d w i t h d i e t was r e t a i n e d . These o b s e r v a t i o n s suggest t h a t h y p e r i c i n may not be r e a d i l y absorbed by the gut but photoactive a t the gut w a l l and r a p i d l y l o s t from the gut by e x c r e t i o n . 2

5 Q


19.

Photodynamic Action of Hypericin

K N O X ET AL.

269

T a b l e I. L e t h a l p h o t o t o x i c i t y o f h y p e r i c i n t o w a r d s Manduca s e x t a larvae. H y p e r i c i n a d m i n i s t e r e d on t o b a c c o l e a f d i s c s . M o r t a l i t y m o n i t o r e d a f t e r 48h o f i r r a d i a t i o n . h y p e r i c i n dose (ug/larva) a

irradiation conditions (Wm~ ) 2

time i n darkness between t r e a t m e n t and i r r a d i a t i o n

percentage mortality 3


ihl 0 0.1 0.3 0.6 1.0 1.5 2.5 2.5

22 " " " " " " DARK

0 " " " " " " "

0 0 6 12 47 89 100 0

2.5 2.5

10 4

" "

15 0

2.0 2.0 2.0 2.0

22 " "

0 2 4

81 34 11 6

"

8

a. average i n i t i a l f r e s h w e i g h t (3rd i n s t a r ) = approx. 60mg. b. a t l e a s t 15 i n s e c t s p e r t r e a t m e n t .

Conclusion These p r e l i m i n a r y o b s e r v a t i o n s d e m o n s t r a t e t h a t o r a l l y a d m i n i s t e r e d h y p e r i c i n i s t o x i c t o l a r v a e o f ML s e x t a , a h e r b i v o r e unaccustomed t o h y p e r i c i n c o n t a i n i n g p l a n t s . This t o x i c i t y has an a b s o l u t e l i g h t dependence a t t h e dose l e v e l s used i n t h i s study, w i t h no m o r t a l i t y o r growth r e t a r d a t i o n o b s e r v e d i n dark m a i n t a i n e d c o n t r o l s . In t h i s c a s e a maximum r a d i a n c e o f 22 Wm was used, c o n s i d e r a b l y l e s s than d a y l i g h t . The L D ^ Q c o u l d t h e r e f o r e be reduced i n a n a t u r a l environment. In t h i s study t h e h y p e r i c i n e q u i v a l e n t t o t h a t c o n t a i n e d i n approx. 50 g l a n d s o f I L h i r s u t u m (10) was l e t h a l t o a t h i r d i n s t a r l a r v a . The l e a f t i s s u e o f ! L p e r f o r a t u m c o n t a i n s h y p e r i c i n a t l e v e l s up t o lmg/g dr.wt. (12). V i s i b l e i r r a d i a t i o n (500-600nm) i s r e q u i r e d for hypericin t o x i c i t y contrasting with that of other plant metabolites capable of p h o t o s e n s i t i z i n g sexta larvae. A t h i o p h e n e , 0 C - t e r t h i e n y l , r e q u i r e d UV i r r a d i a t i o n (320-400nm) f o r i t s a c t i o n (18). The p o s s i b i l i t y t h a t h y p e r i c i n a c t s as a d e t e r r e n t t o phytophagous i n s e c t s r e q u i r e s f u r t h e r t o x i c i t y t e s t s and a survey o f i n s e c t s t h a t u t i l i s e Hypericum s p e c i e s . A beetle, Chrysolina



270

b r u n s v i c e n s i s , does f e e d upon h i r s u t u m , u s i n g h y p e r i c i n as a f e e d i n g cue (19). Other C h r y s o l i n a s p e c i e s have been used s u c c e s s f u l l y as a means o f b i o l o g i c a l c o n t r o l o f IL p e r f o r a t u m i n A u s t r a l i a (5^ 20). An a r e a o f i g n o r a n c e h i g h l i g h t e d by t h i s p o s s i b l e case o f c o e v o l u t i o n , i s t h e means by which o r g a n i s m s a r e a b l e t o t o l e r a t e photodynamic a c t i o n . Photodynamic damage may be reduced by b e h a v i o u r a l o r p h y s i o l o g i c a l mechanisms. The mechanisms whereby b i o l o g i c a l systems c o u l d p r e v e n t t h e g e n e r a t i o n o f s i n g l e t m o l e c u l a r oxygen o r w i t h s t a n d i t s s p e c i f i c b u t d i s r u p t i v e o x i d a t i o n s would be o f e s p e c i a l i n t e r e s t .


Literature cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Blum, H.F. 'Photodynamic action and diseases caused by light'; Reinhold Publishing Company, New York, 1941. Knox, J . P . ; Dodge, A.D. Phytochem. 1985, 24, 889-896. Horsley, C.H. J. Pharmcol. 1934, 50, 310-322. Pace, N. Amer. J. Physiol. 1942, 136, 650-656. Giese, A.C. Photochem. Photobiol. Rev. 1980, 5, 229-255. Towers, G.H.N. Prog. Phytochem. 1980, 6, 183-202. Brockmann, H.H. Prog. Organic Chem. 1952, 1, 64-82. Brockmann, H.H. Proc. Chem. Soc., London 1957, p. 304. Thompson, R.H. 'Naturally occurring Quinones'; Academic:London, 1971. Knox, J.P.;Dodge, A.D. Plant Cell Environ. 1985, 8, 1925. Scheibe, G.; Schöntage, A.Chem.Ber.1942, 75, 20192026. Mathis, C.; Ourisson, G. Phytochem. 1963, 2, 157-171. Walker, E.B.; Lee, T.Y.; Song, P.S. Biochim. Biophys. Acta. 1979, 587, 129-144. Cameron, D.W.; Raverty, W.D. Aus. J. Chem. 1976, 29, 1523-1533. Foote, C.S. In 'Free Radicals in Biology'; Pryor, W.A. Ed; Academic:London, 1976; Vol. II, p.85. Yang, K.C.; Prusti, R.K.; Walker, E.B.; Song, P.S.; Watanabe, M. Furuya, M. Photochem. Photobiol. 1986, 43, 305-310. Arnason, T.; Towers, G.H.N., Philogene, B.J.R.; Lambert, J.D.H. In 'Plant Resistance to Insects'; Hedin, P.A. Ed; ACS Symposium Series No. 208, American Chemical Society: Washington, D.C., 1983, pp. 139-51. Downum, K.R.; Rosenthal, G.A.; Towers, G.H.N. Pest. Biochem. Physiol. 1984, 22, 104-109. Rees, C.J.C. Entomol. Exp. Appl. 1969, 12, 565-583. Clare, N.T. 'Photosensitization in diseases of domestic animals' Commonwealth Agric. Bureaux; England, 1952, pp. 14-15. ;

17.

18. 19. 20.

RECEIVED November 20, 1986


Photodynamic Action of Hypericin - American Chemical Society

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