Tobacco Caterpillar Antifeedent from the Gotti Stem Wood Triterpene Betulinic Acid

Article abstract of DOI:10.1021/jf970768b

Betulinic acid (I), a pentacyclic triterpene, on derivatization gives six compounds: 3β-hydroxylup-20(29)-en-28 oic acid methyl ester (II), 3β-acetoxylup-20(29)-en-28-oic acid (III), 3β-allyloxylup-20(29)-en-28-oic acid (IV), 3β-p-methylcinnamatoxylup-20(29)-en-28-oic acid (V), 3β-p-methoxycinnamatoxylup-20(29)-en-28-oic acid (VI), and 3β-tri-O-methylgallotoxylup-20(29)-en-28-oic acid (VII). Their antifeedent activity against the agricultural pest tobacco caterpillar larvae (Spodoptera litura F) in a no-choice laboratory study showed the active compounds are V, VI, and VII.

Full text of DOI:10.1021/jf970768b

J. Agric. Food Chem. 1998, 46, 2797−2799
2797
Toba cco Ca ter p illa r An tifeed en t fr om th e Gotti Stem Wood
Tr iter p en e Betu lin ic Acid
S. G. J agadeesh, G. L. David Krupadanam, and G. Srimannarayana*
Department of Chemistry, Osmania University, Hyderabad 500 007, India
S. Samba Murthy, Amarjit Kaur, and S. S. Raja
Department of Zoology, Nizam College, Osmania University, Hyderabad 500 001, India
Betulinic acid (I), a pentacyclic triterpene, on derivatization gives six compounds: 3â-hydroxylup-
20(29)-en-28 oic acid methyl ester (II), 3â-acetoxylup-20(29)-en-28-oic acid (III), 3â-allyloxylup-
20(29)-en-28-oic acid (IV), 3â-p-methylcinnamatoxylup-20(29)-en-28-oic acid (V), 3â-p-methoxy-
cinnamatoxylup-20(29)-en-28-oic acid (VI), and 3â-tri-O-methylgallotoxylup-20(29)-en-28-oic acid
(VII). Their antifeedent activity against the agricultural pest tobacco caterpillar larvae (Spodoptera
litura F) in a no-choice laboratory study showed the active compounds are V, VI, and VII.
Keyw or d s: Antifeedent; betulinic acid derivatives; tobacco caterpillar; Spodoptera litura (F)
There is a revival of interest in the isolation of
insecticidally active compounds from indigenous plants
(Benerji et al., 1982). Indeed, to our knowledge neem
(Azadirachta indica) seed extract is the only antifeedent
currently marketed. Plant products have distinct ad-
vantage over commercial organic synthetic insecticides.
Some of these compounds have multiple types of activity
including toxicity (Zehnder et al., 1988), growth regula-
tion activity (Liu et al., 1991), and oviposition deterrence
(Murray et al., 1993; Liu et al., 1989). They also
enhance the activity of other insect control agents such
as Bacillus thuringiensis (Salama and Sharby, 1988).
Antifeedents of natural origin or their derivatives are
specific to target (Benerji et al., 1982; Liu et al., 1989,
1991) and generally do not cause toxicity or residue
problems.
Isolation of pure naturally occurring antifeedents is
tedious and expensive, and these substances are seldom
available from natural sources in the quantities neces-
sary for agricultural applications. Furthermore, the
total synthesis of these compounds on a large scale is
generally econmically impractical. The problem of
economically viable production of antifeedents might be
solved either by simple synthetic transformation, de-
rivatization of easily isolated natural products (Bently
et al., 1995), or by synthesis of natural product model
compounds having structures that mimic the essential
part of the more active and complex natural products.
Ley et al. (1987) have reported the synthesis of model
antifeedents based on azdirachtin and have also re-
ported models based on limonin (Bentley et al., 1990).
reported (Macias et al., 1994) to have allelopathic
activity on monocotyledon species Hordeum vulgara and
Triticum aestivum and dicotyledon species Lactuca
sativa and Lepidium sativum. Here we report the
simple preparation from betulinic acid of antifeedents
for the tobacco caterpillar (Spodoptera litura F).
MATERIALS AND METHODS
NMR spectra were obtained on a Varian Gemini 200 MHz
system and FT-IR spectra on a Perkin-Elmer 1605 system.
Mass spectra were obtained on a Perkin-Elmer Hitachi RMU-
6L and MS-30. Uncorrected melting points were determined
in a sulfuric acid bath. Plant voucher (No. GSN/23/94) was
kept in the Department of Botany, Osmania University,
Hyderabad.
P r ep a r a tion of Ma ter ia ls. Isolation of Betulinic Acid (I).
Betulinic acid was isolated from Zizyphus xylopyrus collected
from the Mannnnoor forest, Mahaboobnager District, AP.
Pulverized, dry stem wood (6 kg) was extracted overnight with
methanol on a Soxhlet apparatus. The extract was evaporated
to dryness under vacuum to give 110 g of crude, which was
purified by column chromatography on silica gel (100-200
mesh) eluted with 5% EtOAc/benzene. One hundred twenty
fractions, each 500 mL, were collected. Fractions 42-65
yielded betulinic acid, which was recrystallized from metha-
nol: pure compound [2 g, 0.033%, mp 280 °C (lit. mp 285 °C
(Ikuta et al., 1988))].
3â-Hydroxylup-20(29)-en-28-oic Acid Methyl Ester (II).
I
(100 mg) was dissolved in 50 mL of freshly prepared ethereal
diazomethane kept at 0-5 °C for 24 h and the ether evapo-
rated. The product was recrystallized from methanol to afford
93 mg (90%) of II: mp 220-222 °C [lit. 223-225 °C (Ritti
Kawaguiti et al., 1940)]; IR (KBr) 3538, 3065, 2941, 2849, 1707,
1639, 1451, 1373, 1167, 1067, and 978 cm-1; 1H NMR (CDCl3)
δ 4.76 and 4.48 (each bs, 2H, 29-H), 3.68 (s, 3H, OCH3), 3.18
(m, 1H, 3R-H), 2.97 (m, 1H, 19-H), 1.70 (s, 3H, 30-H), and
0.97-0.72 (s, 15H, 5 × CH3); MS (EI), m/e (relative intensity)
470 M+ (10), 452 (5), 411 (8), 262 (23), 234 (6), 207 (28), 205
(23), 189 (56), 149 (100), and 135 (30).
We are particularly interested in the study of the
antifeedent activity of plant products (Srimannarayana
et al., 1989) and semisynthetic derivatives of betulinic
acid. These pentacyclic triterpenes of the lupane group
are abundant in the stem wood of Zizyphus xylopyrus
(Gotti) and Diospyros perigrina. Betulinic acid is also
3â-Acetoxylup-20(29)-en-28-oic Acid (III). I (100 mg) was
dissolved in 10 mL of acetic anhydride, two drops of pyridine
was added, and the resulting solution was stirred for 20 h at
room temperature. The solution was poured onto crushed ice
* Author to whom correspondence should be addressed
[telephone (040) 7173331; fax (040) 7018846 (Attn: F-049)].
S0021-8561(97)00768-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/01/1998

2798 J. Agric. Food Chem., Vol. 46, No. 7, 1998
Jagadeesh et al.
3H, 30-H), and 0.98-0.72 (s, 15H, 5 × CH3); MS (EI), m/e
(relative intensity) 496 M+ (1), 438 (4), 411 (30), 393 (4), 220
(25), 207 (50), 189 (89), 175 (30), 135 (56), and 41 (100).
Gen er a l P r oced u r e for P r ep a r a tion of Com p ou n d s
V-VII. A solution of acid (p-methylcinnamic acid, p-methoxy-
cinnamic acid, or tri-O-methylgallic acid, respectively) (1
equiv), DCC (1 equiv), I (1 equiv), and DMAP (0.1 equiv) in
CH2Cl2 (30 mL) was stirred for 24 h at room temperature; DCC
urea was filtered and the filtrate washed with water, 5%
aqueous AcOH, and water (3 × 25 mL) and then dried (MgSO4)
and evaporated under vacuum. The residue was chromato-
graphed by eluting with petroleum ether/EtOAc (6:4 v/v) to
give V, VI, and VII, respectively.
3â-p-Methylcinnamatoxylup-20(29)-en-28-oic Acid (V): yield,
70%; mp 225 °C; IR (KBr) 3269, 2932, 2854, 1708, 1644, 1624,
1596, 1451, 1375, 1152, and 890 cm-1; 1H NMR (CDCl3) δ 7.61
(d, 1H, R-H, J ) 16 Hz), 7.38 (d, 2H, 2′,6′-H, J ) 8 Hz), 7.16
(d, 2H, 3′,5′-H, J ) 8 Hz), 6.66 (d, 1H, â-H, J ) 16 Hz), 4.71
and 4.56 (each bs, 2H, 29-H), 2.37 (s, 3H, 4′-CH3), 1.72 (s, 3H,
30-H), and 0.97-0.72 (s, 15H, 5 × CH3) ppm; MS (EI), m/e
(relative intensity) 600 M+ (2), 509 (5), 368 (18), 288 (6), 243
(17), 205 (6), 160 (38), 145 (100), 117 (25), 115 (25).
F igu r e 1. Structures of betulinic acid derivatives.
3â-p-Methoxycinnamatoxylup-20(29)-en-28-oic Acid (VI): yield,
76%; mp 245 °C; IR (KBr) 3354, 2933, 2855, 1721, 1638, 1604,
1511, 1451, 1304, 1170, 1037, and 977 cm-1; 1H NMR (CDCl3)
δ 7.58 (d, 1H, â-H, J ) 16 Hz), 7.45 (d, 2H, 2′,6′-H, J ) 8 Hz),
6.88 (d, 2H,3′,5′-H, J ) 8 Hz), 6.28 (d, 1H,R-H, J ) 16 Hz),
4.72 and 4.56 (each bs, 2H, 29-H), 3.81 (s, 3H, 4′-OCH3), 1.65
(s, 3H, 30-H), and 0.92-0.71 (s, 15H, 5 × CH3); MS(FAB), m/e
(relative intensity) 616 M+ (3), 438 (8), 393 (12), 367 (25), 248
(10), 221 (48), 207 (56).
3â-Tri-O-methylgallotoxylup-20(29)-en-28-oic Acid (VII): yield,
86%; mp 256 °C; IR (KBr) 3449, 2999, 2854, 1701, 1637, 1508,
1458, 1338, and 938 cm-1; 1H NMR (CDCl3) δ 7.22 (s, 2H, 2′,6′-
H), 4.68 and 4.52 (each bs, 2H, 29-H), 3.88 (s, 6H, 3′,5′-OCH3),
3.83 (s, 3H, 4′-OCH3), 1.68 (s, 3H, 30-H), and 0.92-0.67 (s,
15H, 5 × CH3); MS (EI), m/e (relative intensity) 650 M+ (2),
510 (45), 279 (12), 220 (35), 205 (10), 189 (12), 168 (40), 149
(100), 121 (15).
(100 g), filtered, and washed with water, and the product was
recrystallized from methanol to give III (80 mg, 73%): mp
288-290 °C [lit. 291-293 °C (Ritti Kawaguiti et al., 1940)];
IR (KBr) 2943, 2870, 1735, 1693, 1640, 1367, 1318, 1195, 1026,
and 979 cm-1; 1H NMR (CDCl3) δ 4.67 and 4.46 (each bs, 2H,
29-H), 2.98 (m, 1H, 3R-H), 2.01 (s, 3H, Ac), 1.67 (s, 3H, 30-H),
and 0.99-0.75 (s, 15H, 5 × CH3); MS (EI), m/e (relative
intensity) 498 M+ (3), 454 (4), 438 (12), 248 (6), 189 (25), and
149 (56).
3â-Allyloxylup-20(29)-en-28-oic Acid (IV). A THF (35 mL)
solution of I (100 mg) and NaH (150 mg) was stirred at room
temperature for 1 h, then allyl bromide (1 mL) was added after
24 h, and the excess NaH was decomposed with methanol (10
mL). The methanol was evaporated under vacuum, and the
resulting residue was macerated with ether and washed with
water, 5% AcOH, and water (3 × 50 mL) to afford a residue
that was chromatographed over silica gel eluting with CHCl3
to give IV: 40 mg (37%); IR (KBr) 3071, 2941, 2860, 1689,
1454, 1377, 1190, 1135, 1036, and 943 cm-1; 1H NMR (CDCl3)
δ 5.92 (m, 1H, 2′-H), 5.25 (m, 2H, 3′-H), 4.72 and 4.56 (each
bs, 2H, 29-H), 3.95 (m, 2H, 1′-H), 3.21 (m, 1H, 3R-H), 1.71 (s,
Bioa ssa ys. The antifeedent activity of the compounds was
assessed on tobacco caterpillar (S. litura F). The tobacco
caterpillars were reared on fresh castor leaves (Ricinus com-
munis) grown on the Osmania University campus at 27 ( 1
°C, relative humidity 65 ( 5%, and 14:10 light/dark photo-
F igu r e 2. Antifeedant activity of betulinic acid derivatives.

Tobacco Caterpillar Antifeedant from Betulinic Acid
J. Agric. Food Chem., Vol. 46, No. 7, 1998 2799
period. Freshly molted fourth-instar larvae were used in the
assays. The assays were conducted as described by Ascher
and Rones (1964) in arenas constructed from plastic Petri
dishes (15 × 90 mm). A circle of moistened filter paper (9 cm
diameter) was placed on the floor of each arena. Castor leaf
disks (2 cm diameter) were cut with a cork borer from leaves
with well-developed primary leaflets. Treated leaf disks were
coated on the upper surface with 100 µL of solution having
5% Triton X-100 of the test compound in acetone; control leaf
disks coated with 100 µL of acetone having 5% Triton X-100
only. Acetone was allowed to evaporate before assays were
initiated. For these no-choice assays (J ermy et al., 1968), 10
treated and 10 untreated control disks were run for each test
and each test was replicated three times. In each Petri dish
one prestarved fourth-instar larva was placed. Assays began
4-5 h after the start of the photophase. Arenas were placed
in clear plastic ventilated Crisper boxes containing moist paper
toweling and placed in an environmental chambers at 27 ( 1
°C. The time period of the experiment was 48 h. Leaf
consumption (damaged areas) was measured with the help of
a Planimeter, and the percentage of protection was calculated
using the following formula by adopting the method of Singh
and Panth (1979):
important pest tobacco caterpillar (S. litura F) in the
no-choice laboratory assay.
ACKNOWLEDGMENT
We thank Dr. S. T. Ramchandrachary, Taxonomist,
Director, IICT, Hyderabad, India, for providing spectra.
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×
[
]
(100 - % protection in control)
100
A treatment/control consumption was calculated for each
compound at each dosage level.
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RESULTS AND DISCUSSION
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Betulinic acid (I) (Figure 1) was extracted from stem
wood of Z. xylopyrus with MeOH, followed by chroma-
tography on silica gel and recrystallization from metha-
nol. In feeding assays against S. litura F, betulinic acid
(I) was active only at high dose of application (150 µg/
cm2). Derivatization of betulinic acid by methylation
with diazomethane gave 3â-hydroxylup-20(29)-en-28-oic
acid methyl ester (II), which was also active only at high
dose of application (150 µg/cm2). Derivatization of the
3â-hydroxyl group by acetylation with acetic anhydride/
pyridine gave 3â-acetoxylup-20(29)-en-28-oic acid (III),
and allylation with allyl bromide gave 3â-allyloxylup-
20(29)-en-28-oic acid (IV). Compounds III and IV have
antifeedent activity at both 100 and 50 µg/cm2. Intro-
duction of an aryl group at the 3â-hydroxyl group by
esterification with p-methylcinnamic acid, p-methoxy-
cinnamic acid, and tri-O-methylgallic acid gave 3â-p-
methylcinnamatoxylup-20(29)-en-28-oic acid (V), 3â-p-
methoxycinnamatoxylup-20(29)-en-28-oic acid (VI), and
3â-tri-O-methylgallotoxylup-20(29)-en-28-oic acid (VII)
structures, respectively. These compounds (IV-VII)
showed antifeedent activity at dosages of 100 and 50
µg/cm2. Compounds V-VII displayed antifeedent activ-
ity at even lower dosages of 25 µg/cm2. The EC50 values
for the compounds are 125 µg/cm2 for IV, V is 50 µg/
cm2 for V, 70 µg/cm2 for VI, and 50 µg/cm2 for VII.
Figure 2 showes the antifeedent activity of betulinic acid
and its derivatives at different concentrations. Our
study shows the derivatization of the betulinic acid
structure by the addition of a methylcinnamic, meth-
oxycinnamic, or tri-O-methylgallic acid moiety in the
C-3 position of compound I increases the antifeedent
property of the compounds against the agriculturally
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Received for review September 8, 1997. Revised manuscript
received March 23, 1998. Accepted April 23, 1998. We thank
SAP, UGC, New Delhi, for financial support.
J F970768B