Insect Antifeedants from the Peruvian Plant Alchornea triplinervia

33 Insect Antifeedants from the Peruvian Plant Alchornea triplinervia 1

1

Downloaded by KTH ROYAL INST OF TECHNOLOGY on February 18, 2016 | http://pubs.acs.org Publication Date: April 26, 1985 | doi: 10.1021/bk-1985-0276.ch033

D. HOWARD MILES , BARBARA L. HANKINSON , and SHIRLEY A. RANDLE

2

1

Department of Chemistry, Mississippi State University, Mississippi State, MS 39762 Department of Entomology, Mississippi Agricultural and Forestry Experiment Station, Mississippi State, MS 39762

2

The boll weevil antifeedants, anthranilic acid, gentisic acid, senecioic acid, trans-cinnamic acid, trans-cinnamaldehyde and camphor have been isolated from the Peruvian plant Alchornea triplinervia (Euphorbiaceae). Anthranilic acid and camphor also showed significant inhibition of growth of the tobacco budworm. A l t h o u g h p e s t i c i d e s c o n t i n u e t o be the major a p p r o a c h t o b o l l w e e v i l c o n t r o l , problems r e l a t e d t o t h e i r u s e have l e d t o a s e a r c h f o r a l t e r n a t i v e forms o f p e s t c o n t r o l . These i n c l u d e c h e m i c a l s t h a t m o d i f y b e h a v i o r and/or development, b i o l o g i c a l a g e n t s , and g e n e t i c m a n i p u l a t i o n . A n t i f e e d a n t s appear t o be a p r o m i s i n g a p p r o a c h t o a g r i c u l t u r a l pest c o n t r o l . Kubo e t . a l . ( 1 ) d e f i n e i n s e c t a n t i f e e d a n t s as " s u b s t a n c e s w h i c h when t a s t e d can r e s u l t i n c e s s a t i o n o f f e e d i n g e i t h e r t e m p o r a r i l y o r p e r m a n e n t l y depending upon p o t e n c y . " S i n c e a n t i f e e d a n t s do n o t d i r e c t l y k i l l the i n s e c t , problems a s s o c i a t e d w i t h p e s t i c i d e s , hormones, b i o l o g i c a l a g e n t s , o r g e n e t i c m a n i p u l a t i o n may n o t be o p e r a t i v e ; however, o t h e r problems may be o p e r a t i v e . A n t i f e e d a n t s may be o b t a i n e d by the i s o l a t i o n o f n a t u r a l l y o c c u r r i n g a n t i f e e d a n t s o r by s y n t h e s i s . A n t i f e e d a n t s have been i s o l a t e d w h i c h r e p r e s e n t many b r o a d c l a s s e s o f compounds (2) · S i n c e i t i s w i d e l y a c c e p t e d t h a t t r o p i c a l f l o r a have b u i l t - i n d e f e n s e mechanisms (2) due t o t h e i r c o n s t a n t exposure t o a t t a c k by many t y p e s o f b i o l o g i c a l organisms i n c l u d i n g i n s e c t s , the p l a n t s c h o s e n f o r t h i s s t u d y were s e l e c t e d from the Amazon R i v e r b a s i n o f P e r u and the s o u t h e r n p o r t i o n o f the U n i t e d S t a t e s ( M i s s i s s i p p i ) . The e t h a n o l i c e x t r a c t s o f more than f i v e hundred p l a n t s from P e r u and M i s s i s s i p p i were evaluated f o r potency w i t h a b o l l w e e v i l a n t i f e e d a n t bioassay. Those p l a n t s w h i c h showed t o t a l i n h i b i t i o n o f f e e d i n g were f r a c t i o n a t e d and examined i n a d d i t i o n a l b i o a s s a y s u s i n g the t o b a c c o budworm. T h r e e o f the p l a n t s w h i c h were s e l e c t e d f o r f u r t h e r e x a m i n a t i o n i n c l u d e the P e r u v i a n p l a n t s A l c h o r n e a t r l p l i n e r v l a ( E u p h o r b i a c e a e ) and Machaerium f l o r i b u n d u m Bentham (Leguminosae) and the M i s s i s s i p p i p l a n t H e t e r o t h e c a camporum (Compositae). 0097-6156/85/0276-0469$06.00/0 © 1985 American Chemical Society

In Bioregulators for Pest Control; Hedin, Paul A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

470

BIOREGULATORS FOR PEST CONTROL

Recent p u b l i c a t i o n s (3,4) i n r e g a r d t o t h e l a t t e r two p l a n t s i n d i c a t e t h a t t h e y were a l s o t o x i c t o two b a c t e r i a w h i c h o c c u r i n t h e gut o f t h e tobacco budworm H e l i o t h i s v i r e s c e n s (Fab.) and t h e c o r n earworm H e l i o t h i s z e a (Boddie)· The compounds, rhamnetin 3 - 0 - g l u c o s i d e and i s o q u e r c i t r i n , were i s o l a t e d (3) from H^ camporum based upon t h e i r a c t i v i t y a g a i n s t Pseudomonas m a l t o p h i l i a and E n t e r o b a c t e r c l o a c a e . A l s o , a p r o c y a n i d i n was i s o l a t e d (4) from f l o r i b u n d u m on t h e b a s i s o f a c t i v i t y a g a i n s t P. m a l t o p h i l i a .


B o l l Weevil Antifeedant Bioassay The a g a r - p l u g b i o a s s a y p r o c e d u r e d e v e l o p e d by H e d i n e t . a l . (5) was used w i t h a few m o d i f i c a t i o n s . The agar p l u g s were formed by b o i l i n g 3 grams o f agar and 3 grams o f f r e e z e - d r i e d c o t t o n b o l l s i n 100 ml o f d i s t i l l e d water t o e f f e c t a v i s c o u s s o l . The s o l was poured i n t o 13 mm d i a m e t e r h o l l o w g l a s s r o d s and g e l a t i o n occurred a f t e r c o o l i n g . These g e l a t i n o u s r o d s were c u t i n t o i n d i v i d u a l 3.5 cm p l u g s . The e x t r a c t s o f t h e p l a n t samples were a p p l i e d t o preweighed 4 cm s q u a r e s o f Whatman / / l chromatography paper by d i p p i n g t h e paper i n t o a s o l u t i o n o f t h e e x t r a c t . A f t e r d r y i n g , t h e papers were weighed t o d e t e r m i n e t h e " c o n c e n t r a t i o n " o f t h e e x t r a c t b e i n g t e s t e d . A c o n t r o l paper was p r e p a r e d by d i p p i n g a 4 cm s q u a r e o f paper i n t o t h e s o l v e n t used f o r t h e e x t r a c t and a l l o w i n g the paper t o a i r - d r y . The papers were wrapped around t h e a g a r c o t t o n p l u g s and f a s t e n e d w i t h s t a p l e s . The ends o f t h e p l u g s were s e a l e d w i t h c o r k s . The p l u g s were t h e n p l a c e d s t a p l e - s i d e down i n t h e p e t r i d i s h e s so t h a t t h e b o l l w e e v i l s c o u l d f e e d o n l y by p u n c t u r i n g t h e p a p e r s . Twenty newly emerged b o l l w e e v i l s were p l a c e d i n 14 χ 2 cm p e t r i d i s h e s c o n t a i n i n g t h e t e s t and c o n t r o l p l u g s . The b i o a s s a y was c a r r i e d o u t i n t h e d a r k a t 80°F f o r 4 h o u r s , a f t e r which t h e papers were removed and t h e p u n c t u r e s c o u n t e d . A n t i f e e d a n t a c t i v i t y was e x p r e s s e d a s a %T/C v a l u e , where

%T/C

β

U

N

C

T

U

R

E

S

#P °f t e s t paper (Τ) //punctures o f c o n t r o l paper (C)

χ

^QQ

%T/C v a l u e s o f z e r o r e p r e s e n t e d t o t a l i n h i b i t i o n o f f e e d i n g , w h i l e v a l u e s g r e a t e r t h a n 100 r e p r e s e n t e d f e e d i n g a t t r a c t a n c y . Tobacco Budworm L a r v a l Growth B i o a s s a y The b i o a s s a y was performed a c c o r d i n g t o t h e p r o c e d u r e o f H e d i n e t . a l . (β). A randomized complete b l o c k o f e i g h t r e p l i c a t e s o f f i v e l a r v a e were used f o r e a c h d a t a p o i n t . Compounds were t e s t e d a t 5-6 c o n c e n t r a t i o n s r a n g i n g from 0.02 t o 0.50% o f t h e total diet. L a r v a l w e i g h t s were e x p r e s s e d a s p e r c e n t o f c o n t r o l (%T/C). F o r e s t i m a t i o n o f E D ^ Q v a l u e s , t h e l a r v a l w e i g h t e x p r e s s e d as %T/C was r e g r e s s e d a s t h e p e r c e n t o f t h e compound i n the d i e t u s i n g the l i n e a r p o r t i o n of the curve. The p e r c e n t o f t h e compound t h a t would d e c r e a s e t h e w e i g h t g a i n by 50% ( E D 5 0 ) was then estimated.


33.

MILES ET AL.

E x t r a c t i o n of Alchornea


471

Insect Antifeedants t r i p l i n e r v i a Leaves

The A l c h o r n e a t r i p l i n e r v i a used i n t h i s p r o j e c t was c o l l e c t e d by the I n s t i t u t e f o r B o t a n i c a l E x p l o r a t i o n a t t h e i r r e s e a r c h f a c i l i t y i n I q u i t o s , P e r u , i n the upper Amazon V a l l e y d u r i n g the f i r s t q u a r t e r o f 1980. The p l a n t m a t e r i a l was e x t r a c t e d a c c o r d i n g to the p r o c e d u r e i n F i g u r e 1. The e t h a n o l e x t r a c t [2] ( F i g u r e 1) e x h i b i t e d t o t a l i n h i b i t i o n o f f e e d i n g a g a i n s t the c o t t o n b o l l w e e v i l a t l e v e l s o f 10, 25, and 50 mg ( T a b l e I ) . F u r t h e r f r a c t i o n a t i o n o f the e t h a n o l e x t r a c t [2] showed no abatement o f t h i s a n t i f e e d a n t a c t i v i t y i n e i t h e r the CHC1 [3] o r the aqueous f r a c t i o n [ 4 ] w i t h %T/C v a l u e s o f l e s s than 12% a t the 10-50 mg dose l e v e l s as i n d i c a t e d i n Table I. F u r t h e r f r a c t i o n a t i o n ( F i g u r e 1) and b i o a s s a y ( T a b l e I ) showed t h a t the methanol f r a c t i o n [ 6 ] , and the C H C ^ / e t h a n o l f r a c t i o n [7] demonstrated e x c e l l e n t a n t i f e e d a n t a c t i v i t y . 3

The CHCl-j/ethanol e x t r a c t [ 7 ] was s u c c e s s i v e l y e x t r a c t e d w i t h 5% a c e t i c a c i d , 5% h y d r o c h l o r i c a c i d , 5% sodium b i c a r b o n a t e , 5% sodium c a r b o n a t e , and 5% sodium h y d r o x i d e . Bioassay of these f r a c t i o n s i n d i c a t e d t h a t a n t i f e e d a n t a c t i v i t y r e s i d e d i n the sodium b i c a r b o n a t e and sodium c a r b o n a t e f r a c t i o n s . A c r y s t a l l i n e m a t e r i a l (compound jL, mp 144-145°C) was i s o l a t e d from t h e sodium b i c a r b o n a t e f r a c t i o n by c r y s t a l l i z a t i o n from ether. Compound 1^ was shown t o be a n t h r a n i l i c a c i d a c c o r d i n g to IR, NMR, and UV comparisons w i t h an a u t h e n t i c sample ( A l d r i c h C h e m i c a l Company).

C0 H 2

1

2

4

5

3

6



Alchomea t r i p l i n e r v i a

Spreng. Leaves e x t r a c t e d 16 h o u r s


with

CH C1 2

2

CH C1 2

2

Marc

[1]

e x t r a c t e d 16 h o u r s w i t h EtOH

I EtOH

Marc (discarded)

[2]

p a r t i t i o n e d between CHC1 and H 0 (1:1) 3

CHC1

3

2

H 0

[3]

2

[4] extracted with CHCln-EtOH

partitioned between hexane and Me0H-H 0 (9:1)

(1:1)

2

I Hexane

MeOH-H 0

[5J

[6]'

2

CHCl,-EtOH 3

[7]

H 0 [8] 2

F i g u r e 1. F r a c t i o n a t i o n Scheme f o r the Leaves o f A l c h o m e a t r i p l i n e r v i a Spreng. ( E u p h o r b i a c e a e )


33.

MILES ET AL.

Table I .

B i o a s s a y T e s t R e s u l t s from E x t r a c t i o n F r a c t i o n s o f Alchornea t r i p l i n e r v i a

Layer


Insect Antifeedants

10 mg

%T/C 25 m

S

50 mg

66 161 237

31 100 263

19 219 148

0 0 0

0 0 0

0 0 0

(CHCI3)

3 12 3

0 0 0

0 0 0

4

(H 0)

3 0 11

0 0 1

6 1 0

5

(Hexane)

78 54 51

0 24 1

6 1 7

6

(Methanol)

0 0 0

1 0 0

3 0 0

0 2 0

0 0 1

0 0 0

9 1 0

0 4 0

0 1 0

1

(CH C1 )

2

(Ethanol)

3

7

8

2

2

2

(CHCl /Ethanol) 3

(H 0) 2



474


Column chromatography on s i l i c a g e l o f the sodium c a r b o n a t e f r a c t i o n u t i l i z i n g benzene, c h l o r o f o r m , and methanol as the e l u t i n g s o l v e n t s r e s u l t e d i n the i s o l a t i o n o f compounds 2 (mp 199-200°C) and _3. Compound 2 was shown to be g e n t i s i c a c i d a c c o r d i n g to IR, NMR, UV and mixed m e l t i n g p o i n t comparison w i t h an a u t h e n t i c sample ( A l d r i c h C h e m i c a l Company). Compound 3_ was shown to be s e n e c i o i c a c i d based on IR, NMR, and mixed m e l t i n g p o i n t comparison w i t h an a u t h e n t i c sample ( A l d r i c h C h e m i c a l Company). I n v e s t i g a t i o n o f the c h l o r o f o r m f r a c t i o n [ 3 ] i n F i g u r e 1 r e s u l t e d i n the i s o l a t i o n o f compounds 4_, _5, and 16. T h i s was a c h i e v e d t h r o u g h e x t e n s i v e column chromatography on s i l i c a g e l . Compounds 4^ and _5 were p r e s e n t i n the o i l y c h r o m a t o g r a p h i c f r a c t i o n which had the c h a r a c t e r i s t i c s m e l l o f cinnamon. Compound 4^ was i s o l a t e d as an o i l and i d e n t i f i e d as cinnamaldehyde by f o r m a t i o n o f the 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e d e r i v a t i v e which m e l t e d a t 251-253° [ l i t ( 7 ) m.p. 255°C]. NMR, IR, and UV d a t a were c o n s i s t e n t w i t h t h i s a s s i g n m e n t . F u r t h e r p r o o f was p r o v i d e d by a i r o x i d a t i o n to form a w h i t e c r y s t a l l i n e m a t e r i a l which was. shown to be i d e n t i c a l w i t h compound _5 which upon IR, NMR, and UV comparison w i t h an a u t h e n t i c sample ( A l d r i c h C h e m i c a l Company) was shown t o be c i n n a m i c acid. Compound 6^ was i s o l a t e d v i a r e p e t i t i v e column chromatography o f the CHCl^ f r a c t i o n [ 3 ] . Compound 16 was shown to be i d e n t i c a l upon IR, NMR, and UV comparison w i t h an a u t h e n t i c sample o f camphor. B o l l Weevil Antifeedant Bioassay

Results

The r e s u l t s o f the b o l l w e e v i l a n t i f e e d a n t b i o a s s a y s w i t h a n t h r a n i l i c a c i d , g e n t i s i c a c i d , s e n e c i o i c a c i d , cinnamaldehyde and c i n n a m i c a c i d a r e p r e s e n t e d i n T a b l e I I . Camphor was not b i o a s s a y e d due t o i t s extreme v o l a t i l i t y . However camphor i s a known moth r e p e l l e n t (§) and i s p r o b a b l y a b o l l w e e v i l a n t i f e e d a n t s i n c e the f r a c t i o n from w h i c h i t was i s o l a t e d was a c t i v e . The r e s u l t s w i t h cinnamaldehyde a r e perhaps the most s i g n i f i c a n t s i n c e t o t a l i n h i b i t i o n o f f e e d i n g was demonstrated a t a dose o f 30 mg f o r an e n t i r e a g a r p l u g ( a r e a = 16 c m ) . Moreover the a c t i v i t y remained a t a h i g h l e v e l (96% i n h i b i t i o n ) as the dose was reduced to 20 mg and 10 mg. A dose o f 10 mg f o r the e n t i r e p l u g i s e q u i v a l e n t to a dose o f 0.62 mg p e r cm . Tests w i l l be performed a t even lower doses i n the f u t u r e . 2

2

Tobacco Budworm Growth Study A n t h r a n i l i c a c i d , g e n t i s i c a c i d , s e n e c i o i c a c i d , cinnamic a c i d , and camphor were t e s t e d as i n h i b i t o r s o f tobacco budworm l a r v a l growth. The r e s u l t s o f t h i s s t u d y a r e p r e s e n t e d i n T a b l e I I I . T h i s t a b l e shows t h a t g e n t i s i c a c i d and s e n e c i o i c a c i d d i d not show s i g n i f i c a n t i n h i b i t i o n o f l a r v a l growth o f the tobacco budworm. However a n t h r a n i l i c a c i d , c i n n a m i c a c i d , and camphor showed s i g n i f i c a n t i n h i b i t i o n o f growth w i t h E D Q l e v e l s of 0.68%, 0.38%, and 0.50% r e s p e c t i v e l y . T h i s compares f a i r l y w e l l w i t h t a n n i n s , g o s s y p o l , and a n t h o c y a n i n w h i c h have E D ^ Q v a l u e s o f 0.05-0.07% as r e p o r t e d by H e d i n ( 9 ) . 5


33.

MILES ET

Table I I .

AL.

475

B o l l W e e v i l A n t i f e e d a n t A c t i v i t y o f Compounds I s o l a t e d from A ^ t r i p l i n e r v i a Dose Level

Compound

Average %T/C

%T/C (mg)

10 20 30

12 14 4

11 8 7

13 10 8

12 11 6

5 10 12

48 8 17

95 9 11

51 6 14

65 8 14

Acid

10 15 40

6 15 1

16 14 4

24 21 2

15 17 2

Cinnamaldehyde

10 20 30

3 0 0

1 10 0

9 3 1

4 4 0

Cinnamic A c i d

10 20 30

42 31 9

26 24 46

12 24 21

27 26 25

Anthranilic

Gentisic


Insect Antifeedants

Acid

Acid

Senecioic

Table I I I .

Tobacco Budworm Growth I n h i b i t i o n by from A^ t r i p l i n e r v i a

Compounds I s o l a t e d

D

Compound

^ 50

Anthranilic Acid Gentisic Acid Senecioic Acid Cinnamic A c i d Camphor

0.68 NS° NS 0.38 0.50

a

b

E D c Q = p e r c e n t o f compound r e q u i r e d by 50%. NS = not s i g n i f i c a n t .

b

to r e d u c e w e i g h t

gain

Conclusion T l i i s s t u d y d e s c r i b e s the i s o l a t i o n and b i o a s s a y a g a i n s t i n s e c t s o f s i x compounds from the P e r u v i a n p l a n t A l c h o r n e a t r i p l i n e r v i a . The compounds a n t h r a n i l i c a c i d , c i n n a m i c a c i d , and camphor showed s i g n i f i c a n t i n h i b i t i o n o f the growth o f the t o b a c c o budworm. A n t h r a n i l i c a c i d , g e n t i s i c a c i d , s e n e c i o i c a c i d , c i n n a m i c a c i d , and cinnamaldehyde demonstrated low to moderate a c t i v i t y i n the b o l l weevil antifeedant bioassay. Cinnamaldehyde, a c o n s t i t u e n t o f the s p i c e cinnamon, showed the h i g h e s t l e v e l o f i n h i b i t i o n to b o l l weevil feeding.


476

BIOREGULATORS FOR PEST C O N T R O L

Acknowledgements T h i s r e s e a r c h has been s u p p o r t e d by the M i s s i s s i p p i A g r i c u l t u r a l and F o r e s t r y Experiment S t a t i o n . We a r e g r e a t l y i n d e b t e d t o our c o l l e a g u e , D r . P a u l A. H e d i n , f o r h i s a s s i s t a n c e t h r o u g h o u t t h i s s t u d y and i n the p r e p a r a t i o n o f t h i s m a n u s c r i p t . We a l s o acknowledge Dr. S i d n e y M c D a n i e l o f the I n s t i t u t e f o r B o t a n i c a l Exploration for plant c o l l e c t i o n .


Literature 1. 2.

3. 4. 5. 6.

7. 8. 9.

Cited

I. Kubo, Y.W. Lee, M. Pettei, F. Pilkiewicz, and K. Nakanishi, Chem. Comm., 1013 (1976). I. Kubo and K. Nakanishi. In "Host Plant Resistance to Pests"; Hedin, P.Α., Ed; ACS SYMPOSIUM SERIES No. 62, American Chemical Society: Washington D.C. 1977; pp. 165-178. S.K. Waage and Paul A. Hedin, Phytochemistry, in press, 1984. S.K. Waage and P.A. Hedin, and E. Grimley, Phytochemistry, in press, 1984. P.A. Hedin, A.C. Thompson, and J.P. Minyard, J. Econ. Entomol. 59, 181 (1966). P.A. Hedin, D.H. Collum, W.H. White, W.L. Parrott, H.C. Lane, and J.N. Jenkins, Scientific Papers of the Institute of Organic and Physical Chemistry of Wroclaw Technical University, No. 22, Conferences 7, 1971 (1980). C.D. Hodgman, ed., Tables for Identification of Organic Compounds, Chemical Rubber Publishing Company, 1960, 72. M. Windhoz, ed., Merck Index, 9th Edition, Merck and Co., Inc., 1976, p. 1735. P.A Hedin, J.N. Jenkins, D.H. Collum, W.H. White, W.L. Parrott, and M.W. MacGown, Experientia 39, 799 (1983).

RECEIVED

November 8,

1984


Insect Antifeedants from the Peruvian Plant Alchornea triplinervia

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