Citrus limonoids and their semisynthetic derivatives as antifeedant agents against Spodoptera frugiperda larvae. A structure-activity relationship study

Article abstract of DOI:10.1021/jf020607u

The antifeedant activity of Citrus-derived limonoids limonin (1), nomilin (2), and obacunone (3) and their semisynthetic derivatives 4-26 was evaluated against a commercially important pest, Spodoptera frugiperda. Simple chemical conversions were carried out on the natural limonoids obtained from seeds of Citrus limon. These conversions focused on functional groups considered to be important for the biological activity, namely the C-7 carbonyl and the furan ring. In particular, reduction at C-7 afforded the related alcohols, and from these their acetates, oximes, and methoximes were prepared. Hydrogenation of the furan ring was also performed on limonin and obacunone. The known antifeedant properties of the Citrus limonoids are confirmed. Comparison with previously reported data shows that insect species vary in their behavioral responses to these structural modifications. Highly significant antifeedant activity (P < 0.01) for two natural (1 and 3) and three semisynthetic limonoids (4, 8, and 10) was observed against S. frugiperda.

Full text of DOI:10.1021/jf020607u

6766 J. Agric. Food Chem. 2002, 50, 6766−6774
Citrus Limonoids and Their Semisynthetic Derivatives as
Antifeedant Agents Against Spodoptera frugiperda Larvae. A
Structure−Activity Relationship Study†
GIUSEPPE RUBERTO,*,§ AGATINO RENDA,§ CORRADO TRINGALI,*,#
EDOARDO M. NAPOLI,# AND MONIQUE S. J. SIMMONDS
Istituto del C.N.R. di Chimica BiomolecolaresSezione di Catania,× Via del Santuario 110, I-95028
Valverde (CT), Italy, Dipartimento di Scienze Chimiche, Universita` degli Studi di Catania, Viale A.
Doria, 6, 95125 Catania, Italy, and Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond,
Surrey, United Kingdom, TW9 3DS
The antifeedant activity of Citrus-derived limonoids limonin (1), nomilin (2), and obacunone (3) and
their semisynthetic derivatives 4-26 was evaluated against a commercially important pest, Spodoptera
frugiperda. Simple chemical conversions were carried out on the natural limonoids obtained from
seeds of Citrus limon. These conversions focused on functional groups considered to be important
for the biological activity, namely the C-7 carbonyl and the furan ring. In particular, reduction at C-7
afforded the related alcohols, and from these their acetates, oximes, and methoximes were prepared.
Hydrogenation of the furan ring was also performed on limonin and obacunone. The known antifeedant
properties of the Citrus limonoids are confirmed. Comparison with previously reported data shows
that insect species vary in their behavioral responses to these structural modifications. Highly significant
antifeedant activity (P < 0.01) for two natural (1 and 3) and three semisynthetic limonoids (4, 8, and
10) was observed against S. frugiperda.
KEYWORDS: Citrus limon; semisynthetic limonoids; antifeedant activity; Spodoptera frugiperda
INTRODUCTION
Limonoids are the most representative class of secondary
metabolites in the order Rutales, which includes the families
Rutaceae, Meliaceae, and Simaroubaceae. They are tetranor-
triterpenoids with a 4,4,8-trimethyl-17-furanylsteroidal skeleton,
bearing several oxygenated functions. These secondary me-
tabolites exhibit a wide range of biological activities, including
insect antifeedant activity (1) as well as antibacterial, antifungal,
antiviral, and cytotoxic activity (2). Some have potent anti-insect
properties and thus show potential for development as natural
pesticides. The best known example is azadirachtin, a potent
antifeedant and insect growth-regulator isolated from the seed
of the Indian neem tree Azadirachta indica A. Juss (Meliaceae)
(3-5).
In the Citrus genus, limonoids are present as aglycons and
glucosides. To date, 36 aglycons and 17 glucosides have been
isolated from Citrus and its hybrids (6-9). Limonin (1), nomilin
(2), and obacunone (3) (Figure 1) are the main limonoid
Figure 1. Natural Citrus limonoids.
aglycons, whereas limonin 17â-D-glucopyranoside is the pre-
dominant glucoside. Limonoid aglycons are responsible for the
delayed bitterness of Citrus juices, due to hydrolysis of the
corresponding glucosides (10).
To date, research has focused on the medicinal and insect-
antifeedant properties of Citrus-derived limonoids. Their anti-
tumoral properties were studied using both in Vitro (11-16)
and in ViVo assays (17-19). The limonoids limonin (1), nomilin
(2), and obacunone (3) are reported to deter insect feeding (20-
† Dedicated to the memory of Prof. Guido Sodano.
* To whom correspondence should be addressed. Tel.: +39 0957212136.
Fax: +39 0957212141. E-mail: ruberto@issn.ct.cnr.it; ctringali@
dipchi.unict.it.
§ ICB-CNR.
# Universita` di Catania.
Royal Botanic Gardens.
× Formerly Istituto del C.N.R. per lo Studio delle Sostanze Naturali di
Interesse Alimentare e Chimico-Farmaceutico.
10.1021/jf020607u CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/09/2002

Citrus Limonoids as Antifeedant Agents
J. Agric. Food Chem., Vol. 50, No. 23, 2002 6767
a mixture of R- and â-isomers, which were separated by column
chromatography. The procedure is exemplified by the reduction of
limonin.
23). However, previous data on the antifeedant activity of Citrus
limonoids are difficult to compare, due to the heterogeneity of
protocols and species of insects used (1, 24-27), as well as the
fact that structure-activity studies are rare. Data from larvae
of Leptinotarsa decemlineata suggest that the functional groups
important for the antifeedant activity are the epoxy and furan
rings, while reduction of the carbonyl function at C-7 should
enhance activity (20-22, 28). In contrast, the complete hydro-
genation of the furan ring caused a loss of activity toward L.
decemlineata, even at higher doses. This latter evidence seems
in contrast with data reported for other limonoids (1).
These natural products are readily available from waste
products (seeds, peel, molasses, etc.) of the Citrus-processing
industry, and there is increasing commercial interest in their
exploitation. Brazil and the United States of America, the two
main Citrus-processing countries, produce ca. 2 × 106 tons of
waste products annually (28). In Southern Italy, one of the
principal Citrus-processing countries of the Mediterranean basin,
ca. 500 000 tons/year of waste are produced (30).
Limonin (1, 240 mg) was dissolved in CH2Cl2 (10 mL), and 1.6 mL
of a NaBH4 solution (110 mg in 5 mL of anhydrous CH3OH) was added.
The mixture was continuously stirred at -5 °C for 24 h. The reaction
was stopped by adding dilute HCl, and then the mixture was extracted
with CH2Cl2 and the residue purified by HPLC (Hypersil ODS column,
5 µm, 8.0 mm i.d.; H2O:CH3CN 6:4, 3 mL/min) to give limonol (15
mg) and epi-limonol (108 mg).
Limonol (4): [R]25D ) -57.28 (c ) 0.30 in CHCl3); HREIMS [M+]
472.5347 (calcd for C26H32O8 472.5353); UV λmax (in CH3CN) 210
nm (ꢀ ) 3800); IR νmax (in CH2Cl2) 3453, 1745 cm-1
.
Epi-limonol (5): [R]25 ) +7.16 (c ) 0.80 in CHCl3); HREIMS
D
[M+] 472.5344 (calcd for C26H32O8 472.5353); UV λmax (in CH3CN)
206 nm (ꢀ ) 5900); IR νmax (in CH2Cl2) 3450, 1750 cm-1
.
7R-Nomilol (13): amorphous solid prepared by reduction of nomilin;
[R]25D ) -8.79 (c ) 0.33 in CHCl3); HREIMS [M+] 516.5879 (calcd
for C28H36O9 516.5884); UV λmax (in CH3CN) 205 nm (ꢀ ) 6000); IR
νmax (in CH2Cl2) 3448, 1753 cm-1
.
7â-Nomilol (14): amorphous solid prepared by reduction of nomilin;
[R]25D ) +23.82 (c ) 0.78 in CHCl3); HREIMS [M+] 516.5877 (calcd
for C28H36O9 516.5884); UV λmax (in CH3CN) 206 nm (ꢀ ) 4590); IR
The present study investigates whether three limonoids (1-
3) that occur in Citrus-processing waste influence the feeding
behavior of larvae of Spodoptera frugiperda and whether their
activity can be enhanced by simple chemical transformations.
νmax (in CH2Cl2) 3450, 1750 cm-1
.
7R-Obacunol (19): [R]25D ) +43.83 (c ) 0.40 in CH2Cl2); HREIMS
[M+] 456.5351 (calcd for C26H32O7 456.5359); UV λmax (in CH2Cl2)
249 nm (ꢀ ) 340); IR νmax (in CHCl3) 3470, 1736, 1692 cm-1
.
MATERIALS AND METHODS
7â-Obacunol (20): amorphous; [R]25D ) +48.41 (c ) 0.76 in CH2-
Cl2); HREIMS [M+] 456.5350 (calcd for C26H32O7 456.5359); UV λmax
(in CH2Cl2) 229.5 nm (ꢀ ) 8812); IR νmax (in CHCl3) 3468, 1736,
General Experimental Procedures. High-resolution electron impact
mass spectra (HREIMS) were obtained on a Kratos M50S mass
spectrometer. UV and IR spectra were recorded on Beckman model
DU-65 and Perkin-Elmer model 684 spectrophotometers, respectively.
Optical rotations were measured at 25 °C on a JASCO 135 instrument.
1H NMR spectra were measured on a Varian INOVA operating at 500
MHz and a Bruker ARX-250 at 250 MHz, whereas 13C NMR spectra
were run at 63 MHz on a Bruker ARX-250 instrument. Multiplicities
of 13C signals were determined by distortionless enhancement of
polarization transfer (DEPT) experiments. Two-dimensional (2D) NMR
experiments, including correlation spectroscopy (COSY), nuclear
Overhauser enhancement spectroscopy (NOESY), rotational Overhauser
enhancement spectroscopy (ROESY), heteronuclear multiple-quantum
correlation (HMQC), and heteronuclear multiple-bond correlation
(HMBC), were performed using standard Varian and Bruker software.
All NMR experiments were carried out at constant temperature (298
K). Chemical shifts (δ) are indirectly referred to TMS using residual
solvent signal. High-performance liquid chromatography (HPLC) was
conducted on a Varian LC Star chromatographic system equipped with
a UV-vis detector (model 9050). Preparative liquid chromatography
(PLC) was conducted on a Jobin-Yvon instrument. Thin-layer chro-
matography (TLC) was carried out using precoated silica gel plates
(Merck).
1696 cm-1
.
Reaction of Limonin (1), Nomilin (2), and Obacunone (3) with
Hydroxylamine. Limonoids were reacted with hydroxylamine chlo-
ridrate as described below for 1.
Limonin (186 mg) and hydroxylamine hydrochloride (200 mg) were
dissolved in C5H5N (4 mL) and absolute C2H5OH (4 mL), and the
solution was refluxed for 4 h. The reaction was cooled and a saturated
solution of NaCl added. The mixture was then extracted with AcOEt
to obtain pure limonin-7-oxime (184 mg, 94% yield).
Limonin-7-oxime (8): amorphous solid; [R]25D ) -159.46 (c ) 1.72
in CH2Cl2); HREIMS [Μ+] 485.5334 (calcd for C26H31NO8 485.5341);
UV λmax (in CH2Cl2) 230 nm (ꢀ ) 525); IR νmax (in CHCl3) 3460,
1741, 1597 cm-1. NMR data are given in Table 1.
Nomilin-7-oxime (16): obtained from nomilin as amorphous solid
in 97% yield; [R]25D ) -141.62 (c ) 0.74 in CH2Cl2); HREIMS [M+]
529.5865 (calcd for C28H35NO9 529.5872); UV λmax (in CH2Cl2) 229.5
nm (ꢀ ) 1064); IR νmax (in CHCl3) 3464, 1748, 1597 cm-1. NMR data
are given in Table 1.
Obacunone-7-oxime (23): amorphous solid obtained from obacunone
in 93% yield; [R]25 ) -26.69 (c ) 0.34 in CH2Cl2); HREIMS [M+]
D
469.5339 (calcd for C26H31NO7 469.5347); UV λmax (in CH2Cl2) 248
nm (ꢀ ) 401); IR νmax (in CHCl3) 3468, 1736, 1698, 1602 cm-1. NMR
data are given in Table 1.
Plant Material. Seeds of Citrus limon were kindly supplied by Ditta
Lucchesi, Aci Catena (CT), Italy.
Extraction of Citrus Limonoids. Seeds were washed and then freeze
dried. Dry material (150 g) was ground and then Soxhlet extracted,
first with C6H14 (1 L) and then with CH2Cl2 (1 L). This latter extract
was evaporated to dryness, and the residue was dissolved in C6H14 and
kept at 5 °C for 24 h. The precipitate, consisting mainly of limonoids,
was collected by filtration and subjected to several column chroma-
tography separations on silica gel using increasing amounts of CH2Cl2
in Et2O as the eluant. Limonin (1), nomilin (2), and obacunone (3)
were obtained in that order and with the following yields: 0.24%,
0.20%, and 0.13%, respectively.
Preparation of Limonoid Derivatives. Limonoid derivatives were
prepared by conventional methods and purified by chromatography.
Known compounds were identified on the basis of comparison of
physical data with those reported in the literature (31-37). The
structures of compounds not previously described were deduced
essentially from MS and NMR data.
Reaction of Limonin (1), Nomilin (2), and Obacunone (3) with
Methylhydroxylamine. Limonoid methoximes were prepared by
reaction of the parent compounds with methylhydroxylamine, following
the procedure illustrated below for limonin.
Limonin (95 mg) was dissolved in CH2Cl2 (10 mL), and then
methylhydroxylamine (17 mg) dissolved in 2 mL of absolute C2H5OH
and 2 mL of C5H5N was added. The mixture was kept under reflux
and continuous stirring for 4 h. After the mixture cooled, a saturated
solution of NaCl was added and the organic matter extracted with
AcOEt. The residue was purified by preparative liquid chromatography
(PLC) (silica gel, CH2Cl2:Et2O 6:4) to obtain limonin-7-methoxime
(73.8 mg, 73% yield).
Limonin-7-methoxime (9): amorphous solid; [R]25
) -134.65 (c
D
+
) 0.945 in CH
2Cl2); HREIMS [M ] 499.5602 (calcd for C27H33NO8
499.5610); UV λ
max (in CH2Cl2) 240 nm (ꢀ ) 25); IR νmax (in CHCl3)
1730, 1633, 1600 cm-1
. NMR data are given in Table 2.
NaBH4 Reduction of Limonoids 1, 2, and 3. Limonin (1), nomilin
(2), and obacunone (3) were reduced with NaBH4 to give, in each case,
Nomilin-7-methoxime (18): amorphous solid prepared from nomilin
in 60% yield; [R]25 ) -87.21 (c ) 0.26 in CH2Cl2); HREIMS [M+]
D

6768 J. Agric. Food Chem., Vol. 50, No. 23, 2002
Ruberto et al.
Table 1. NMR Data of Oximes 8, 16, and 23a
limonin-7-oxime (8)
nomilin-7-oxime (16)
1H
obacunone-7-oxime (23)
position
1H
13C
13C
1H
13C
1
2
4.04 bs
2.95 dd (17, 3)
2.74 d (17)
79.6 d
36.3 t
5.02 d (7.0)
3.22 d (15)
3.08 dd (15, 7.2)
71.2 d
35.7 t
6.53 d (12)
5.91 d (12)
158.8 d
122.9 d
3
4
5
6
170.8 s
81.1 s
60.4 d
19.1 t
170.1 s
85.8 s
50.4 d
21.4 t
168.0 s
85.5 s
56.1 d
23.0 t
1.98 m
3.57 m
2.00 m
2.34 dd (14, 3.2)
3.74 dd (14, 3.2)
1.94 m
2.36 dd (14, 4)
3.44 dd (14.2, 4)
2.18 m
7
8
9
10
11
159.1 s
46.1 s
50.1 d
46.5 s
19.8 t
159.6 s
46.6 s
45.9 d
44.7 s
18.0 t
158.6 s
46.4 s
50.3 d
43.7 s
20.2 t
2.45 m
1.82 m
2.44 m
2.10 m
1.60 m
1.20 m
1.80 m
1.20 m
1.80 m
1.45 m
1.85 m
12
1.80 m
1.50 m
33.2 t
34.0 t
34.2 t
13
14
15
16
17
18
19
38.3 s
65.7 s
54.6 d
168.2 s
78.8 d
21.6 q
66.3 t
38.2 s
65.8 s
54.3 d
168.3 s
78.9 d
21.9 q
16.9 q
38.6 s
65.8 s
54.4 d
168.1 s
78.8 d
21.9 q
16.6 q
3.82 s
3.73 s
3.73 s
5.47 s
1.23 s
4.40 d (13)
4.20 d (13)
5.45 s
1.20 s
1.24 s
5.51 s
1.21 s
1.40 s
20
21
22
23
28
29
30
120.5 s
141.4 d
110.1 d
143.6 d
21.7 q
120.6 s
141.3 d
110.1 d
143.6 d
24.6 q
120.7 s
141.3 d
110.2 d
143.4 d
27.3 q
7.40 bs
6.34 bs
7.40 bs
1.19 s
1.33 s
0.96 s
7.42 bs
6.36 bs
7.39 bs
1.57 s
1.59 s
1.09 s
7.41 bs
6.36 bs
7.41 bs
1.54 s
1.53 s
1.13 s
30.7 q
18.7 q
34.1 q
17.8 q
32.7 q
17.5 q
CH CO
CH CO
3
169.8 s
21.2 q
3
2.03 s
a
1H NMR (250 MHz) and 13C NMR (63 MHz) spectra were run in CDCl3 (ppm from TMS). 13C multiplicities were obtained by DEPT experiments.
543.6135 (calcd for C29H37NO9 543.6141); UV λmax (in CHCl3) 240
nm (ꢀ ) 633); IR νmax (in CHCl3) 1741, 1650, 1595 cm-1. NMR data
are given in Table 2.
7R-Obacunyl acetate (21): obtained from 7R-obacunol in 84% yield;
[R]25D ) +22.97 (c ) 0.18 in CH2Cl2); HREIMS [M+] 498.5727 (calcd
for C28H34O8 498.5732); UV λmax (in CH2Cl2) 240 nm (ꢀ ) 808); IR
νmax (in CHCl3) 1742 cm-1
.
Obacunone-7-methoxime (25): obtained from obacunone as amor-
phous solid in 68% yield; [R]25 ) -27.87 (c ) 1.94 in CH2Cl2);
D
7â-Obacunyl acetate (22): amorphous powder by acetylation of 7-â-
HREIMS [M+] 483.5610 (calcd for C27H35NO7 483.5616); UV λmax
(in CH2Cl2) 244 nm (ꢀ ) 6047); IR νmax (in CHCl3) 1733, 1645, 1607
cm-1. NMR data are given in Table 2.
obacunol in 61% yield; [R]25 ) +30.59 (c ) 0.09 in CH2Cl2);
D
HREIMS [M+] 498.5726 (calcd for C28H34O8 498.5732); UV λmax (in
CH2Cl2) 225.5 nm (ꢀ ) 1547); IR νmax (in CHCl3) 1736, 1701 cm-1
.
Acetylation. The starting compound (usually 10 mg or more) was
treated with C5H5N (1 mL) and (CH3CO)2O (1 mL). The mixtures were
continuously stirred for 24 h at room temperature, and then the solvents
were removed and the residue was purified by chromatography.
Limonyl acetate (6): prepared by acetylation of limonol in 90% yield;
[R]25D ) -54.31 (c ) 0.23 in CH2Cl2); HREIMS [M+] 514.5719 (calcd
for C28H34O9 514.5726); UV λmax (in CH2Cl2) 232 nm (ꢀ ) 544); IR
Obacunone-7-oxime acetate (24): amorphous solid from obacunone-
7-oxime in 98% yield; [R]25D ) -21.85 (c ) 0.41 in CH2Cl2); HREIMS
[M+] 511.5712 (calcd for C28H35NO8 511.5720); UV λmax (in CH2Cl2)
248 nm (ꢀ ) 376); IR νmax (in CHCl3) 1747, 1702, 1601 cm-1
.
Preparation of Perhydrolimonin (11) and Epi-perhydrolimonin
(12). Limonin (100 mg) and palladium (10 wt %) on activated charcoal
were suspended in CH2Cl2 (10 mL) and subjected to H2 pressure (2
atm) for 48 h. After filtration, the resulting mixture was purified by
PLC (silica gel, gradient of tetrahydrofuran in cyclohexane from 40%
to 85%) to obtain 32 mg (32% yield) of perhydrolimonin and 15.5 mg
(16% yield) of epi-perhydrolimonin.
νmax (in CH2Cl2) 1744 cm-1
.
Epi-limonyl acetate (7): obtained as amorphous solid by acetylation
of epi-limonol; [R]25D ) +10.00 (c ) 0.07 in CH2Cl2); HREIMS [M+]
514.5718 (calcd for C28H34O9 514.5726); UV λmax (in CH2Cl2) 230 nm
(ꢀ ) 1370); IR νmax (in CH2Cl2) 1744 cm-1
.
Perhydrolimonin: (11): amorphous powder; [R]25 ) -116.35 (c
D
Limonin-7-oxime acetate (10): obtained as amorphous solid from
) 0.85 in CH2Cl2); HREIMS [M+] 474.5505 (calcd for C26H34O8
474.5512); UV λmax (in CHCl3) 240 nm (ꢀ ) 1887). NMR data are
given in Table 3.
limonin-7-oxime in 95% yield; [R]25 ) -116.89 (c ) 1.59 in CH2-
D
Cl2); HREIMS [M+] 527.5708 (calcd for C28H33NO9 527.5714); UV
λmax (in CH2Cl2) 248 nm (ꢀ ) 99); IR νmax (in CHCl3) 1747, 1601
Epi-perhydrolimonin (12): amorphous powder; [R]25 ) -120.26
cm-1
.
D
(c ) 1.36 in CH2Cl2); HREIMS [M+] 474.5507 (calcd for C26H34O8
474.5512); UV λmax (in CHCl3) 282 nm (ꢀ ) 126). NMR data are given
in Table 3.
7â-Nomilyl acetate (15): amorphous solid prepared from 7â-nomilol
in 80% yield; [R]25 ) +25.88 (c ) 0.68 in CH2Cl2); HREIMS [M+]
D
558.6249 (calcd for C30H38O10 558.6257); UV λmax (in CH2Cl2) 227
nm (ꢀ ) 298); IR νmax (in CHCl3) 1752 cm-1
.
Preparation of 1,2-Didehydroobacunone (26). Obacunone (18 mg)
and palladium (10 wt %) on activated charcoal were suspended in CH2-
Cl2 (3 mL) and subjected to H2 pressure (1 atm) for 20 min. After
filtration, the mixture was subjected to PLC (silica gel, CH2Cl2 as the
eluant) to yield 1,2-didehydroobacunone (7 mg, 40% yield).
Nomilin-7-oxime acetate (17): prepared in 98% yield as amorphous
solid from nomilin-7-oxime; [R]25D ) -102.78 (c ) 0.40 in CH2Cl2);
HREIMS [M+] 571.4238 (calcd for C30H37NO10 571.6245); UV λmax
(in CH2Cl2) 240.5 nm (ꢀ ) 422); IR νmax (in CHCl3) 1732, 1601 cm-1
.

Citrus Limonoids as Antifeedant Agents
J. Agric. Food Chem., Vol. 50, No. 23, 2002 6769
obacunone-7-methoxime (25)
Table 2. NMR Data of Methoximes 9, 18, and 25a
limonin-7-methoxime (9)
nomilin-7-methoxime (18)
position
1H
13C
1H
13C
1H
13C
1
2
3.96 bs
2.95 dd (16, 4)
2.66 dd (16, 1.5)
79.2 d
35.8 t
5.08 d (7)
3.18 dd (16, 1.5)
3.04 dd (16, 7)
71.3 d
35.7 t
6.50 d (12)
5.91 d (12)
158.3 d
123.0 d
3
4
5
6
169.3 s
80.4 s
60.4 d
19.4 t
169.7s
85.4 s
50.5 d
21.2 t
167.6 s
85.2 s
56.3 d
23.8 t
1.93 m
3.43 d (12)
1.95 d (12)
2.29 dd (12, 3.5)
3.60 dd (17, 3.5)
1.92 m
2.30 dd (12.5, 4)
3.21 dd (15, 4)
2.15 m
7
8
9
10
11
157.7 s
46.1 s
49.8 d
45.8 s
19.5 t
158.3 s
46.6 s
45.8 d
44.7 s
19.9 t
158.5s
46.4 s
50.2 d
43.6 s
20.2 t
2.39 d (10)
1.74 m
2.39 d (9)
1.95 m
1.52 m
1.20 m
1.80 m
1.17 m
1.80 m
1.45 m
1.87 m
12
1.50 m
1.30 m
33.0 t
34.0 t
34.0 t
13
14
15
16
17
18
19
37.9 s
64.8 s
54.2 d
166.9 s
78.2 d
21.3 q
65.7 t
38.8 s
65.6 s
54.4 d
169.6 s
78.8 d
22.0 q
17.0 q
38.4 s
65.6 s
54.4 d
167.6 s
78.8 d
22.1 q
16.6 q
3.82 s
3.72 s
3.70 s
5.46 s
1.24 s
4.66 d (13)
4.32 d (13)
5.46 s
1.20 s
1.21 s
5.50 s
1.24 s
1.39 s
20
21
22
23
28
29
30
OCH
CH CO
CH CO
120.2 s
140.9 d
109.7 d
143.1 d
21.4 q
30.2 q
18.0 q
62.1 q
120.7 s
141.3 d
110.1 d
143.6 d
24.3 q
18.1 q
17.7 q
62.4 q
169.6 s
21.2 q
120.8 s
141.3 d
110.2 d
143.5 d
17.3 q
32.7 q
17.4 q
62.3 q
7.41 bs
6.35 bs
7.39 bs
1.10 s
1.30 s
0.94 s
3.85 s
7.39 bs
6.34 bs
7.39 bs
1.52 s
1.54 s
1.12 s
3.84 s
7.42 bs
6.38 bs
7.42 bs
1.51 s
1.50 s
1.11 s
3.82 s
3
3
2.00 s
3
a
1H NMR (250 MHz) and 13C NMR (63 MHz) spectra were run in CDCl3 (ppm from TMS). 13C multiplicities were obtained by DEPT experiments.
1,2-Didehydroobacunone (26): amorphous solid; [R]25D ) -79.76
(c ) 0.09 in CH2Cl2); HREIMS [M+] 456.5351 (calcd for C26H32O7
456.5359); UV λmax (in CH2Cl2) 229.5 nm (ꢀ ) 1234); IR νmax (in
CHCl3) 1720, 1718 cm-1. NMR data are given in Table 3.
scale applications. The conversions focused mainly on modifica-
tion of the carbonyl function at C-7, namely reduction to
epimeric alcohols, conversion to the corresponding oxime and
methoxime, and preparation of their acetates. Catalytic hydro-
genation of the furan ring was also carried out, to verify its
role in the antifeedant activity of limonin.
Antifeedant Activity Bioassay. Spodoptera frugiperda larvae were
reared on a wheatgerm-based diet (38). Final stadium larvae aged 24-
36 h were used in the binary choice bioassay. They were deprived of
food for 2 h before being placed individually into Petri dishes, which
contained control and treatment glass-fiber disks (Whatman GF/A, 2.1
cm diameter). All disks were treated with the phagostimulant sucrose
(100 µL of a 0.05 M solution) and then left to dry. An aliquot (100
µL) of one of the test compounds in (CH3)2CO was applied to 10
treatment disks. Acetone alone (100 µL) was applied to the control
disks. The disks were then dried and weighed prior to bioassay. The
bioassays were observed every 30 min, and each experiment was
terminated after 8 h, the time before larvae had eaten 50% of either
control or treatment disks. Larvae were then removed from the Petri
dishes. The disks were allowed to dry and then reweighed. The amount
of control (C) and treatment (T) disk eaten in each Petri dish was used
to calculate the feeding index, [(C - T)/(C + T)]%. The potency of
each compound was analyzed using the Wilcoxon matched-pairs test,
and the Mann-Whitney U test was used to determine whether the
semisynthetic derivatives were more potent than the parent natural
compounds (39).
Limonin (1) Derivatives. The reduction of the carbonyl
function at C-7 in 1 was carried out through mild NaBH4
treatment, as previously described for various limonoids (31,
36, 37). The two epimeric alcohols limonol (4) and epi-limonol
(5) (Figure 2) were obtained with a 1:30 (R:â) ratio. These
results could be explained by the strong steric hindrance on the
two faces of the carbonyl function, due to the spatial location
of the A and A′ rings of limonin. A number of 7R-hydroxy
natural limonoids are known; among them, limonol (4) is present
in seeds of Citrus paradisi (31). Compounds 4 and 5 were
subjected to acetylation, which afforded limonyl acetate (6) and
epi-limonyl acetate (7), respectively (Figure 2). Limonyl acetate
(6) is known as a natural product, as it occurs in fruit of a
Citrus-Poncirus hybrid (31).
To verify the effect on the antifeedant activity of a further
increase of lipophilicity at C-7, attempts were made to obtain
palmitate, valerate, or benzoate esters, but all these reactions
showed negligible yields, probably owing to the steric hindrance
of the 7-hydroxy group.
RESULTS AND DISCUSSION
A range of three Citrus-derived limonoids (1-3) and 23
semisynthetic derivatives (4-26) were evaluated for antifeedant
activity against larvae of the fall armyworm, S. frugiperda. The
derivatives were obtained through simple chemical modifications
of the natural limonoids and are suitable for possible large-
The preparation of limonin-7-oxime (8, Figure 2) from 1 was
carried out as a slight modification of a previously reported
protocol (37), which enhanced the yield and substantially
reduced the reaction time. The product was identified by its

6770 J. Agric. Food Chem., Vol. 50, No. 23, 2002
Ruberto et al.
Table 3. NMR Data of Hydrogenated Derivatives 11, 12, and 26a
perhydrolimonin (11)
epi-perhydrolimonin (12)
1,2-didehydrobacunone (26)
1H
position
1
1H
13C
1H
13C
13C
4.08 bs
79.1 d
4.08 bs
79.5 d
1.96 m
1.48 m
2.65 m
33.2 t
2
3.00 dd (15, 4)
2.70 dd (15, 2)
35.7 t
3.01 dd (15, 4)
2.71 dd (15, 1.5)
36.1 t
39.8 t
3
4
5
6
168.9s
80.2 s
60.9 d
36.4 t
169.6 s
80.7 s
61.2 d
36.8 t
166.6 s
84.4 s
54.6 d
37.6 t
2.22 dd (15, 3)
2.83 dd (15, 14.5)
2.45 dd (15, 3)
2.23 dd (15.7, 3)
2.85 dd (15, 14.5)
2.46 dd (15, 3.5)
2.32 dd (13.5, 5)
2.94 dd (13.5, 13.5)
2.42 dd (13.5, 5)
7
8
9
206.1 s
51.6 s
48.2 d
46.0 s
19.2 t
206.5 s
51.9 s
48.5 d
46.4 s
19.5 t
207.4 s
51.3 s
53.0 d
40.2 s
18.6 t
2.53 dd (10, 3)
2.55 dd (15, 2)
2.04 m
10
11
12
13
14
15
16
17
18
19
1.79 m
1.61 m
1.80 m
1.75 m
1.80 m
1.40 m
30.1 t
30.6 t
32.3 t
37.7 s
64.9 s
53.1 d
166.4 s
82.0 d
21.5 q
65.4 t
38.2 s
65.6 s
53.6 d
166.8 s
84.3 d
21.7 q
65.8 t
40.1 s
65.5 s
53.6 d
166.9 s
78.5 d
19.4 q
21.6 q
3.80 s
3.83 s
3.66 s
4.32 d (6)
1.21 s
4.77 d (13)
4.43 d (13)
2.35 m
3.86 m
3.38 m
2.06 m
1.94 m
4.45 d (7)
1.19 s
5.45 s
1.24 s
1.21 s
4.77 d (13)
4.44 d (13)
2.39 m
4.02 m
3.62 m
1.94 m
1.74 m
3.86 m
3.75 m
1.02 s
20
21
39.4 d
70.5 t
39.6 d
70.3 t
120.7 s
141.4 d
7.41 bs
6.36 bs
7.41 bs
22
23
31.7 t
67.9 t
31.3 t
68.1 t
110.2 d
143.5 d
3.82 m
28
29
30
1.00 s
1.30 s
1.18 s
17.2 q
28.8 q
21.3 q
17.6 q
29.5 q
21.8 q
1.12 s
1.51 s
1.48 s
18.9 q
30.7 q
27.2 q
1.30 s
1.19 s
a
1H NMR (250 MHz) and 13C NMR (63 MHz) spectra were run in CDCl3 (ppm from TMS). 13C multiplicities were obtained by DEPT experiments.
influenced by the dNsOH group. In principle, the formation
of the oxime should afford two geometric isomers at the CdN
double bond. We could not observe the formation of a cis/trans
mixture; indeed, the analysis of NMR spectra showed signals
associated with a single product.
To distinguish between E and Z configuration, the limonin-
7-oxime (8) was subjected to methylation with CH3I to obtain
the corresponding limonin-7-methoxime (9, Figure 2). The
1
analysis of H and 13C NMR spectra (Table 2) confirmed that
the expected derivative has been obtained, showing the new
methoxy signals at δ 3.85 (1H) and δ 62.4 (13C). A 2D NMR
1
study allowed complete assignment of both H and 13C NMR
spectra (Table 2). A ROESY experiment on this compound
showed a strong correlation between the methoxy group at δ
3.85 and the signal at δ 3.43, assigned to one of the methylene
protons in C-6. This is consistent only with an E configuration
for the CdN double bond.
Furthermore, applying the same methodology to obtain the
oxime (8), the reaction of limonin (1) with methylhydroxylamine
allowed us to obtain directly the limonin-7-methoxime (9),
whose spectral features were perfectly coincident with those of
the methoxime obtained from the corresponding oxime. In both
cases, a strong driving force ascribable to a considerable steric
hindrance of one side of the CdO double bond of limonin (as
well as of the other limonoids) led to the exclusive formation
of only one geometric isomer.
Figure 2. Limonin derivatives.
As previously described for limonols, various esterifications
of the oxime function have been attempted, but only the acetate
was obtained in good yield. Limonin-7-oxime acetate (10,
Figure 2) was purified and fully characterized.
spectroscopic features. In particular, the NMR data (Table 1)
showed the expected shifts of the proton and carbon signals

Citrus Limonoids as Antifeedant Agents
J. Agric. Food Chem., Vol. 50, No. 23, 2002 6771
Figure 3. Nomilin derivatives.
Catalytic hydrogenation of 1 afforded, as expected, a mixture
of C-20 epimeric perhydro derivatives, namely perhydrolimonin
(11) and epi-perhydrolimonin (12) (Figure 2). Although dif-
ficult, the separation of these epimers was achieved through
HPLC. Compound 11 showed signals easily attributable to the
Figure 4. Obacunone derivatives.
1
tetrahydrofuran ring in the aliphatic region of the H and 13C
spectral analysis. Nomilin-7-oxime (16, Figure 3) was obtained
by applying the procedure used for limonin. Spectral charac-
terization of this new product confirmed its structure (Table
1). In analogy to the reaction with limonin, the formation of a
cis/trans mixture was not observed. Acetylation of 16 gave the
corresponding nomilin-7-oxime acetate (17, Figure 3), in
agreement with spectral analysis.
The procedure used to obtain 9 allowed us to prepare nomilin-
7-methoxime (18, Figure 3) with 60% yield, but a further
product could be isolated from the reaction mixture. The main
methoxime 18 was fully characterized through homo- and
heteronuclear 2D NMR experiments. Analogously, MS and
NMR studies established the minor product as obacunon-7-
methoxime (25, Figure 4), thus confirming the easy conversion
of nomilin to obacunone.
Obacunone (3) Derivatives. Reduction of C-7 carbonyl in
3, carried out in analogy to the reductions of 1 and 2, gave the
expected alcohols, 7R-obacunol (19) and 7â-obacunol (20)
(Figure 4), with an R:â ratio ca. 1:1. The physical data of 19
were in agreement with those reported for the natural 7R-
obacunol previously isolated from C. paradisi seeds (31).
Standard acetylation of both 19 and 20 afforded, respectively,
7R-obacunyl acetate (21) and 7â-obacunyl acetate (22) (Figure
4).
The procedures reported above for the preparation of oximes
were applied to obacunone, and obacunone-7-oxime (23, Figure
4) was obtained and submitted to spectral analysis. Compound
23 showed NMR data (Table 1) compatible with the expected
structure. Acetylation of 23 afforded obacunone-7-oxime acetate
(24, Figure 4). The reaction of 3 with methylhydroxylamine
yielded obacunone-7-methoxime (25, Figure 4), characterized
on the basis of NMR study (Table 2). Both 23 and 25 were
obtained only as the trans geometrical isomer at the CdN double
bond, as expected from the above-reported preparations of
oximes and methoximes from 1 and 2.
NMR spectra (Table 3). The reported assignments were based
on the integrated analysis of homo- and heteronuclear 2D NMR
spectra, establishing the structure of 11, apart from the C-20
configuration. Unambiguous determination of the relative ster-
eochemistry at C-20 in 11 and 12 was not a trivial task, due to
the conformational mobility of the tetrahydrofuran ring with
respect to the core of the limonin skeleton. Thus, MM2 (87)
calculations were used to minimize the structures of both
epimers. The torsional energy profile of the H17-C17-C20-
H20 bond was analyzed by using the dihedral driver tool of
Macromodel (Version 7.2), setting a 0-360° interval and a 5°
increment. From the results of this study, omitted here for the
sake of brevity, we obtained for each compound a couple of
very similar energy minimum structures, assumed as preferred
conformations (Supporting Information). Two NMR ROESY
experiments were carried out on 11 and 12, and the resulting
data (Supporting Information) were compared with the above-
determined stereostructures, checking selected internuclear
distances for each couple of conformers. This study allowed
unambiguous assignment of C-20 relative configuration for 11
and 12. In particular, a strong ROE correlation between the H2-
12 signal at δ 1.61 and the H2-21 methylene at δ 3.86 and 3.31
established the relative configuration of the C-20 position, as
reported in structure 11. In contrast to 11, and in agreement
with the epimeric C-20 configuration, 12 showed a correlation
of the methyl group at C-18 (δ 1.19) with one of the H2-22
signals (δ 1.94).
Nomilin (2) Derivatives. Reactions carried out on 2 and some
of its derivatives were adversely affected by the low stability
of nomilin. A low total conversion was observed during the
NaBH4 reduction of 2, affording two epimeric 7-hydroxyderiva-
tives, namely 7R-nomilol (13) and 7â-nomilol (14) (Figure 3)
in an R:â ratio of ca. 1:2. The acetylation of 14 afforded the
expected 7â-nomilyl acetate (15, Figure 3), as confirmed by
spectral analysis and comparison with data reported for the
natural 7â-nomilyl acetate isolated from Dictamnus albus (31).
When submitted to standard acetylation conditions, the epimer
13 gave 7R-obacunyl acetate (21, Figure 4), as confirmed by
Catalytic hydrogenation of 3 afforded, similarly to the
hydrogenation of 2, an unresolvable mixture of two C-20
epimers. After spectroscopic examination, this mixture revealed

6772 J. Agric. Food Chem., Vol. 50, No. 23, 2002
Ruberto et al.
be due to primary activity or via some secondary feedback
associated with the toxicity of the test compound.
Table 4. Antifeedant Activity of Citrus Limonoids and Their
Semisynthetic Derivatives against Fifth Instar Larvae Spodoptera
frugiperdaa
In our experiments, limonol (4) was active but less so than
limonin, whereas limonol decreased the consumption and growth
rates of L. decemlineata more than limonin (24). In fact, Liu
and co-workers (25) showed that the derivatives of limonin with
a more polar function at either C-7 or C-6 depressed feeding of
L. decemlineata. In addition, we found that limonol (4) was
more active toward S. frugiperda than its epimer epi-limonol
(5), whereas epi-limonol has been reported to be more active
than limonol against L. decemlineata (21). Limonin-7-oxime
(8) was as active as limonol, whereas acetates 6, 7, and 10 are
less active than the parent compounds. Interestingly, limonin-
7-methoxime (9) shows the highest activity (FI ) 76%) among
all the semisynthetic derivatives tested. Both the epimers 11
and 12, with hydrogenation of the furan moiety, have antifeedant
activity against S. frugiperda. This is in contrast to data reported
for L. decemlineata, that showed that perhydro derivatives are
devoid of any antifeedant activity (21).
compound
feeding indexb (sem)
Limonin and Derivatives
limonin (1)
limonol (4)
87 (5.7)**a
68 (4.0)**b
56 (4.8)*c
45 (4.8)*c
26 (13.8)c
68 (5.8)**b
45 (2.5)*c
76 (12.7)**a
29 (4.8)*c
45 (7.8)*c
epi-limonol (5)
limonyl acetate (6)
epi-limonyl acetate (7)
limonin-7-oxime (8)
limonin-7-oxime acetate (10)
limonin-7-methoxime (9)
perhydrolimonin (11)
epi-perhydrolimonin (12)
Nomilin and Derivatives
nomilin (2)
56 (4.9)*a
46 (12.8)*a
35 (8.7)*b
46 (11.8)*a
35 (21.9)*b
41 (5.4)*b
38 (11.7)*b
7-R-nomilol (13)
7-â-nomilol (14)
7-â-nomilyl acetate (15)
nomilin-7-oxime (16)
nomilin-7-oxime acetate (17)
nomilin-7-methoxime (18)
Nomilin (2) displays the lowest activity among the three main
natural Citrus limonoids (Table 4). The importance of the
configuration and composition of the substituent at C-7 is
unclear. The reduction of the C-7 carbonyl function did not
always result in a decrease in activity, as 7-nomilol (13) and
7-nomilyl acetate (14) are as potent as nomilin, whereas the
other derivatives are less active. It appears that, with nomilin,
modification of the functional group at C-7 did not influence
the antifeedant activity of the compound as much as occurred
with the derivatives of either limonin (1) or obacunone (3).
Obacunone and Derivatives
obacunone (3)
7-R-obacunol (19)
7-â-obacunol (20)
7-R-obacunyl acetate (21)
7-â-obacunyl acetate (22)
obacunone-7-oxime (23)
obacunone-7-oxime acetate (24)
obacunone-7-methoxime(25)
1,2-didehydro-obacunone (26)
68 (4.8)**a
53 (7.9)*b
35 (3.8)*c
24 (12.8)c
26 (16.4)c
25 (12.5)c
21 (10.2)c
29 (8.8)*c
53 (12.9)*b
Obacunone (3) shows significant antifeedant activity toward
S. frugiperda, but all the derivatives were less active (Table
4). Reduction of the C-7 carbonyl function caused a decrease
in activity, and the configuration of the hydroxy group at C-7
influenced activity, as the 7R epimer (19) was more active (FI
) 53%) than the 7â (20) (FI ) 35%). Acetylation of the polar
function at C-7 in 21, 22, and 24 resulted in a loss of activity.
Interestingly, the reduction in activity associated with hydro-
genation of the furan ring is not observed in the partially
hydrogenated 1,2-didehydroobacunone (26), that is as active as
7R-obacunol (19).
These data represent the results from the first homogeneous
structure-activity study into the antifeedant activity of the three
main Citrus limonoids, 1-3, and their semisynthetic derivatives.
Limonin (1), nomilin (2), and obacunone (3) displayed anti-
feedant activity against S. frugiperda, thus confirming their
probable role as chemical defense agents in Citrus-herbivore
interactions. In comparison with previously reported data, these
results show that different insect species may have different
responses to structural modifications (1, 21, 25), in which case
structure-activity relations may be highly species-specific.
Thus, the assessment that the introduction of polar groups in
the limonoid skeleton at C-7 would result in an enhancement
of antifeedant activity was not confirmed for S. frugiperda, as
indicated for instance by comparison of FI values for limonin
(1), limonin-7-oxime (8), and limonin-7-methoxime (9). The
last one was the most active semisynthetic compound.
a Expressed as feeding index, [(C − T)/(C + T)]%, with standard error of the
mean (SEM) in parentheses. All compounds were tested at 100 ppm and n ) 10.
b Significance of feeding index: **, P < 0.01 and *, P < 0.05, Wilcoxon matched-
pairs test. Within each group of compounds the values of the feeding indexes
have been compared with that of the corresponding natural compound; values
followed by a similar letter do not differ; a > b ) P < 0.05, a > c ) P < 0.01,
Mann−Whitney U test.
that the 1,2 double bond had also been saturated. Using milder
hydrogenation conditions, it was possible to get the 1,2-
didehydroobacunone (26, Figure 4), as confirmed by spectral
analysis. In fact, the 1H NMR spectrum of 26 (Table 3) showed
the absence of the 1,2 double bond signals and the presence of
new signals accounting for two methylenes groups, resonating
at δ 1.96-1.48 (H2-1) and 2.65 (H2-2). Analogously, in the 13
C
NMR spectrum (Table 3) two CH2 signals were observed at δ
33.2 and 39.8.
Antifeedant Activity of Natural and Semisynthetic Li-
monoids. The antifeedant activities of the natural Citrus
limonoids 1-3 and their semisynthetic derivatives 4-26 at 100
ppm are summarized in Table 4. The natural limonoids showed
potent antifeedant activity, with limonin (1) being the most
active against S. frugiperda. This contrasts with the data for S.
frugiperda of Champagne et al. (1), who reported the ED50
values for limonin, nomilin, and obacunone as 756, 72, and 70
ppm, respectively. However, the results are not directly com-
parable, as we have used an inert substrate treated with sucrose
as a test compound to evaluate the activity of the compounds,
whereas other authors have applied or incorporated the com-
pounds into insect food. Our short-term assay was designed to
evaluate whether plant-derived compounds play a primary role
in modulating feeding behavior. Activity in these assays often
correlates with neural input from gustatory sensilla. When
compounds are incorporated into food, the behavior output could
In conclusion, we obtained significant antifeedant activity (P
< 0.01) for two natural (1 and 3) and three semisynthetic
limonoids (4, 8, and 9) against the commercially important pest,
S. frugiperda. Although the antifeedant activity of natural Citrus
limonoids could not be enhanced through chemical modification,
the derivative might exhibit potent antifeedant activity against
other pests. The limonoids justify further exploitation as pest

Citrus Limonoids as Antifeedant Agents
J. Agric. Food Chem., Vol. 50, No. 23, 2002 6773
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J. M.; Hasegawa, S.; Lam, L. K. T.; Sunahura, G. I. Inhibition
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Hasegawa, S. Citrus limonoids: Increasing importance as
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agriculture and foods; Berhow, M. A., Hasegawa, S., Manners,
G. D., Eds.; ACS Symposium Series 758; American Chemical
Society: Washington, DC, 2000; pp 132-144.
(16) Guthrie, N.; Morley, K.; Hasegawa, S.; Manners, G. D.;
Vandenberg, T. Inhibition of human breast cancer cells by citrus
limonoids. In Citrus limonoidssFunctional chemicals in agri-
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control agents, especially as they are readily available as waste
products of the Citrus industry.
ACKNOWLEDGMENT
We thank Dr. Angela Patti (ICB-CNR, Valverde) for carrying
out the MM2 calculations and for her helpful suggestions. We
also thank Dr. Agata Scuderi and Dr. Stefania Sapienza for their
skilful technical assistance and Mrs. Concetta Rocco (ICB-CNR,
Valverde) for NMR measurements.
Supporting Information Available: Summary list of natural
Citrus limonoids and their semisynthetic derivatives; NMR data
of 5, 7, 10, 13, 14, 15, 17, 20, 22, 24; ROESY data of 11 and
12; energy profile as a function of torsion angle (H17-C17-
C20-H20); and stereoviews of the lowest-energy conformers for
11 and 12. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Received for review May 29, 2002. Revised manuscript received August
8, 2002. Accepted August 8, 2002. This work was financially supported
by the Regione Siciliana, Assessorato Beni Culturali e Ambientali
(Grant No. 77504-1999), and by Ministero della Pubblica Istruzione,
Universita` e Ricerca Scientifica (PRIN, Rome, Italy). Consiglio Nazio-
nale delle Ricerche (C.N.R.-Rome) and University of Catania (Progetti
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