Glycosides: The Interface Between Plant Secondary and Insect

activity and from the subsequent glycolysis of alpha-D- glucose. (6). An ... of alpha-chitin (1,4 ..... Keeler, R.F., Eds.; Queensland Poisonous Plant...
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Chapter 27

Glycosides: The Interface Between Plant Secondary and Insect Primary Metabolism Kevin C. Spencer

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Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago, IL 60612

Plant secondary glycosides are proposed to have evolved as specific toxins toward enzymes of herbivore primary digestive metabolism. Insect herbivore digestive enzymes are optimized to process plant foods. An essential component of their digestive effort is the breakdown of sugar-containing plant materials by glycosidases. This essential activity has provided a target for plant toxins, which are delivered as glycosides. Such toxic action against glycosidases is described for cyanogenic glycosides, glucosinolates, iridoid glycosides, phenol glycosides and triterpene glycosides. The toxicity of individual glycosides depends upon their specific interaction with specific glycosidases, wherein they function as inhibitory substrates or release toxic aglycones. This great specificity in delivery and manifestation of toxicity can be measured for given sets of plants and herbivores, and may possibly be exploited in the design of effective, species-specific insecticides. A p l a n t i s , i n i t s essence, a c o l l e c t i o n o f chemicals. A d i v e r s i t y o f s e n s o r y m o d a l i t i e s e x i s t i n an i n s e c t , collectively constituting a mechanism f o r mating i t s digestive specialization with host searching, s e l e c t i o n and a c c e p t a n c e (1). The a b i l i t y o f an i n s e c t t o acquire and i n t e r n a l i z e a p p r o p r i a t e l e v e l s and t y p e s o f n u t r i t i o n a l f a c t o r s i s c r i t i c a l t o i t s s u c c e s s ( 2 ) . These n u t r i t i o n a l f a c t o r s a r e related t o d e f e n s i v e a l l e l o c h e m i c a l regimes i n two ways: f i r s t , as the effectiveness o f t h e d e f e n s e impinges upon t h e v a l u e of the n u t r i e n t s , and second, as t h e q u a l i t y o f t h e n u t r i e n t s may p e r m i t t h e t o l e r a n c e o f s e c o n d a r y c h e m i c a l s (3). The quantitative and q u a l i t a t i v e variation expressed in allelochemical factors i s a powerful force against herbivore specialization ( 4 ) . The p r e s e n c e o f such v a r i a b i l i t y i n h o s t - p l a n t populations i s evidence f o r the existence of a strong selection potential p r o m o t i n g s p e c i a l i z a t i o n o f i n s e c t s upon i n d i v i d u a l plant chemotypes. E c o l o g i c a l f a c t o r s a r e a l s o i m p o r t a n t , and c o n s i d e r a t i o n

c

0097-6156/88/0380-0403S06.00/0 1988 American Chemical Society

Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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of the o v e r a l l p h y s i o l o g i c a l e f f i c i e n c y o f f e e d i n g (5) i s a f a r more useful c o n s t r u c t f o r e v a l u a t i n g i n s e c t h o s t - p l a n t i n t e r a c t i o n s than narrower feeding specialization hypotheses. Whatever their evolutionary r e l a t i o n s h i p to plant defensive chemistries, herbivorous insects have been under evolutionary pressure f o r m i l l e n i a to optimize their digestive systems for efficient extraction of nutrients from t h e i r h o s t p l a n t s . Their primary metabolism having thus been adapted t o p l a n t n u t r i e n t c o m p o s i t i o n , i t seems r e a s o n a b l e to hypothesize that i n s e c t d i g e s t i v e systems have been forced to adapt t o p l a n t s e c o n d a r y compounds as w e l l . In the f o l l o w i n g p a r a g r a p h s the e s s e n t i a l s o f insect primary metabolism o f g l u c o s e and i t s e x t r a c t i o n from p l a n t s o u r c e s through the a c t i o n of glycosidases i s discussed. These and other sugarprocessing enzymes are c o n s i d e r e d i n view o f t h e i r activity upon potentially toxic plant glycosides. P o s s i b l e i n t e r f e r e n c e with the biological activity of glycoside h y d r o l y s i s products in situ by insect d e t o x i f i c a t i o n enzymes i s e x p l o r e d , and interference with normal insect glycosidase activity by specialized plant-derived inhibitory s u b s t a n c e s i s found t o o c c u r . Finally, the r e s u l t s of experimental t e s t s of the hypothesis that plant glycosides are elaborated p r i m a r i l y as t o x i n s t a r g e t e d against essential insect glycosidases are given, and t h e c o n c l u s i o n i s r e a c h e d t h a t t h i s may indeed be the case. If so, the consideration of plant glycoside/insect glycosidase i n t e r a c t i o n s may be fundamentally i m p o r t a n t i n o p t i m i z i n g t h e d e s i g n and e f f i c a c y o f n o v e l and s p e c i f i c pesticides. Insect Primary Metabolism The energy needed to d r i v e i n s e c t primary metabolism is derived primarily from the u p t a k e o f g l u c o s e from the d i e t t h r o u g h c a t a b o l i c glycosidase a c t i v i t y and from the subsequent g l y c o l y s i s o f alpha-Dglucose (6). An especially efficient glucose transport and transformation a b i l i t y i s r e q u i r e d by i n s e c t s . W h i l e t h e i r primary metabolism i s e s s e n t i a l l y s i m i l a r to that of other animals, insects are s u b j e c t t o extreme p h y s i o l o g i c a l s t r e s s e s d u r i n g metamorphosis, a c q u i s i t i o n of cold-hardiness, o v i p o s i t i o n and f l i g h t . D e v e l o p m e n t a l processes require the r a p i d and specific depolymerization and repolymerization of a l p h a - c h i t i n (1,4 linked 2-deoxy-2-acetamidobeta-D-glucose polymer). A c q u i s i t i o n of c o l d - h a r d i n e s s r e q u i r e s the rapid production of g l y c o l . O v i p o s i t i o n i n v o l v e s the p r o d u c t i o n of storage glycoproteins and flight depends upon the efficient degradation of t r e h a l o s e . Insect Glucose

Digestion

G l u c o s e uptake and a b s o r p t i o n t a k e s p l a c e i n the midgut as a very rapid and o s m o t i c a l l y - r e g u l a t e d p r o c e s s . Increased sugar or other solute concentrations i n t h e haemolymph d e c r e a s e the r a t e o f cropemptying, i n c r e a s e d s u g a r c o n c e n t r a t i o n s i n t h e gut i n c r e a s e the r a t e of crop-emptying, and c r o p d i s t e n s i o n r e g u l a t e s the r a t e o f uptake o f f o o d . The f u n c t i o n a l p r o c e s s o f g l u c o s e t r a n s p o r t a c r o s s membranes is probably a f a c i l i t a t e d d i f f u s i o n which does not depend upon an a c t i v e c a r r i e r mechanism · The u p t a k e o f g l u c o s e can be p o t e n t i a t e d by t r e h a l o s e formation

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27. SPENCER

Glycosides: Plant and Insect Metabolism

(Figure 1). An a c t i v e t r a n s p o r t system involving Malpighian reabsorption has been reported f o r Locusta migratoria (8) and Calliphora vomitoria (9), which may permit maintenance o f an exceptionally high g l u c o s e - t o - t r e h a l o s e r a t i o i n t h e haemolymph of these insects, relative t o i n s e c t s shown t o depend upon passive d i f f u s i o n o n l y (1_) .

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F i g u r e 1. Trehalose.

Trehalose (alpha-D-glucopyranosyl-alpha-D-glucopyranoside) i s the major form of ready-reserve stored energy i n i n s e c t s , not g l y c o g e n as i n mammals (10^). C o n c e n t r a t i o n o f t h e d i s a c c h a r i d e i n t h e haemolymph can r e a c h 2% w/v, more t h a n enough t o a c c o u n t f o r the extreme energy r e q u i r e m e n t s o f f l i g h t t h r o u g h t h e d i r e c t hydrolysis of trehalose to glucose. The e n e r g e t i c s o f c o n v e r s i o n a r e s u f f i c i e n t t o o f f s e t t h e i n e f f i c i e n t i n s e c t c i r c u l a t o r y system (11). Trehalase, the enzyme c a t a l y z i n g t h e h y d r o l y s i s o f t h e h i g h - e n e r g y storage product, i s localized i n t h e f o r e g u t - and m i d g u t - e p i t h e l i a . The l a t t e r contains the highest t i t e r o f t r e h a l a s e a c t i v i t y , and i s a l s o responsible f o r t h e h i g h e s t r a t e o f a b s o r p t i o n o f g l u c o s e from t h e d i e t ( i n t h e midgut c a e c a ) (12). I n s e c t D i g e s t i v e Enzymes : G l y c o s i d a s e s Insect beta-glucosidase a c t i v i t y i s l o c a l i z e d i n t h e f o r e g u t - and m i d g u t - e p i t h e l i a ( 1 3 ) . I n t h e c o t t o n s e e d f e e d e r Dysdercus c i n g u l a t u s , the highest titers a r e found i n t h e f o r e g u t and i n c r e a s e with continued feeding (14). Phytophagous i n s e c t s produce digestive enzymes a t l e v e l s c o n s i s t e n t w i t h q u a n t i t a t i v e s t i m u l a t i o n by f o o d i n the gut, probably v i a a p r o t e i n - s e n s i t i v e neurosecretory pathway (15,16). A l l o f t h e s e c r e t e d midgut enzymes may v a r y q u a n t i t a t i v e l y together, as was found f o r 3 c a r b o h y d r a s e s , t r e h a l a s e and t r y p s i n i n L o c u s t a m i g r a t o r i a ( 1 5 ) . The h i g h pH o f many i n s e c t d i g e s t i v e system maximizes t h e a c t i v i t y o f a l k a l i n e - t o l e r a n t enzymes (glycosidases, oxygenases) and p e r m i t s a high rate of passive uptake o f sugars t h r o u g h p o l a r c h a n n e l s (1_). C o m p a r t m e n t a l i z a t i o n and r e g u l a t i o n o f t h e s e a c t i v i t i e s has been observed. Digestive glycosidase a c t i v i t i e s of Schistocerca gregaria disappear, and a r e p o s s i b l y i n some way i n a c t i v a t e d as t h e y p a s s t o the h i n d g u t ( 1 3 ) . I t i s n o t known whether t h e y a r e i n a c t i v a t e d and excreted, reabsorbed, d i g e s t e d as p a r t o f t h e i n a c t i v a t i o n p r o c e s s , or even retained, by an unknown p r o c e s s , proximally above t h e hindgut. Insect g u t m i c r o f l o r a may a l s o be important sources of glycosidases, as t h e y a r e c e l l u l a s e s ( Γ 7 ) . These may be e s p e c i a l l y significant factors i n t h e d e t o x i f i c a t i o n and d e g r a d a t i o n o f p l a n t glycosides (18). F i v e b e t a - g l y c o s i d a s e s i s o l a t e d and p a r t i a l l y p u r i f i e d from t h e generalist f e e d e r L o c u s t a m i g r a t o r i a (19) showed h y d r o l y t i c a c t i v i t y toward c e l l o b i o s e , g e n t i o b i o s e and m e t h y l - b e t a - g l u c o s i d e . While a b r o a d range o f h y d r o l y t i c c a p a b i l i t y was p o s t u l a t e d f o r t h i s s u i t e o f

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enzymes, i t i s more p r o b a b l e t h a t each o f the five activities r e p r e s e n t s a p a r t i a l l y - s e p a r a t e d m i x t u r e o f more s p e c i f i c a c t i v i t i e s . Cellobiases a r e c r i t i c a l i n the n u t r i t i o n o f wood-devouring i n s e c t s . These g l y c o s i d a s e s c a t a l y z e the t e r m i n a l d e g r a d a t i v e step in the digestion o f wood c e l l u l o s e (12). However, the t e r m i n a l c e l l u l o s i c activity i s dependent upon the p r e s e n c e o f an initial cellulase activity: c e l l o b i a s e alone does not c o n f e r the a b i l i t y to accept cellulose i n the d i e t . Insect carbohydrases, including amylases, transglycosidases, phosphorylases and glucosidases, exhibit specificity o f a c t i o n depending upon s u b s t r a t e l i n k a g e conformation (alpha vs beta), conformation (D vs L) and type and number of monosaccharide u n i t s (10). Substrate specificity has been established for plant g l y c o s i d a s e s which h y d r o l y z e p h e n o l g l y c o s i d e s , s t e r o i d a l g l y c o s i d e s , coumarins, flavonoids, cyanogenic glycosides, thioglucosides and oligosaccharides (20-23). Glycosidases have been shown t o exhibit s u b s t r a t e s p e c i f i c i t y dependent upon t h e s t r u c t u r e o f the a g l y c o n e o r t h e t y p e and number o f s u g a r m o i e t i e s (20-23). This s p e c i f i c i t y can be exclusive (24), but i s generally more "relaxed" (2j5). The admission of xenosubstrates to glycosidase sugar b i n d i n g sites c r e a t e s a p o t e n t i a l f o r the i n h i b i t i o n o f t h e s e enzymes. I n s e c t D e t o x i f i c a t i o n Systems Before we can p o s t u l a t e an e f f e c t i v e i n h i b i t o r y i n t e r a c t i o n between p l a n t t o x i c g l y c o s i d e s and i n s e c t g l y c o s i d a s e s , we must c o n s i d e r t h e l i k e l i h o o d t h a t i n s e c t d e t o x i f i c a t i o n systems w i l l degrade g l y c o s i d e s before they can reach t h e i r p u t a t i v e s i t e s of action. The major enzymes u t i l i z e d by insects i n detoxification reactions are the m i c r o s o m a l monooxygenases (mixed f u n c t i o n o x i d a s e s o r MFO). Insect MFO a c t i v i t y i s a multienzyme 02/NADPH-requiring system capable of catalyzing o x i d a t i o n o f many s u b s t r a t e s ( E q u a t i o n 1 ) . R represents t h e s u b s t r a t e and X the e l e c t r o n donor. RH

+ 0

2

+ HX 2



ROH

+ X + HO

(1)

Reactions catalyzed include hydroxylations, dealkylations, oxidations, epoxidations, d e s u l f u r a t i o n s and d e h a l o g e n a t i o n s (26). These v a r i e d r e a c t i o n s a r e c a r r i e d out by i n d i v i d u a l h e m o p r o t e i n s , P450 cytochrome t e r m i n a l o x i d a s e isozymes ( 2 7 ) , which a r e p r o d u c e d i n response to the p r e s e n c e o f s p e c i f i c s u b s t r a t e s i n the gut (28)S p e c i f i c inducers, i n h i b i t o r s and s y n e r g i s t s o f a c t i v i t y a r e known. The e x p r e s s e d a c t i v i t y o f t h e s e isozymes i s q u i t e v a r i a b l e , and is r e s p o n s i v e t o many e c o l o g i c a l , e d a p h i c , and o r g a n i s m a l f a c t o r s ( 2 9 ) . Of the many isozymes p o s t u l a t e d , o n l y 3 from i n s e c t s and 6 from mammals have been c h a r a c t e r i z e d . MFO a c t i v i t y can be quantitatively much g r e a t e r i n mammals t h a n i n i n s e c t s on a p e r u n i t p r o t e i n basis (30). Insects are adept at synthesizing glycosides through the addition of glucose to a substrate v i a UDP-glucosyltransferase (31), t h e same p r o c e s s as o c c u r s i n p l a n t s . I n s e c t s a r e known t o s y n t h e s i z e mainly 0-glucosides and t o a l e s s e r e x t e n t S-glucosides, whereas plants a l s o synthesize N-glucosides. G l y c o s i d a t i o n i s an effective d e t o x i f i c a t i o n mechanism ( 3 2 ) , especially for phenolics, coumarins, quinones and o t h e r c o n j u g a t e d a r o m a t i c compounds containing active

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Glycosides: Plant and Insect Metabolism

407

oxygens. Destructive f r e e - r a d i c a l formation from these source compounds can be p r e v e n t e d through c o n j u g a t i o n o f g l u c o s e at the a c t i v e center. Other detoxification pathways of importance include s u l f o t r a n s f e r a s e s and g l u t a t h i o n e - S - t r a n s f e r a s e s ( 3 3 ) . Insect g l y c o s i d a s e a c t i v i t y seems t o be p r e s e n t e a r l i e r i n t h e d i g e s t i v e sequence t h a n MFO a c t i v i t y ( f o r e g u t / m i d g u t v s m i d g u t ) , and hydrolysis often has t o t a k e p l a c e before t o x i c aglycones a r e r e l e a s e d and a c t t o i n d u c e MFO s y n t h e s i s . G l y c o s i d a s e a c t i v i t y may be greater t h a n MFO a c t i v i t y as a g e n e r a l consequence o f t h e g r e a t e r frequency o f o c c u r r e n c e o f t h e m o l e c u l a r e v e n t s o f sugar digestion than those of t o x i n degradation. I t seems l i k e l y t h a t glycosidase activity w i l l p r e c e d e MFO a c t i v i t y because o f p h y s i c a l location of enzymes, o f d i f f e r e n c e s i n r e l a t i v e a c t i v i t i e s , and because i t would be unreasonable t o e x p e c t MFO a c t i v i t y t o be needed b e f o r e toxic aglycones a r e l i b e r a t e d . A f u r t h e r r e a s o n i s found i n t h e r e l a x e d specificity shown by some g l y c o s i d a s e s . F o r example, a betaglucosidase with generalized activity isolated from Phoracantha semipunctata (34) i n c r e a s e s g l u c o s e a v a i l a b i l i t y t h r o u g h c e l l o b i o s e hydrolysis w h i l e a t t h e same time r e l e a s i n g t o x i c a g l y c o n e s i n o t h e r h y d r o l y t i c r e a c t i o n s (Applebaum, 1985). MFO a c t i v i t y t a r g e t e d a g a i n s t such toxins would a l s o undoubtedly interfere with glucose assimilation. Glycosidase

Inhibitors

The r a t e o f uptake o f glucose, being a m o l a r - d i f f u s i v e process, is dependent upon t h e c o n c e n t r a t i o n o f g l u c o s e i n t h e midgut. I n h i b i t i o n o f g l y c o s i d a s e a c t i v i t y w i l l l i m i t t h e amount o f f r e e g l u c o s e i n t h e lumen and d e c r e a s e t h e r a t e o f uptake o f g l u c o s e i n t o haemolymph as t e r h a l o s e ( 3 5 ) , which c r e a t e s c o n d i t i o n s o f g l u c o s e s t a r v a t i o n . The insect w i l l r e s p o n d by emptying t h e c r o p f a s t e r (2[6) and e a t i n g more (_37)In a d d i t i o n , s i n c e i n s e c t d i g e s t i v e enzymes seem o f t e n t o be produced i n response t o the stimulus of p r o t e i n i n food as a p r e p a c k a g e d s e t o f i n v a r i a n t c o m p o s i t i o n ( 1 6 ) , d e f e a t o f one c r i t i c a l enzyme system will effectively diminish an extended range of digestive a c t i v i t i e s . I n h i b i t i o n o f h e r b i v o r e g l y c o s i d a s e a c t i v i t y by p l a n t g l y c o s i d e s has been found i n the case of castanospermine (1,6,7,8tetrahydroxyoctahydroindolizidine) isolated from Castanospermum a u s t r a l e A. Cunn. (Fabaceae) ( 3 8 ) . T h i s compound i n h i b i t s several insect and mammalian a l p h a - and b e t a - g l y c o s i d a s e s (39), often c o m p e t i t i v e l y (40). I n h i b i t i o n i s e f f i c i e n t a t h i g h pH, and d i s r u p t s g l y c o p r o t e i n p r o c e s s i n g (4_1). Castanospermine was found t o be a f e e d i n g d e t e r r e n t t o aphids (42) and L o c u s t a m i g r a t o r i a L. b u t n o t t o S c h i s t o c e r c a gregaria Forsk. ( 4 3 ) , and t o be t o x i c i n a dose-dependent manner t o Callosobruchus maculatus ( 4 4 ) . I n t h i s i n s e c t and i n T r i b o l i u m confusum t o x i c i t y was c a u s e d by i n h i b i t i o n of alpha-D-glucosidase, beta-D-glucosidase and beta-D-galactosidase activities in the a l i m e n t a r y t r a c t ( 4 5 ) . Castanospermine appears t o a c t as a s t r u c t u r a l analogue o f glucose ( 4 3 ) . A s i m i l a r compound, s w a i n s o n i n e (an i n d o l i z i d i n e triol), was shown to inhibit alpha-mannosidase (46,47). O t h e r p y r a n o s e and furanose sugar analogues a r e known t o be p o t e n t inhibitors of

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glycosidases, and i n a d d i t i o n , of glucosytransferase (43). One furanose analogue, l,5-dideoxy-l,5-imino-D-mannitol, has been found to i n h i b i t t r e h a l a s e (48). These data on sugar analogue action prompted the o b s e r v a t i o n t h a t i n h i b i t o r s o f g l y c o s i d a s e s may be an i m p o r t a n t and common d e f e n s i v e mechanism i n p l a n t s ( 4 3 ) .

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Plant Glycosides

as I n h i b i t o r s o f

Glycosidases

Plant glycosides which r e l e a s e t o x i c a g l y c o n e s upon h y d r o l y s i s may have evolved to target the e s s e n t i a l process by which insect h e r b i v o r e s d i g e s t p l a n t f o o d s and i n t e r n a l i z e energy. T h i s h y p o t h e s i s is t e s t e d i n t h e e x p e r i m e n t s d e s c r i b e d i n the f o l l o w i n g section. A summary o f the s t r u c t u r a l c l a s s e s o f t o x i c g l y c o s i d e s i s g i v e n in T a b l e I ( a f t e r 49-51).

Table

I. S t r u c t u r a l Classes

of B i o a c t i v e Plant

Terpene g l y c o s i d e s Steroidal glycosides Saponins Phenolic glycosides Flavonoids Stilbene glycosides Xanthone g l y c o s i d e s Lignan glycosides

Glycosides

Glucosinolates Lactone g l y c o s i d e s Glycoalkaloids Cyanogenic g l y c o s i d e s Glycoproteins Iridoid glycosides Quinone g l y c o s i d e s Benzoxazine g l y c o s i d e s

Plant g l y c o s i d e s u t i l i z e d i n t e s t s o f i n h i b i t i o n were selected to r e p r e s e n t t h e commonly-studied t o x i c g l y c o s i d e s , and i n c l u d e d a thioglucoside, cyanogenic glucoside, iridoid glycoside, phenol g l y c o s i d e , t r i t e r p e n e g l y c o s i d e and b e n z o x a z i n e ( F i g u r e 2 ) . E x p e r i m e n t a l T e s t s Of I n h i b i t i o n Glycosidase preparations were made by f l a s h - f r e e z i n g whole starved insect larvae under liquid nitrogen, b i s e c t i n g the larvae and extracting t h e f r o z e n midgut under a s t e r e o m i c r o s c o p e , and dipping the e x t r a c t e d t i s s u e i n t o a c o l d m i c r o c e l l c o n t a i n i n g 2.0 mL of pH 6.8 phosphate b u f f e r . T h i s i n i t i a l enzyme m i x t u r e was a c c r e t e d u n t i l protein-assay aliquots ( m i c r o - L o w r y / b i u r e t ) showed 10-100 pg total p r o t e i n . The m i x t u r e was t h e n p a r t i a l l y p u r i f i e d by p a s s a g e t h r o u g h a series o f s m a l l Sephadex columns (G-25, G-100, G-200) i n b u f f e r . Beta-glycosidase activity was a s s a y e d using 4-nitrophenyl-beta-Dg l u c o s i d e as a s u b s t r a t e ( 4 0 ) . S t a n d a r d r e a c t i o n c o n d i t i o n s c o n s i s t e d of a 1.0 pg s o l u t i o n o f g l y c o s i d a s e i n 1.0 mL o f b u f f e r t o which was added 5.0 umol o f n i t r o p h e n y l g l u c o s i d e . The m i x t u r e was incubated f o r 30 min a t 37 ° C , t h e n 3.0 mL o f pH 10.4 g l y c i n e b u f f e r was added t o slow the r e a c t i o n and a l l o w q u a n t i t a t i v e c o l o r i m e t r i c d e t e c t i o n o f 4-nitrophenol released a t 410 nm. The f i n a l p r o d u c t s o f isolation had, on average, o n l y 10 t i m e s t h e a c t i v i t y o f t h e i n i t i a l enzyme mixture, but were f r e e o f p r o t e a s e s and t i s s u e p a r t i c l e s , and c o u l d be s t o r e d i n the r e f r i g e r a t o r f o r s e v e r a l weeks. T e s t s f o r i n h i b i t i o n o f i n s e c t g l u c o s i d a s e a c t i v i t y were c a r r i e d

Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Glycoside

Aglycone

F i g u r e 2. P l a n t g l y c o s i d e s and a g l y c o n e s t e s t e d f o r effects against insect glucosidase fractions.

Number

inhibitory

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Reference

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out by a d d i n g 1.0 μg/mL o f p l a n t g l y c o s i d e t o the above reaction mixture. When n e c e s s a r y , small amounts o f d i m e t h y l sulfoxide or e t h a n o l were added t o improve s o l u b i l i t y . C o n t r o l r e a c t i o n s were a l s o run with these s o l v e n t s . R e a c t i o n s were c o n d u c t e d as above, and a t the end of the i n c u b a t i o n period, the amount o f 4-nitrophenyl glucoside released was compared w i t h t h a t of the control, and e x p r e s s e d as a p e r c e n t a g e . S e p a r a t e t e s t s o f a g l y c o n e a c t i v i t y were a l s o c a r r i e d o u t , under the assumption that not a l l plant glycoside/insect glycosidase i n t e r a c t i o n s p r o c e e d t o h y d r o l y s i s . A g l y c o n e s were p r o d u c e d u s i n g a s p e c i a l i z e d microware a p p a r a t u s d e p i c t e d i n F i g u r e 3. The p r o d u c t i o n chamber i s made o f a d i a l y s i s t u b i n g sac a f f i x e d t o t h e d i s t a l end o f a thistle tube, i n t o which could be added b u f f e r and plant glycoside/plant glycosidase p a i r s optimal f o r hydrolysis. A long needle i n s e r t e d i n t o the tube p r o v i d e s a s t r e a m of nitrogen for mixing. The production chamber (1.0 mL volume) was h e l d below the surface o f a tube h o l d i n g 1.0 mL o f b u f f e r . T h i s tube contains the insect test glycosidase fraction, and i s s t i r r e d c o n s t a n t l y w i t h a m i c r o b a r magnetic s t i r r e r . H y d r o l y s i s o f the p l a n t g l y c o s i d e takes place efficiently i n the production chamber, and the aglycone produced diffuses efficiently a c r o s s t h e membrane i n t o the test solution. During the r e a c t i o n experiment, s u f f i c i e n t glycoside is i n t r o d u c e d i n t o t h e r e a c t i o n chamber t o y i e l d a p p r o x i m a t e l y 1.0 ug/mL of a g l y c o n e a t e q u i l i b r i u m ( u s u a l l y 2.0 mg o f a g l y c o n e as glycoside molar equivalent). The pH o f t h e b u f f e r was sometimes lowered to prevent b i n d i n g o f the a g l y c o n e t o the membrane, and to stabilize ketone p r o d u c t s . Samples drawn from t h e t e s t s o l u t i o n a f t e r 30 min a t 37 °C were a s s a y e d f o r r e l a t i v e g l u c o s i d a s e a c t i v i t y as above. Glucosidase p r e p a r a t i o n s from n i n e i n s e c t s p e c i e s were u t i l i z e d i n the present experiments. These i n s e c t s were c h o s e n t o r e p r e s e n t a range o f f e e d i n g s p e c i a l i s t s and g e n e r a l i s t s , i n c l u d i n g specialists known t o be t o l e r a n t o f c e r t a i n o f t h e t e s t p l a n t g l y c o s i d e s (Table II). The purpose o f combining i n d i v i d u a l g l y c o s i d e s w i t h individual

Table

Insect

I I . T e s t P a i r s Of I n s e c t s And T h e i r H o s t p l a n t s Used In A n a l y s e s Of I n h i b i t i o n Of I n s e c t G l u c o s i d a s e s By Plant Glycosides Species

Host

P i e r i s b r a s s i c a e L. H e l i c o n i u s e r a t o L. Ceratomia c a t a l p a B o i s . P a p i l i o glaucus canadensis Drosophila mojavensis H e l i o t h i s zea L.

L.

S p o d o p t e r a e r i d a n i a Cramer L o c u s t a m i g r a t o r i a L. D r o s o p h i l a m e l a n o g a s t e r L.

Plant

Brassicaceae Passifloraceae Catalpa speciosa L. S a l i c a c e a e Cactaceae Zea mays L.

Interaction Specialist Specialist Specialist L Generalist Salicin-tolerant Specialist Generalist Specialist mays Generalist Generalist Generalist

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27. SPENCER

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Plant glycoside

~

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Plant glycosidase ^

N

1.0 mL tubing

0

exit • 2.0 mL tube containing insect glycosidase solution 1 pH 6.8 P 0 buffer

dialysis sac

4

magnetic

stirbar

F i g u r e 3. Apparatus f o r the d o n a t i o n o f a g l y c o n e h y d r o l y s i s p r o d u c t s from a p l a n t g l u c o s i d e / p l a n t g l u c o s i d a s e m i x t u r e t o a test insect glucosidase s o l u t i o n .

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glucosidase f r a c t i o n s i n t h e above r e a c t i o n system was t o d e t e r m i n e the following: a) Do p l a n t g l y c o s i d e s i n h i b i t insect glucosidase activities i n v i t r o ? b) I f such i n h i b i t i o n i s d e t e c t e d , does i t derive from competitive o r noncompetitive behavior? c) If noncompetitive i n h i b i t i o n takes p l a c e , what i s t h e mode o f a c t i o n o f the i n h i b i t o r ? d) When i n h i b i t i o n i s o b s e r v e d , i s t h e r e a component of specificity o f a c t i o n which c a n be r e l a t e d t o i n s e c t host-plant specialization? The r e s u l t s o f t h e s e e x p e r i m e n t s a r e summarized i n Table I I I . All plant glycosides t e s t e d reduced insect glucosidase activity towards 4-nitrophenyl g l u c o s i d e t o some degree in vitro. Enzyme f r a c t i o n s from s p e c i a l i s t s were most p o t e n t l y i n h i b i t e d by g l y c o s i d e s not p r e s e n t i n t h e i r h o s t p l a n t s : e r a t o by t h i o g l u c o s i d e 2, P.

Table

I I I . I n h i b i t o r y E f f e c t s Of P l a n t G l y c o s i d e s And A g l y c o n e s (A) Upon I n s e c t G l y c o s i d a s e F r a c t i o n s

Source o f Glycosidase

Glycoside 1

1A

2

2A

(Number R e f e r s 3

% Activity

ÇL Σ\ EL ÎL_

EL

brassicae erato catalpae glaucus can. mojavensis zea eridania migratoria melanogaster

80 80 90 80 90 80 80 100 100

90 70 80 70 90 90 90 100 90

90 40 90 90 90 70 60 70 90

60 50 50 60 60 60 60 50 50

40 90 80 60 90 70 70 80 90

3A

4

To F i g u r e 2) 4A

5

5A

6

6A

100 90 90 90 100 80 80 90 90

100 80 70 80 100 70 50 80 90

R e l a t i v e to Control 40 30 30 40 30 30 40 40 20

70 70 50 90 90 80 80 90 100

70 60 60 70 70 80 70 70 80

100 90 90 90 40 100 100 100 90

100 100 100 100 80 100 100 100 90

b r a s s i c a e by c y a n o g e n i c g l y c o s i d e 3, C^ c a t a l p a e by s a l i c i n 4, and P. g l a u c u s by c y a n o g e n i c g l y c o s i d e 3. T h i s s t r o n g l y i n d i c a t e s t h a t t h e s e insects are able t o avoid the h y d r o l y s i s products corresponding to the compounds found i n t h e i r p r e f e r r e d h o s t p l a n t s , which a r e seen t o be effective inhibitors, by n o t a c c e p t i n g the glycoside as a substrate for t h e i r glycosidases. D^ m o j a v e n s i s i s an e x c e p t i o n i n responding t o i n h i b i t i o n by a t r i t e r p e n e g l y c o s i d e 5 found in i t s host plant, but t h i s species p r o b a b l y does not encounter this compound i n i t s normal d i e t as i t i s f i r s t p r o c e s s e d t o t h e a g l y c o n e by yeasts ( 2 1 ) . The t r i t e r p e n e 5 i s seen t o have l i t t l e e f f e c t i n other species. The t h i o g l u c o s i d e 2 i s n o t an e f f e c t i v e i n h i b i t o r o f g l u c o s i d a s e s , as i s e x p e c t e d from i t s b e i n g an S - g l u c o s i d e , e x c e p t i n H. e r a t o and t h r e e g e n e r a l i s t s p e c i e s . A l l o f t h e s e were found t o possess t h i o g l u c o s i d a s e a c t i v i t y s u f f i c i e n t t o produce the aglycone product 2A. T h i s was found t o be a n o n c o m p e t i t i v e inhibitor of glucosidase activity, p o s s i b l y through a l k y l a t i o n o f a c i d i c active s i t e s by t h i o c y a n a t e ( 4 0 ) . The cyclopentenoid aglycone 3A i s a p o t e n t a l k y l a t o r and diminished enzyme a c t i v i t y wherever p r e s e n t (21,22). Some degree o f

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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 f o r c a t a l p o l 1, s a l i c i n 4 and DIMBOA 6, as t h e r e c o v e r a b l e t i t e r o f g l y c o s i d e was detectably smaller than e x p e c t e d a f t e r r e a c t i o n s were r u n . Retention of the c o r r e s p o n d i n g a g l y c o n e s 1A, 4A, 6A was a l s o o b s e r v e d from r e a c t i o n s run using the aglycone donation apparatus, b u t q u a n t i f i c a t i o n was d i f f i c u l t . I t appears t h a t b o t h n o n c o m p e t i t i v e b i n d i n g and a l k y l a t i o n o c c u r w i t h t h e s e compounds, and p o s s i b l y w i t h t h e t h i o g l u c o s i d e 2 and cyanogenic glucoside 3 as w e l l . Alkylation under certain c i r c u m s t a n c e s seems t o be a r a t i o n a l p o s s i b i l i t y , as c a n be i n f e r r e d from c o n s i d e r a t i o n o f t h e s t r u c t u r e s o f a g l y c o n e s shown i n F i g u r e 2. It should be s t r e s s e d that e x t r a p o l a t i o n o f these results to toxicity i n v i v o s h o u l d be done w i t h care. We do know that tetraphyllin Β i s usually toxic i n proportion to i t s alkylating a b i l i t y t o H e l i c o n i u s sp. and t o KL_ z e a (21,22). B e n z y l i s o t h i o c y a n a t e 2 i s a t o x i n o r d e t e r r e n t t o many s p e c i e s (52J, and DIMBOA 6 has been shown t o reduce f i t n e s s i n S p o d o p t e r a s p . , b u t i t s e f f e c t upon H. zea i s less c l e a r (3). Triterpene glycosides 5 are frequently bioactive, b u t s p e c i f i c i t y o f a c t i o n a g a i n s t d e s e r t f r u i t f l i e s has only r e c e n t l y been s t u d i e d (21). Both c a t a l p o l 1 and s a l i c i n 4 a r e such e f f e c t i v e f e e d i n g d e t e r r e n t s (29,53) t h a t d e t e r m i n a t i o n o f modes of a c t i o n requires s p e c i a l i z e d techniques. The o r a l t o x i c i t y o f a l l these compounds toward i n s e c t s i s c u r r e n t l y under i n v e s t i g a t i o n i n this and o t h e r l a b o r a t o r i e s , and i t i s hoped t h a t t h e s e in vitro studies w i l l p r o v i d e some g u i d a n c e i n e x p e r i m e n t a l d e s i g n . Work i s a l s o underway t o f u r t h e r p u r i f y i n s e c t b e t a - g l u c o s i d a s e f r a c t i o n s , t o acquire alpha-glucosidase fractions, and t o p r o v i d e adequate r e p l i c a t i o n f o r t h e s e r e s u l t s . O t h e r t e s t s u b s t r a t e s and enzymes a r e a l s o being studied. Summary The p r i m a r y m e t a b o l i s m o f i n s e c t s depends upon e f f i c i e n t f u n c t i o n o f glycosidases optimized f o r e x t r a c t i o n o f g l u c o s e from p l a n t food sources i n t h e d i e t . The e l a b o r a t i o n by p l a n t s o f substances inhibitory to glycosidases, such as t h e e f f e c t i v e t o x i n and feeding deterren castanospermine, s u g g e s t t h a t i n s e c t d i g e s t i v e enzymes c a n be targets of plant defensive chemistry. Insect d e t o x i f i c a t i o n mechanisms a r e p r o b a b l y not able t o e f f i c i e n t l y i n t e r f e r e with glycoside hydrolysis events. Bioactive plant glycosides are ubiquitous i n t h e p l a n t kingdom, and i t i s p r o p o s e d t h a t t h e s e have arisen i n e v o l u t i o n as s p e c i f i c t o x i n s toward insect glycosidases. Preliminary d a t a a r e p r e s e n t e d which s u p p o r t t h i s hypothesis. They demonstrate: a) i n h i b i t i o n of a selection of insect glucosidase f r a c t i o n s by d i v e r s e p l a n t g l y c o s i d e s t r u c t u r e s , b) t h e e x i s t e n c e o f biochemical mechanisms o f t o x i c i t y i n c l u d i n g b o t h competitive and n o n c o m p e t i t i v e modes o f i n h i b i t i o n , c ) s e l e c t i v i t y and s p e c i f i c i t y o f action i n inhibitory effect related to insect s p e c i a l i z a t i o n upon host p l a n t s . Exploitability

In P e s t i c i d e Design

In the ongoing effort to eliminate major crop pests, special attention has been g i v e n t o c h e m i c a l control, including compounds derived from p l a n t sources. Many o f t h e s e p l a n t - d e r i v e d natural products are glycosides. We have i n t h e p a s t assumed toxicity for

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many of these compounds whereas they are in fact toxic only upon hydrolysis by glycosidases. Work in this laboratory and others has shown that these glycosidases are highly specific in action, and may be inhibited either by other glycosidases or glycosides. The determination of toxicity of glycosides will therefore depend upon an assessment of the ability of the enzyme complement of the targeted organism to either potentiate or inhibit hydrolysis and hence toxification. Literature Cited 1. Miller, J.R.; Strickler, K.L. In Chemical Ecology of Insects; Bell, W.J.; Cardé, R.T., Eds.; Sinauer Associates: Sunderland, MA, 1984; p. 127. 2. Slansky, F., Jr.; Scriber, J.M. In Comprehensive Insect Physiology, Biochemistry and Pharmacology; Kerkut, G.A.; Gilbert, L.I., Eds.; Pergamon Press: New York, 1985; Vol. 4, p. 87. 3. Scriber, J.M. In Chemical Ecology of Insects; Bell, W.J.; Cardé, R.T., Eds.; Sinauer Associates: Sunderland, MA, 1984; p. 159. 4. Cates, R.G.; Redak, R.A. Terpene chemistry of Douglas-fir and its relationship to western spruce budworm success. In Chemical Mediation of Coevolution; Spencer, K.C., Ed.; Academic Press: New York, 1988; in press. 5. Scriber, J.M. In Variable Plants and Herbivores in Natural and Managed Systems; Denno, R.F.; McClure, M.S., Eds.; Academic Press: New York, 1983; p. 373. 6. Candy, D.J. In Comprehensive Insect Physiology, Biochemistry and Pharmacology; Kerkut, G.A.; Gilbert, L.I., Eds.; Pergamon Press: New York, 1985; Vol. 10, p. 1. 7. Turunen, S. In Comprehensive Insect Physiology, Biochemistry and Pharmacology; Kerkut, G.A.; Gilbert, L.I., Eds.; Pergamon Press: New York, 1985; Vol. 4, p. 241. 8. Rafaeli-Bernstein, Α.; Mordue, W. J. Insect Physiol. 1979, 25, 241-47. 9. Knowles, G. J. Exp. Biol. 1975, 62, 327-40. 10. Chefurka, W. In The Physiology of Insecta; Rockstein, M., Ed.; Academic Press: New York, 1965; p. 581. 11. Friedman, S. In Comprehensive Insect Physiology, Biochemistry and Pharmacology; Kerkut, G.A.; Gilbert, L.I., Eds; Pergamon Press: New York, 1985; Vol. 10, p. 43. 12. Applebaum, S.W. In Comprehensive Insect Physiology, Biochemistry and Pharmacology; Kerkut, G.A.; Gilbert, L.I., Eds.; Pergamon Press: New York, 1985; Vol. 4, p. 279. 13. Evans, W.A.L.; Payne, D.W. J. Insect Physiol. 1964, 10, 657-74. 14. Sláma, K.; Nĕmec, V. Acta Ent. Bohem. 1981, 78, 1-9. 15. Anstee, J.H.; Charnley, A.K. J. Insect Physiol. 1977, 23, 965-74. 16. Chapman, R.F. In Comprehensive Insect Physiology, Biochemistry and Pharmacology; Kerkut, G.A.; Gilbert, L.I., Eds.; Pergamon Press: New York, 1985; Vol. 4, p. 213. 17. McBee, R.H. Ann. Rev. Ecol. Syst. 1971, 2, 165-76. 18. Scheline, R.R. Mammalian metabolism of Plant Xenobiotics; Academic Press: New York, 1978. 19. Chippendale, G.M. In Biochemistry of Insects; Rockstein, M. Ed.; Academic Press: New York, 1978; p. 1. 20. Hosel, W. In The Biochemistry of Plants ; Stumpf, P.K.; Conn, E.E., Eds.; Academic Press: New York, 1981; Vol. 7, p. 725.

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26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

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