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Effect of sunlight on
azadirachtin: Antifeeding
potency
a a
J. B. Stokes & R. E. Redfern
a
Agricultural Research Service, USDA , Biologically
Active Natural Products Laboratory and Livestock
Insects Laboratory , Beltsville, MD, 20705
Published online: 15 Dec 2008.

To cite this article: J. B. Stokes & R. E. Redfern (1982) Effect of sunlight on

azadirachtin: Antifeeding potency, Journal of Environmental Science and Health .
Part A: Environmental Science and Engineering: Toxic/Hazardous Substances and
Environmental Engineering, 17:1, 57-65, DOI: 10.1080/10934528209375019

To link to this article: http://dx.doi.org/10.1080/10934528209375019

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 J . ENVIRON. SCI. HEALTH, A 1 7 ( l ) , 57-65 (1982)

EFFECT OF SUNLIGHT ON AZADIRACHTIN: ANTIFEEDING POTENCY

Key words: a n t i f e e d i n g potency, a z a d l r a c h t l n , f a l l armyworm

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Spodoptera f r u g i p e r d a , s u n l i g h t degradation

J . B. Stokes and R. E. Redfern 2

B i o l o g i c a l l y Active Natural Products Laboratory and

Livestock I n s e c t s Laboratory,
A g r i c u l t u r a l Research S e r v i c e , USDA, B e l t s v i l l e , MD 20705

ABSTRACT

Exposure of a z a d i r a c h t i n to s u n l i g h t caused a rapid decrease

i n a n t i f e e d i n g potency a g a i n s t newly emerged f i r s t i n s t a r (0.046

mg) f a l l armyworm, Spodoptera frugiperda ( J . E. Smith), as

compared to unexposed a z a d i r a c h t i n . Acetone s o l u t i o n s of

a z a d i r a c h t i n exposed for seven days gave more than a 50 percent

reduction i n a c t i v i t y while a z a d i r a c h t i n exposed for s i x t e e n days

gave complete reduction i n a c t i v i t y with d e s t r u c t i o n of the

compound. p-Aminobenzoic acid, a uv-absorbing a d d i t i v e , gave

s l i g h t p r o t e c t i o n to a z a d i r a c h t i n from photodegradation.

Azadirachtin, mixed with various plant o i l s , was exposed t o

s u n l i g h t for two weeks. Bioassay of these mixtures showed t h a t

neem, a n g e l i c a , c a s t o r , and calamus o i l s provided moderate

p r o t e c t i o n (<25 p e r c e n t ) to a z a d i r a c h t i n from degradation by

sunlight.

57

Copyright © 1982 by Marcel Dekker, Inc.

 58 STOKES AND REDFERN

INTRODUCTION

Insect feeding deterrents have great potential as alternative

insect control agents to insecticides in protecting food and fiber

crops from harmful insects. One source of insect feeding

inhibitors i s the neem tree, Azadirachta indica A. Juss.

Extracts of the seeds, leaves, flowers, or fruits of the neem tree

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contain three major antifeedant triterpenoids, i.e., meliantriol,

salannin, » and azadirachtin. » » The most active triterpenoid,

for a variety of insects, i s azadirachtin. Recently, a method for

the gram-scale isolation of azadirachtin from neem seeds was

developed. » The Isolated azadirachtin was effective as a

feeding deterrent against f i r s t instar larvae of the f a l l

armyworm, Spodoptera frugiperda (J. E. Smith), at 0.35 ppm in an

artificial diet12.

Utilization of azadirachtin as an insect control chemical

requires a study of i t s stability to natural elements in field

applications. We examined the antifeeding potency of azadirachtin

exposed to sunlight with a laboratory bioassay against the f i r s t

instar larvae of the f a l l armyworm. The effect of sunlight on

azadirachtin when mixed with various plant oils was investigated

so as to find ways to prevent degradation by sunlight. Our

results are described in this report.

METHODS AND MATERIALS

Bioassay

The f a l l armyworms were laboratory-reared on an a r t i f i c i a l

d i e t . ^ The sun-exposed samples were incorporated into 100 g of
 EFFECT OF SUNLIGHT ON AZADIKACHTIN 59

a r t i f i c i a l d i e t a t the appropriate concentrations. Treated d i e t s

and untreated control d i e t s were poured i n t o 1-oz j e l l y cups;

there were ten r e p l i c a t e s for each d i e t . One newly emerged f i r s t

i n s t a r (0.046 mg) larva was placed i n each cup. The cups were

f i t t e d with prepunched l i d s and held a t 27° ± 1°C and 50 ± 5

percent r e l a t i v e humidity. After seven days the larvae were

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weighed and observed for m o r t a l i t y .

Sample preparation and s u n l i g h t exposure

Azadirachtin samples (1 ug/1 vL acetone), i n small capped

quartz tubes, were exposed to d i r e c t sunlight (approximately eight

hours per day) for seven t o s i x t e e n days. Unexposed samples of

azadirachtin (1 ug/1 uL) were stored i n the dark a t 5°C.

Additional 500 yL samples of a z a d i r a c h t i n (1 vg/1 vh), containing

1 percent chemical a d d i t i v e ( c i t r i c acid, BHT, t r i o c t a n o i n , or

jr-aminobenzoic a c i d ) , were exposed t o s u n l i g h t for s i m i l a r

periods. The samples were d i l u t e d with acetone for bioassay.

A commercial plant o i l (5 yL) and 25 vL of an a z a d i r a c h t i n

solution (1 ug/1 P L ) were applied t o a microscope s l i d e . The

mixture was spread t h i n l y on the s l i d e with a small g l a s s rod. An

untreated sample of the plant o i l (5 PL) was spread on another

s l i d e f o r use as a standard. After a p p l i c a t i o n of the o i l s i t h e

g l a s s s l i d e s were placed i n an aluminum f o i l - l i n e d , wooden box,

and protected from the r a i n . Following exposure t o the sun for

two weeks, the samples were removed from the s l i d e s , f i r s t by

treatment with 50 uL dimethyl sulfoxide (DMSO), then by a thorough

r i n s i n g with reagent grade acetone. Each sample was concentrated

to a f i n a l volume of 200 pL t h a t was used f o r bioassay.

 60 STOKES AND REDFERN

Hplc analyses

In a d d i t i o n t o bioassay of acetone solutions of a z a d i r a c h t i n ,

with o r without chemical a d d i t i v e s , t h e sunlight-exposed samples

were analyzed f o r a z a d i r a c h t i n with a Waters Associates ALC-100

l i q u i d chromatograph, equipped with two M-6000 pumps, a U6K

i n j e c t o r , and a Schoeffel SF 770 multi-wavelength UV d e t e c t o r . An

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a l i q u o t of each s o l u t i o n was i n j e c t e d onto a 30 x 0.39 cm ID

liBondapak C^g column eluted with 50 percent (v/v) me thanol-water

at 1.0 mL/min. The e l u a t e was monitored a t 217 nm and compared

with a z a d i r a c h t i n standards.

RESULTS AND DISCUSSION

The antifeeding potency of a z a d i r a c h t i n decreased r a p i d l y

a f t e r exposure t o s u n l i g h t for seven t o s i x t e e n days (Table 1 ) ,

whereas a z a d i r a c h t i n t h a t was stored i n the dark f o r s i m i l a r

periods retained i t s potency. Bioassay against the f a l l armyworm

showed t h a t the antifeeding potency decreased more than 50 percent

a f t e r exposure f o r seven days. Hplc analyses of the a z a d i r a c h t i n

solutions confirmed these r e s u l t s by showing t h a t l e s s than 50

percent of t h e i n i t i a l a z a d i r a c h t i n concentration remained a f t e r

the seven day exposure. When exposure was extended t o s i x t e e n

days, a z a d i r a c h t i n was completely destroyed; bioassay of t h e

resultant solutions gave no antifeeding inhibition against the

f a l l armyworm. Hplc analyses of the exposed solutions showed no

traces of azadirachtin. Hplc analyses of unexposed solutions

showed no changes of i n i t i a l azadirachtin concentrations.

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TABLE 1

Bioassay of Sunlight Exposed Azadlrachtin In Acetone Solutions

At 0.3 ppm Against F i r s t Ins t a r F a l l Anayworm Larvae

Chemical Exposure % I n i t i a l AZ ^ A c t i v i t y % I n i t i a l AZ Concentration §

Additive (1%) Time (days) After Exposure — After Exposure c '

none 7 <50 40 E

CHT
none 16 0 0
c i t r i c acid 14 0 0 H
a
BHT ~l 14 0 0
trioctanoin 14 0 5
£-aminobenzole 14 <30 20
acid

BJ Azadlrachtin
—' Determined by bioassay and compared to same concentrations of unexposed azadirachtin
—' Determined by hplc analyses and compared to same concentrations of unexposed azadirachtin
i/ 2,6-Bis(l,l-dimethylethyl)-4-methylphenol
 N3
TABLE 2
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Bioassay of Sunlight Exposed Azadlrachtln i n Plant Oils

At 0.5 ppm Against F i r s t Instar Fall Armyworm Larvae

Average Weight (mg) of Larvae (+ S.D.) After 7 Days SJ % I n i t i a l AZ Activity

Oil Without AZ_ & With AZ After Exposure —'
soybean 64 ± 14 53 ± 7 * .d/
tangerine 80 ± 23 71 + 35 0
neem —' 78 ± 19 46 + 4 ** <25
angelica 83 ± 18 52 ± 6 ** <25
sesame 85 ± 28 70 + 10 ** <10
castor 1.06 ± 27 44 ± 245 ** <25
linseed 126 ± 30 128 + 0
corn 85 ± 32 113 + 28 0
calamus 124 ± 27 62 ± 9 ** <25
olive 122 ± 32 121 ± 30 f/
0
AZ exposed — 73
28
± 14 tl
AZ unexposed ± 5
check 89 ± 24

—' 0% mortality; ten larvae per t e s t en

8
1 / Azadirachtin 3
—I Determined by bioassay and compared to same concentration of unexposed azadirachtin
i/ " t " test; *, significant at 5 % level; **, significant at 1 % level
—' A sample shown to be inactive In earlier tests
U Without o i l
 EFFECT OF SUNLIGHT ON AZADIRACHTIN 63

Bioassay of azadirachtin solutions, containing 1 percent

chemical additive ( c i t r i c acid, BHT, t r i o c t a n o i n , or

jv-aminobenzoic a c i d ) , exposed to sunlight f o r fourteen days showed

that jv-aminobenzoic acid s l i g h t l y prevented the degradation of

azadirachtin i n sunlight. Hplc analyses confirmed that

jv-aminobenzoic acid, gave some protection from photodegradation:

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approximately 15-20 percent of the antifeedant remained. Bioassay

and hplc analyses of the trioctanoin-azadlrachtin solution

revealed t h a t trioctanoin gave l i t t l e protection to azadirachtin

from sunlight. C i t r i c acid and BHT gave no protection.

The protective effect of various plant o i l s i s shown i n

Table 2. S t a t i s t i c a l analyses with a " t " t e s t of these data show

that the most effective o i l s for protecting the antifeeding

potency of azadirachtin a r e neem, angelica, castor, and calamus.

After a two week exposure i n these o i l s the photodegradation of

azadirachtin was s l i g h t l y i n h i b i t e d ; the biological a c t i v i t y was

at l e a s t twice t h a t of a sample exposed without o i l . Of these

four o i l s , neem and castor gave the greatest amount of

protection. Soybean and sesame o i l s afforded l i t t l e protection

to azadirachtin since less than 10 percent of the compound

remained a f t e r exposure, a s with azadirachtin without o i l .

Tangerine, linseed, corn, and olive o i l s gave no protection from

photodegradation.

In general, crude neem e x t r a c t s or f r a c t i o n s , and not

azadirachtin, have been used i n laboratory o r f i e l d applications

1q

as feeding deterrents. Ladd et_jal_. applied solvent extracts of

neem to soybean leaves, Glycine max (L.), Merr., in field tests to

 64 STOKES AND REDFERN

deter feeding by Japanese b e e t l e s , P o p i l l i a japonica Newman.

Differences between t r e a t e d and untreated p l a n t s were

significant. Following a p p l i c a t i o n of a 1 percent neem e x t r a c t

(emulsion containing 0.0625 percent Tween-20v->'as a s u r f a c t a n t ) ,

t r e a t e d plants were only s l i g h t l y damaged (10-30 percent) a f t e r 14

days compared t o untreated p l a n t s (70-90 percent). By day 18,

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damage t o the t r e a t e d p l a n t s increased (30-50 p e r c e n t ) . Some l o s s

of antifeeding a c t i v i t y may be a t t r i b u t e d t o runoff, but our

r e s u l t s show t h a t a d d i t i o n a l l o s s may also a r i s e from

photodegradation of a z a d i r a c h t i n .

In our s t u d i e s of a z a d i r a c h t i n i n plant o i l s , neem and c a s t o r

appear t o provide more p r o t e c t i o n than other o i l s ; however, even

then a s u b s t a n t i a l amount of the antifeeding potency of

azadirachtin was l o s t a f t e r two weeks. Consequently, i f neem

extracts or azadirachtin are to be used in field applications,

special formulations for protection from sunlight will be

necessary for extension of the antifeeding potency of

azadirachtin.

ACKNOWLEDGEMENTS

We thank J. D. Warthen, J r . and E. C. Uebel of BANPL f o r a

sample of a z a d i r a c h t i n and f o r v a l u a b l e comments and s u g g e s t i o n s ,
and G. D. M i l l s , J r . of LIL, f o r t e c h n i c a l a s s i s t a n c e w i t h
bioassays a g a i n s t f a l l armyworm.

REFERENCES

1. To whom r e p r i n t r e q u e s t s should be d i r e c t e d a t t h e
B i o l o g i c a l l y Active Natural Products Laboratory,
A g r i c u l t u r a l Environmental Q u a l i t y I n s t i t u t e . BARC-East,
 EFFECT OF SUNLIGHT ON AZADIRACHTIN 65

B e l t s v l l l e , MD 20705. Mention of a commerical or

proprietary product in t h i s paper does not c o n s t i t u t e an
endorsement of that product by the USDA.
2. Livestock Insects Laboratory, Agricultural Environmental
Qualitity I n s t i t u t e , BARC-East, B e l t s v i l l e , MD 20705.
3. J. D. Warthen, J r , USDA Sci. and Educ. Adm. ARM-NE-4,
21 pp. (1979).
4. D. Lavie, M. K. Jain, and S. R. Shpan-Gabrielith, Chem.
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Commun., 910. (1967).

5. R. Henderson, R. Crlndle, and K. H. Overton, Tetrahedron

Lett., 52, 3969. (1964).
6. R. Henderson, R. McCrindle, A. Melera, and K. H. Overton,
Tetrahedron, 24, 1525. (1968).

7. J. H. Butterworth and E. D. Morgan, Chem. Commun., 23.

(1968).
8. J. H. Butterworth, E. D. Morgan, and G. R. Percy, J. Chem.
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9. K. Nakanishi, Recent Adv. Phytochem., 9, 283. (1975).

10. J. D. Warthen, J r . , R. E. Redfern, E. C. Uebel, and G. D.

Mills, J r . , USDA Sci. and Educ. Adm. ARR-NE-1, 9 pp.
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11. E. C. Uebel, J. D. Warthen, J r . , and M. Jacobson, J. Liquid

Chromatog., 2, 875. (1979).
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Received: July 28, 1981

Accepted: September 1, 1981