1) The study examines the insecticidal properties of extracts and compounds isolated from the roots of Hippocratea excelsa and Hippocratea celastroides against the stored grain pest Sitophilus zeamais. 2) All extracts of H. excelsa showed strong antifeedant effects and moderate mortality against S. zeamais, while extracts of H. celastroides showed only moderate antifeedant effects. 3) The compounds pristimerin and a mixture of sesquiterpene alkaloids, isolated from H. excelsa extracts, strongly reduced insect feeding, while other isolated compounds showed little or no activity.

1. NATURAL INSECTICIDES FROM HIPPOCRATEA EXCELSA AND
HIPPOCRATEA CELASTROIDES1
RICARDO REYES-CHILPA, MANUEL JIME´NEZ-ESTRADA,
ELIZABETH CRISTO´ BAL-TELE´
SFORO, LETICIA TORRES-COLI´
N,
MIGUEL ANGEL VILLAVICENCIO, BLANCA ESTELA PE´
REZ-ESCANDO´ N,
AND ROBERTO MERCADO-GONZA´ LEZ
Reyes-Chilpa, Ricardo, Manuel Jime´nez-Estrada, Elizabeth Cristo´bal-Tele´sforo, (Instituto
de Quı´mica, Universidad Nacional Auto´noma de Me´xico. Circuito Exterior, Ciudad Universi-taria,
Coyoaca´n, 04510, Me´xico D.F.), Leticia Torres-Colı´n, (Instituto de Biologı´a, Univer-sidad
Nacional Auto´noma de Me´xico), Miguel Angel Villavicencio, Blanca Estela Pe´rez-
Escando´n, (Centro de Investigaciones Biolo´gicas. Universidad Auto´noma de Hidalgo, Carre-tera
Pachuca-Tulancingo s/n, Pachuca, Hidalgo, Me´xico), and Roberto Mercado-Gonza´lez
(Novartis Farmaceu´tica S.A. de C.V. Calzada de Tlalpan No. 1779, Col. San Diego Churubusca
04120, Mexico D.F.). NATURAL INSECTICIDES FROM HIPPOCRATEA EXCELSA AND HIPPOCRATEA CE-LASTROIDES.
Economic Botany 57(1):54–64, 2003. Hippocratea excelsa and Hippocratea celas-troides
have therapeutic and insecticide applications in Mexican traditional medicine. The
toxicity of H. excelsa root cortex has been previously demonstrated against the stored grain
pest Sitophilus zeamais. To identify the active compounds, several extracts (petroleum ether,
CH2Cl2, acetone, methanol, and water) and compounds were obtained from the roots, and tested
(1% w/w) with a force-feeding assay against S. zeamais. All H. excelsa extracts showed high
antifeedant activity, and elicited moderate mortality. The triterpenoid pristimerin and a mixture
of sesquiterpene evoninoate alkaloids, isolated from the hexane and methanol extracts, respec-tively,
strongly reduced the insect feeding capacity. Other triterpenoids (friedelin, b-sitosterol,
canophyllol) isolated from the hexane extract, and the alditol galactitol obtained from the water
extract, were innocuous or its activity was not statistically significant. The organic extracts
from H. celastroides only showed moderate antifeedant activity, while the water extract was
innocuous. Galactitol was also obtained from this extract.
INSECTICIDAS NATURALES DE HIPPOCRATEA EXCELSA E HIPPOCRATEA CELASTROIDES. A las plantas
citadas, se les atribuye en Me´xico propiedades medicinales e insecticidas. Estudios previos han
demostrado que las raı´ces (corteza) de Hippocratea excelsa poseen actividad insecticida contra
la plaga de granos almacenados Sithophilus zeamais. Para identificar las substancias activas
de las raı´ces, se obtuvieron diversos extractos (e´ter de petro´leo, CH2Cl2, acetona, metanol y
agua) y compuestos; estos fueron ensayados mediante pruebas de alimentacio´n obligada (1%
p/p) con S. zeamais. Todos los extractos de H. excelsa redujeron dra´sticamente la alimentacio´n
e incrementaron moderadamente la mortalidad. De los extractos de hexano y metanol se ais-laron
el triterpeno pristimerina y una mezcla de alcaloides sesquiterpe´nicos, respectivamente;
dichas substancias presentaron actividad antialimentaria alta. Otros triterpenoides aislados del
extracto hexa´nico (friedelina, b-sitosterol, canofilol) y el alditol galactitol obtenido del extracto
acuoso resultaron inocuos, o bien, su actividad no fue significativa estadı´sticamente. Los ex-tractos
orga´nicos de H. celastroides presentaron actividad antialimentaria moderada, en tanto
el extracto acuoso resulto´ inocuo. De dicho extracto tambie´n se obtuvo galactitol.
Key Words: Hippocratea excelsa, Hippocratea celastroides, Sitophilus zeamais, insecticidal
plants, medicinal plants, botanical insecticides, stored grain pests, alkaloids, triterpenes, alditols,
polyols, pristimerin, galactitol, friedelin, canophyllol.
Higher plants synthesize chemical substances
that can be toxic, repellent or inhibitory to the
1 Received 20 July 2000; accepted 21 November 2002.
growth and feeding of insects. Knowledge of
these species is ancient and they are used world
wide nowadays, especially by peasants and na-tive
ethnic groups, for the control of insect pests
(Secoy and Smith 1983). During the first half of
Economic Botany 57(1) pp. 54–64. 2003
q 2003 by The New York Botanical Garden Press, Bronx, NY 10458-5126 U.S.A.

2. 2003] REYES-CHILPA ET AL.: NATURAL INSECTICIDES 55
TABLE 1. VERNACULAR NAMES USED FOR HIPPOCRATEA SPECIES IN MEXICO.
Species Vernacular name
Hippocratea celastroides (5Hippo-cratea
acapulcensis) barajilla, barajita, bejuco de piojo, cucaracho, hierba del piojo,
ixcate, ixcate cimarro´n, izcate blanco, mata piojo, piojoso, quina
Hippocratea excelsa cancerina, hierba del piojo, ixcate, izcate rojo, mata piojo
the 20th century, several botanical insecticides,
generally used as plant dusts or extracts, were
available in the international markets. These
comprise pyrethrins from Chrysanthemum ciner-riaefolium
Vis. (Asteraceae), rotenone from
Lonchocarpus and Derris spp. (Leguminosae),
nicotine and anabasine from Nicotiana glauca
Graham and N. tabacum L. (Solanaceae), quas-sin
from Quassia amara L. and Aeschrion ex-celsa
Kuntze (Simaroubaceae), ryanodine from
Ryania speciosa Vahl (Flacourtiaceae), and cev-eratrum
alkaloids from Schoenocaulon and Ve-ratrum
spp. (Liliaceae) (Benner 1993; Jacobson
1982, 1989).
Most botanical insecticides languished after
1950, but pyrethrins and neem oil from Azadi-rachta
indica A. Juss. (Meliaceae) are still com-mercial
products (Benner 1993; Jacobson 1982).
Renewed interest in insecticide phytochemicals
has surged because these compounds could
serve as models for developing new synthetic
insecticides that may prove to be safer for hu-man
health and environment (Benner 1993).
From another perspective, research, develop-ment,
and utilization of insecticide plants have
been proposed as part of the agricultural tech-nology
support directed toward poor farmers of
less developed countries (Lagunes and Rodrı´-
guez 1989, 1990).
In Mexico, Hippocratea excelsa Kunth and H.
celastroides Kunth are used due to their medic-inal
and insecticide properties (INI 1994; Mar-tı
´nez 1959; Soto Nu´n˜ez and Sousa 1995; Stand-ley
and Steyermark 1949). Hippocratea excelsa
root cortex, known as cancerina, is commonly
found in popular markets all over the country
(Hersch-Martı´nez 1995, 1997). Experimental ev-idence
has demonstrated cancerina antifeeding
activity against four stored-grain insect pests, in-cluding
Sitophilus zeamais Mots (Lagunes and
Rodrı´guez 1989), but its active principles have
not yet been determined. Therefore, we decided
to examine the effects of the root extracts from
both species and several isolated compounds on
the feeding and survival of S. zeamais. A pre-liminary
analysis of cancerina water decoction
and capsules containing cancerina is also pre-sented.
The botany, ethnobotany, chemistry, and
biological activity of H. excelsa and H. celas-troides
are briefly reviewed.
BOTANY
The Hippocrateaceae comprises approximate-ly
115 species distributed pantropically. In
America, it is distributed from South Florida,
Mexico, Central America, the Antilles, Bolivia,
Argentina, and Paraguay to southeast Brazil
(Smith 1940). The Hippocratea complex is dif-ficult
to distinguish and its taxonomy is confus-ing
due to incomplete information and, in some
cases, errors in collection labels. The same ver-nacular
names are applied to different taxa con-tributing
to confusion (Table 1).
In the first monograph for the family, Smith
(1940) described the genera Pristimera and
Hemiangium, which include two species of in-terest
to the present study Pristimera celastro-ides
(5 Hippocratea celastroides) and Hemian-gium
excelsum (5 Hippocratea excelsa). The
author distinguished these species only by inflo-rescence
characters. Standley and Steyermark
(1949) in their contribution to The Flora of Gua-temala
included Hippocratea excelsa and H. ce-lastroides
in the same genus. These species were
distinguished by their growth, size and mor-phology
of the leaves, inflorescence, and color
of the flowers. Fonseca (1995), in her floristic
and taxonomic study of the Hippocrateaceae of
the State of Guerrero, Mexico, also considered
both species in the same genus and distinguished
them by fruit characters and flower size.
Both species are climbing vines, glabrous or
puberulent with opposite leaves, persistent, el-liptical
or oblong, coriaceous. In the case of H.
celastroides, the flowers are 5 mm in diameter
with green glabrous sepals and green-yellow
petals. The inflorescences are axillary; the fruit
lobules are separated from the base. Regarding

3. 56 ECONOMIC BOTANY [VOL. 57
Fig. 1. Hippocratea excelsa root cortex (canceri-na).
Upper right: Roots from a collected plant. Lower
right: Raw cancerina as expended in Mexican markets.
Left: Cancerina capsules.
H. excelsa, the flowers are 10 mm in diameter
with pulverulent greenish sepals and green-yel-low
petals; the fruit lobules are united until half
of their length. In both species flowering and
fruiting are long lasting, but plants both with
fruits and flowers are seldom found. The collec-tion
of samples with flowers is important in the
delimitation of these species.
ETHNOBOTANY AND ETHNOPHARMACOLOGY
Several Hippocratea species are known in
Mexico for their presumed medicinal and insec-ticide
properties. Among the Maya people of the
State of Yucatan, H. celastroides has been used
as a sedative (Smith 1940; Standley and Stey-ermark
1949; Dı´az 1976) and as a remedy
against dysentery (Sanabria-Diago 1986). In this
state, one of its vernacular names is matapiojo
(lice killer—Table 1) because a paste made of
the ground seeds or the whole fruit is applied to
kill head lice (Standley and Steyermark 1949).
Insecticide application is also widespread in
Central America (Standley and Steyermark
1949) and extends to other species and regions
of Mexico. For instance, peasants of the states
of Michoacan and Guerrero apply a paste made
of the grounded seeds of H. excelsa, H. acapul-censis
(5 H. celastroides), or H. uniflora as a
remedy against lice and other insect and mite
skin parasites (Soto-Nu´n˜ez and Sousa 1995).
Hippocratea excelsa is also known as matapiojo
in El Salvador (Standley and Steyermark 1949).
In the State of Mexico, H. celastroides (espe-cially
the seeds) is used against intestinal para-sites
and skin mites, as a purgative, antiseptic,
and disinfectant, and to mitigate cough (INI
1994). It is also purported to be useful in the
treatment of gynecological ailments, cancer,
wounds, and sores (Legorreta 1989). Cough is
also mitigated with an infusion of the leaves
(Dı´az 1976; Martı´nez 1959).
In Mexico, H. excelsa is the most important
species of the genus from an economic point of
view. The root cortex is popularly known as can-cerina.
It has a characteristic pink-orange color
with yellow strips and is somewhat elastic, mak-ing
it easy to recognize (Fig. 1). In folk medi-cine,
cancerina is prepared as a water decoction
which is used for the treatment of gastritis, gas-tric
ulcers, and as an anti-inflammatory and cic-atrizant
agent (INI 1994). Recent reports also
point out the application of cancerina in the
treatment of cancer (Popoca et al. 1998). Can-cerina
medicinal properties have been poorly in-vestigated.
Nevertheless, pharmacological stud-ies
have reported that the water (Germes-Lo´pez
and Basurto-Dorantes 1985) and ethanol extracts
(Pe´rez et al. 1995) exhibit anti-inflammatory ac-tivity
using animals models of experimental ede-ma.
The petroleum ether extract has also shown
high cytotoxicity against KB, UISO-SQC-1, and
HCT carcinoma cell lines (Popoca et al. 1998).
Hippocratea excelsa root cortex, but not the
whole roots or other organs, is collected in the
wild and sold in markets and herbal stores in
rural and urban centers in Mexico (Hersch-Mar-tı
´nez 1995, 1997). New releases, such as pack-aged
brands and capsules presumably containing
cancerina (Fig. 1) have appeared, but the
claimed botanical origin of these products has
not been verified. The medicinal applications
and mode of preparation have been provided by
the sellers in the markets, but several brands
now include this information on the labels. For
example, the label on the commercially prepared
cancerina (20 g presentation, Plantas Seleccion-adas
in Ixmiquilpan, State of Hidalgo, Mexico)
instructs one to: ‘‘Boil two soup spoons of can-cerina
in 1 liter of water for 3 minutes. Let it
cool to room temperature, and drink it during
the day instead of water’’ (agua de tiempo). On
the other hand, H. celastroides is not an object
of trade; it is only known, collected, and con-sumed
by native people and local peasants.
INSECTICIDE ACTIVITY
The insecticide properties attributed to can-cerina
(H. excelsa root cortex) were first inves-

4. 2003] REYES-CHILPA ET AL.: NATURAL INSECTICIDES 57
tigated by a Mexican entomological research
group during 1985–1990. This group tested 387
plant species under laboratory conditions against
four stored-grain pests: Acanthocelides obtectus
Say, Prostephanus truncatus Horn, Zabrotes su-bfasciatus
Boh., and Sitophilus zeamais Mots.
(Lagunes and Rodrı´guez 1989). A dozen prom-ising
active plants against each pest were iden-tified;
interestingly cancerina was effective
against all four species.
This study also involved field tests in grana-ries,
where cancerina was also effective. There-fore
it was proposed as a valuable alternative to
control stored-grain pests in rustic granaries of
poor rural areas (Lagunes and Rodrı´guez 1989).
Cancerina recommended dose in granaries was
1 g/kg of seed against A. obtectus and P. trun-catus.
In the cases of Z. subfasciatus and S. zea-mais
the recommended dose was 100 g/kg of
seed (Lagunes and Rodrı´guez 1989). Prelimi-nary
data also indicated that Hippocratea excel-sa
(cancerina) was among the 64 more promis-ing
plants against the plant maize pest Spodop-tera
frugiperda. Cancerina water extract or de-coction
(5% w/v) caused 40% mortality rate of
S. frugiperda first instar larvae (Lagunes and
Rodrı´guez 1990).
CHEMISTRY
Hippocratea excelsa root cortex chemistry
has been thoroughly studied. The petroleum
ether extract contains several triterpenoids, such
as friedelin (I), canophyllol (II), canophyllal and
canophyllic acid as well as the methylene-qui-nones
pristimerin (III), celastrol (IV), tingenone,
and excelsin (Calzada et al. 1991) (Fig. 2). The
chloroform extract is known to contain b-sitos-terol
and high amounts of trans polyisoprene
(Palacios et al. 1989). The yield of this com-pound
is similar to that of Parthenium argen-tatum
(Asteraceae) and has been suggested as a
source of natural rubber (Palacios et al. 1989).
The methanol extract contains five evoninoate
sesquiterpene alkaloids: hippocrateine I, II and
III (VI–VIII), as well as emarginatine A and
mayteine (Calzada and Mata 1995; Mata et al.
1990). Recently, H. celastroides roots were in-vestigated
and two unusual diels alder adducts,
named as celastroidine A and B, were isolated
from the methylene chloride extract (Jime´nez-
Estrada et al. 2000). Celastroidine A is presum-ably
produced by a fusion of a triterpene and
diterpene, whereas celastroidine B is a diterpene
dimer. The leaves of H. celastroides are known
to contain triterpenes of the friedelin and lupane
types, such as: friedelin (I), friedelan-3b-ol (epi-friedelinol),
lup-20-en-3b,30-diol, and 3-oxo-lup-
20-en-30-ol (Gonza´lez et al. 1989).
METHODS AND MATERIALS
PLANT MATERIALS
Cancerina samples were bought during 1996
at the Mercado de Sonora located in Mexico
City, and at the Central de Abastos of Iguala
City, State of Guerrero, Mexico. Both samples
were positively identified as Hippocratea excel-sa
root cortex by comparison with an authentic
specimen collected at Chamela Biological Sta-tion,
State of Jalisco, Mexico (voucher MEXU
830,828). Hippocratea celastroides was collect-ed
near Ticuman in the State of Morelos, Mex-ico
(vouchers MEXU 702,365; 702,366;
702,368). Identity of the collected plants was de-termined
following botanical keys (Fonseca
1995; Standley and Steyermark 1949). Capsules
containing a pink powder and labeled ‘‘Cancer-ina’’
(brand PROSA) also were purchased at the
Central de Abastos in Iguala, Mexico (Fig. 1).
CHEMICAL ANALYSIS
Hippocratea excelsa root cortex (1000 g, So-nora
market sample) was ground and sequen-tially
extracted at room temperature with petro-leum
ether, methylene chloride, acetone, meth-anol,
and finally water. Extraction with each or-ganic
solvent (3 l, 48 hours) was repeated three
times, and the extracts were pooled and then
concentrated in a rotary evaporator. Extraction
with water (24 hours) was done twice; the ex-tract
was concentrated by evaporation in a vapor
bath. The hexane, acetone, methanol, and water
extract yield was 7.3, 9.4, 41.9, and 74.8 g, re-spectively.
The methylene chloride extract yield
was not determined; it appeared as a gummy liq-uid
that polymerized into an amorphous brown
solid, which was soluble in metacresol.
The petroleum ether extract (7.3 g) was dis-solved
in CH2Cl2, and then treated with cold
methanol. The precipitated long chain hydrocar-bons
were removed by vacuum filtration. The
remaining extract was concentrated (6.38 g) and
subjected to column chromatography (CC) over
Silica Gel 60 (Merck 180 g). Elution was carried
out with mixtures of petroleum ether-ethyl ace-tate
in order of increasing polarity. Fractions
were first analyzed by thin layer chromatogra-

5. 58 ECONOMIC BOTANY [VOL. 57
Fig. 2. Compounds isolated from Hippocratea excelsa.
phy (TLC) using Silica Gel plates (Merck, 0.25
mm) with different elution systems. Developed
TLC plates were observed under UV light; and
afterwards sprayed with a reagent (Cerium IV
tetrahydrate sulfate—Merck—1% in 2N H2SO4)
and warmed up on a hot plate (1508C 1 min).
Fractions with similar TLC pattern were pooled.
The identity of the isolated compounds was de-termined
by their physical and 1HNMR, IR, UV,
and MS spectroscopic data.
In the case of the petroleum ether extract, the
first CC fractions eluted with a mixture of hex-ane-
ethyl acetate (9.5:0.5) afforded several tri-terpenoids:
friedelin (I) (4 mg), b-sitosterol (857
mg), and canophyllol (II) (8 mg) (Table 2). Fur-ther
fractions eluted with the same solvent mix-ture
afforded a red syrup, which after prepara-tive
TLC (Silica Gel 2 mm; hexane-ethyl acetate
8:2), yielded a red-orange oil identified as pris-timerin
(III) (159 mg).
Part of the methanol extract (12 g) was sub-jected
to CC over Silica Gel 60 (360 g) with

6. 2003] REYES-CHILPA ET AL.: NATURAL INSECTICIDES 59
CH2Cl2, acetone, and mixtures of these solvents
in order of increasing polarity. Presence of al-kaloids
in the CC fractions was detected by TLC
(Silica Gel) spraying the plates with Draggen-dorff
reagent. Fractions 1 to 154 were devoid of
alkaloids, whereas fractions 155 to 171 eluted
with a solvent mixture CH2Cl2-acetone (8:2) re-sulted
positively. These fractions showed a sim-ilar
TLC profile and were pooled obtaining a
pale brown powder (m.p.112–1208C). The con-centrated
water extract was a syrup, which after
treating it with methanol yielded a white powder
(6.7 g) that was further identified by its spectro-scopic
data as the alditol, galactitol (V).
To investigate whether the Cancerina capsules
contained H. excelsa root cortex, the pink pow-der
was extracted with petroleum ether, and the
extract was then analyzed by TLC (Silica Gel
0.25 mm; petroleum ether-ethyl acetate 8:2).
Several triterpenoids isolated from an authentic
H. excelsa sample (see above), were used as
standards: canophyllol (I) (Rf 5 0.48), pristi-merin
(III) (Rf 5 0.33, red spot without spray
reagent). Finally, the chemical composition of a
traditional cancerina water decoction was inves-tigated.
For this purpose a decoction was pre-pared
(12 g, boiled for 10 min in 1 l of water).
Half of the decoction was treated as previously
described for the water extract, obtaining again
galactitol (V) (1.43 g). The remaining decoction
was extracted three times with CH2Cl2. The or-ganic
phase was dried with Na2SO4, concentrat-ed,
and analyzed by TLC, as above described.
Hippocratea celastroides roots (1610 g) were
ground and extracted sequentially at room tem-perature
with petroleum ether, methylene chlo-ride,
acetone, methanol, and water. The extracts
were prepared and concentrated as previously
described. The petroleum ether, methylene chlo-ride,
acetone, and methanol yields were 6.18
14.1, 4.1 and 21.6 g, respectively. The methy-lene
chloride and methanol extracts afforded a
white precipitate after treatment with acetone.
The acetone extract was dissolved with metha-nol
and also yielded a precipitate. Finally, the
water extract was concentrated as described pre-viously
(49.5 g); during this process galactitol
(V) precipitated spontaneously (2.48 g).
BIOLOGICAL TESTS
The antifeeding activity and induced mortality
of the plant extracts, fractions, or compounds
were evaluated in a force feeding test with Sith-ophilus
zeamais as described by Villavicencio
and Pe´rez-Escando´n (1993). The chemical in so-lution
was mixed with commercial maize flour
(Brand Maizena) and water. The paste was cut
into tablets (9 mm diameter, 2 mm height) and
dried (608C, 10 min.) in an oven. Control tablets
were treated only with the solvents used for dis-solving
the samples. Positive control tablets
were also prepared using rotenone (ICN). The
final concentration of all the extracts, fractions,
or compounds in the tablets was 1% (w/w). Each
tablet was deposited in a petri dish along with
10 adult insects. These were taken from colonies
raised on corn grains and kept in glass flasks.
Ten replicates (tablets) per treatment were run
simultaneously. Mortality and feeding (as indi-cated
by number of excreta) were evaluated after
five days. Results were expressed as Antifeeding
Activity Index (AAI) and Corrected Mortality
(CM). Where: AAI 5 100 2 (number of excreta
in treatment/number of excreta in control)100,
and CM 5 (Y 2 X/100 2 X)100. Y 5 treatment
mortality, X 5 control mortality.
RESULTS
Hippocratea excelsa root cortex (cancerina)
was easily found in the Mercado de Sonora, the
biggest herbal market of Mexico City, and in the
main market of Iguala City, Mexico. In both
markets, the average price for cancerina was US
$9.0/kg during 1996. This species could not be
collected in the wild in nearby locations within
the State of Morelos, where, according to her-barium
labels, it was present several years ago.
This is in agreement with the warnings that
overexploitation and inadequate gathering prac-tices
are leading to H. excelsa extinction in
southeastern Morelos and in the neighboring
southwestern area of the State of Puebla
(Hersch-Martı´nez 1995, 1997). The hypothesis
that excessive demand is threatening H. excelsa
survival in those regions is also supported by the
fact that large populations of H. celastroides (a
species without commercial value) are found
within the State of Morelos, even next to urban
developments.
CHEMICAL ANALYSIS AND
INSECTICIDE TESTS
All the extracts from the root cortex of H.
excelsa tested at 1% reduced the survival and
feeding of Sitophilus zeamais (Table 2). The or-ganic
extracts were the best, inhibiting the feed-

7. 60 ECONOMIC BOTANY [VOL. 57
TABLE 2. ANTIFEEDING ACTIVITY INDEX (AAI) AND MORTALITY (M) OF SITOPHILUS ZEAMAIS CAUSED
BY HIPPOCRATEA EXCELSA EXTRACTS, AND FRACTIONS (1% W/W). MEAN 6 S.E. OF 10 REPLICATES.
Extract, fraction (F) or compound eluent1 % AAI % M
Control
Rotenone (positive control)
Petroleum ether
Friedelin (F 10-17) 9.5:0.5
0.0
88.6 6 0.3**
83.8 6 0.8**
30.0 6 4.0
0.0
22.6 6 0.1*
68.0 6 0.4**
3.0 6 0.22
b-sitosterol (F 26-33) 9.5:0.5
Canofilol (F 37-39) 9.5:0.5
Pristimerin (F 49-53) 9.5:0.5
F 65-71 9.5:0.5
22.0 6 5.1
0.0 6 6.7
89.2 6 0.5**
44.2 6 1.59*
2.0 6 0.22
0.0 6 0.02
16.0 6 0.3
17.9 6 0.2
F 72-79 9:1
F 80-87 8.5:1.5
F 88-97 8.5:1.5
F 98-106 8:2
F 107-115 7:3
F 116-124 6:4
21.2 6 2.6
34.3 6 2.2
74.2 6 1.5**
76.6 6 1.6**
81.5 6 1.6**
88.2 6 0.7**
5.3 6 0.2
13.8 6 0.4
35.8 6 0.2**
40.0 6 0.5**
83.5 6 0.7**
73.6 6 0.7**
F 125-130 5:5
F 131-140 4:6
Methylene chloride
Acetone
80.9 6 1.3**
83.5 6 0.7**
84.5 6 2.9**
89.2 6 1.1**
61.5 6 0.6**
46.8 6 0.4**
55.7 6 0.3**
23.4 6 0.5*
Methanol
Alkaloids (F 155-171)
Water
Galactitol
93.1 6 0.8**
93.8 6 0.5**
68.5 6 2.4**
33.8 6 3.2
25.5 6 0.4*
64.0 6 0.1**
21.3 6 0.3*
1.0 6 0.2
1 Mobile phase in column chromatography: petroleum ether-ethyl acetate.
Significantly different (*p , 0.01, **p , 0.01) from control values by Mann-Whitney U test.
ing capacity of the insects 83–93%. The water
extract caused only a 68% inhibition. The petro-leum
ether extract elicited the highest mortality
(68%), followed by the methylene chloride ex-tract
(55.7%); mortality figures for the remaining
extracts were less than 25.5%.
The petroleum ether extract was subjected to
CC, and afforded several fractions and pure
compounds, which were in turn examined using
S. zeamais (Table 2). Four triterpenoids were
isolated: b-sitosterol, friedelin (I), canophyllol
(II) and pristimerin (III). None of these com-pounds
significantly increased the mortality rate;
but pristimerin (IV) showed high antifeedant ac-tivity
(89.2%). This value was similar to that
exhibited by the reference insecticide rotenone.
Although friedelin (I) and b-sitosterol exhibited
mild antifeedant activity (22–30% inhibition),
these figures were not statistically significant.
Canophyllol (II) was completely harmless. The
most polar fractions (F 107–140) obtained by
CC exhibited high antifeedant activity (.80%)
and increased (.46%) insect mortality (Table 2).
The chemical composition of these fractions is
currently under investigation.
The methanol extract showed the highest an-tifeeding
activity among all extracts (Table 2)
and was subjected to CC. Several alkaloid pos-itive
fractions (155–171) were obtained and
pooled. Its 1HNMR (200 MHZ) spectrum clearly
indicated a mixture of sesquiterpene evoninoate
alkaloids, identified as: hippocrateine I, II, and
III (VI–VIII), as well as emarginatine (Calzada
and Mata 1995; Mata et al. 1970). The intensity
of a singlet at 9.00 ppm assigned to H-20 of the
nicotinic residue of hippocrateine III, indicated
this compound was the most abundant in the
mixture. No attempt was done to further purify
the individual components. The alkaloid mixture
tested with the insects exhibited high antifeeding
activity (93.8%) and increased the mortality of
S. zeamais 64%.
The water extract yielded an alditol that was
identified as galactitol (V) (Voelter et al. 1973).
This compound was not toxic to the insects, but
was able to inhibit their feeding by 33.8%; nev-ertheless,
this figure was not statistically signif-icant
(Table 2).
Galactitol (Dulcitol) (V). White powder, m.p.
186–1888 (reported 188.58, Voelter et al. 1973).

8. 2003] REYES-CHILPA ET AL.: NATURAL INSECTICIDES 61
TABLE 3. ANTIFEEDING ACTIVITY INDEX (AAI) AND MORTALITY (M) OF SITOPHILUS ZEAMAIS CAUSED
BY HIPPOCRATEA CELASTROIDES ROOT EXTRACTS (1% W/W). MEAN 6 S.E. OF 10 REPLICATES.
Extract % AAI % M
Control
Hexane
Methylene chloride
Acetone (soluble part)
0.0
67.8 6 3.7**
70.3 6 3.3**
72.3 6 2.7**
0.0
7.3 6 0.4
9.4 6 0.8
12.5 6 0.4
Precipitate
Methanol (soluble part)
Precipitate
Water (soluble part)
73.9 6 2.5**
44.4 6 4.0*
49.0 6 4.8**
0.0 6 4.5
0.0 6 0.1
6.2 6 0.2
5.2 6 0.2
0.0 6 0.2
Significantly different (*p , 0.01, **p , 0.001) from control values by Mann-Whitney U-test.
IR n max (KBr): 3365, 3316, 3252, 2943, 1458,
1377, 1118, 1078, 1050, 1030. 1HNMR (D2O,
200 MHz) d ppm: 3.53 (d, 6H, J 5 4.6 Hz) H-
1, H-6, H-3, and H-4; 3.81 (t, 2H, J 5 4.2 Hz)
H-2 and H-5. 13CNMR (D2O, 50 MHz) d ppm:
64.2 (CH2) C-1 and C-6, 70.3 (CH) C-2 and C-
5, 71.2 (CH) C-3 and C-4. CIMS 70 ev (m/z):
183 M1 1 H (100%) [C6H14O6 1 H]1, 165
(10%) [183- H2O]1, 147 (18%) [183- 2H2O]1,
129 (45%) [183- 3H2O]1, 111 (10%) [183-
4H2O]1, 99 (6%), 81 (6.5%).
Because scarcity of medicinal plants may lead
to adulteration, it was interesting for us to ex-amine
chemically the new releases (or fashions)
in folk therapeutics, such as the cancerina cap-sules,
which claimed botanical origin was con-firmed
by TLC. The petroleum ether extract pro-file
from the capsules powder was identical with
an extract prepared from an authentic sample of
H. excelsa root cortex, and clearly showed the
presence of canophyllol (II) and pristimerin (III).
A preliminary analysis of the cancerina tradi-tional
water decoction indicated it contains high
amounts of galactitol (V), but also the low po-larity
compounds friedelin and pristimerin were
detected in the organic phase by TLC.
In the case of Hippocratea celastroides, only
the organic extracts from the roots showed re-markable
biological activity. These extracts in-hibited
the feeding of the insects 44.4–73.9%,
but did not significantly increase the mortality
rate. (Table 3). The highest antifeedant activity
was elicited by the precipitate obtained from the
acetone extract, this was closely followed by the
acetone extract (soluble part), and then by the
methylene chloride and hexane extracts. The
methanol extract (both the soluble part and the
precipitate) showed the lowest activity. The wa-ter
extract was completely innocuous. Galactitol
(V) was also obtained from this extract.
DISCUSSION
The triterpenoid pristimerin (III) and a mix-ture
of sesquiterpene evoninoate alkaloids were
isolated from the hexane and methanol extracts,
respectively, and were found in part responsible
for the antifeeding and toxic properties of Hip-pocratea
excelsa root cortex against the insect
pest Sitophilus zeamais. Because each of the ex-tracts
showed high antifeedant activity and mod-erate
mortality (Table 2), other active com-pounds
may need to be further characterized.
For example, many fractions (pristimerin free)
from the CC of the hexane extract were also
active. On the other hand, only the organic ex-tracts
of H. celastroides exhibited mild antifee-dant
activity (Table 3), indicating that active
compounds may be circumscribed to the low
and medium polarity extracts.
Both pristimerin, and the mixture of sesqui-terpene
evoninoate alkaloids, (with hippocrat-eine
III as the main constituent) strongly re-duced
the feeding capacity of the insect to a sim-ilar
extent of the well-known botanical insecti-cide
rotenone (Table 2). Moreover, the alkaloid
mixture was estimated to be 2.8 times more tox-ic
than rotenone. This is noteworthy, considering
that rotenone has been reported as the best nat-ural
antifeedant compound so far tested against
several insect storage pests, including Sitophilus
granarius L. (Nawrot et al. 1989; Nawrot and
Harmatha 1994). Interestingly, the insecticide
activity of Trypterigium wilfordii Hook (Celas-traceae),
a Chinese related species with a pesti-cide
reputation, also has been tracked to the al-kaloid
fraction of the ether extract (Acree and

9. 62 ECONOMIC BOTANY [VOL. 57
Haller 1950; Beroza 1951). To date, approxi-mately
10 sesquiterpene alkaloids have been iso-lated
from this species (Ya, Strunz, and Calhoun
1990), but further evaluation of their insecticide
properties is needed.
To our best knowledge, pristimerin (III) pre-viously
has not been reported as a stored-grain
pest antifeedant. Celastrol, known also as trip-terine-(
IV), a methylenequinone closely related
to pristimerin, was once proposed as the active
principle of T. wilfordii Hook (Schechter and
Haller 1942), but afterwards it was found to be
insecticide inert (Acree and Haller 1950). Struc-ture
activity relationship may be keen, because
pristimerin differs from celastrol by only an es-ter
instead of a carboxyl functional group.
Galactitol (V) is an abundant component of
H. excelsa and H. celastroides root water ex-tracts.
This compound did not show insecticide
or antifeeding activity (Table 2). Nevertheless, it
could play a physiological role as an organic os-molyte,
such as has been described for galactitol
in Bostrychia tenella, a epiphytic red algae of
mangrove trees, which is subjected to long pe-riods
of desiccation (Karsten et al. 1996). Ga-lactitol
(V) has not been reported previously as
a constituent of H. excelsa and H. celastroides.
However, galactitol has been found commonly
in the Hippocrataceae (Pristimera, Salacia, and
Tontelea) and Celastraceae, for example, Trip-terygium
wilfordii (Acree and Haller 1950).
Therefore, the presence of galactitol has taxo-nomic
value, and confirms the parentage be-tween
the Hippocrateaceae and Celastraceae
(Plouvier 1963). Galactitol has also been found
in species of the Lauraceae, Saxifragaceae, and
Scrophulariaceae (Plouvier 1963).
Hippocratea excelsa water decoction, which
is currently consumed for medicinal purposes in
Mexico, contains high concentrations of galac-titol
(V), but also low polarity compounds, such
as canophyllol (II) and pristimerin (III). The
presence of these compounds should be taken
into account in the evaluation of its medicinal
and/or potential toxic properties. For example,
pristimerin (III) is known to exhibit antibacterial
activity (Bhatnagar and Divekar 1951). This
property could be relevant, because the bacteria
Helicobacter pylori is implicated as a cause of
chronic gastritis, peptic ulcer, and probably in
the etiology of gastric cancer (Goodman 1997).
Information concerning the metabolism of ex-ogenous
galactitol (V) in mammals is scant;
nevertheless, it is known that galactitol is slowly
absorbed in rats fed with high oral doses of this
polyol (Ma¨kinen and Ha¨ma¨la¨inen 1985). In
these experiments, galactitol also elicited some
interesting metabolic effects, such as retarded
growth rate, and low levels of blood glucose,
serum total cholesterol, and liver ascorbic acid,
as compared with normal-fed rats. Finally, ga-lactitol
accumulation in lens fibers has been re-lated
to early development of cataracts in pa-tients
suffering galactosemia, a disease that aris-es
from the genetic inability to metabolize ga-lactose
to glucose (Strombolian 1988). It would
be interesting to investigate if galactitol metab-olism
in humans is similar to that reported for
rats, to assess any risk from the H. excelsa de-coction.
ACKNOWLEDGMENTS
This research was possible with the economic support provided by the
Program for Economic Botany in Latin America and the Caribbean (PRE-BELAC)
The New York Botanical Garden, and DGAPA-UNAM
(IN214996). We are also grateful with Leticia Paul, Adriana Ramı´rez,
and Dagoberto Alavez for field and laboratory assistance. Thanks to Wil-ber
Matus, Luis Velasco, Javier Pe´rez, and Rocio Patin˜o for recording
the spectra, and to Mazahiro Tanikawa for photographic work.
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