2003 natural insecticides from hippocratea

Economic Botany 57(1) pp. 54–64. 2003q 2003 by The New York Botanical Garden Press, Bronx, NY 10458-5126 U.S.A.

NATURAL INSECTICIDES FROM HIPPOCRATEA EXCELSA AND

HIPPOCRATEA CELASTROIDES1

RICARDO REYES-CHILPA, MANUEL JIMENEZ-ESTRADA,ELIZABETH CRISTOBAL-TELESFORO, LETICIA TORRES-COLIN,MIGUEL ANGEL VILLAVICENCIO, BLANCA ESTELA PEREZ-ESCANDON,AND ROBERTO MERCADO-GONZALEZ

Reyes-Chilpa, Ricardo, Manuel Jimenez-Estrada, Elizabeth Cristobal-Telesforo, (Institutode Quımica, Universidad Nacional Autonoma de Mexico. Circuito Exterior, Ciudad Universi-taria, Coyoacan, 04510, Mexico D.F.), Leticia Torres-Colın, (Instituto de Biologıa, Univer-sidad Nacional Autonoma de Mexico), Miguel Angel Villavicencio, Blanca Estela Perez-Escandon, (Centro de Investigaciones Biologicas. Universidad Autonoma de Hidalgo, Carre-tera Pachuca-Tulancingo s/n, Pachuca, Hidalgo, Mexico), and Roberto Mercado-Gonzalez(Novartis Farmaceutica S.A. de C.V. Calzada de Tlalpan No. 1779, Col. San Diego Churubusca04120, 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. Thetoxicity of H. excelsa root cortex has been previously demonstrated against the stored grainpest 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 highantifeedant activity, and elicited moderate mortality. The triterpenoid pristimerin and a mixtureof 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 waterextract, were innocuous or its activity was not statistically significant. The organic extractsfrom H. celastroides only showed moderate antifeedant activity, while the water extract wasinnocuous. Galactitol was also obtained from this extract.

INSECTICIDAS NATURALES DE HIPPOCRATEA EXCELSA E HIPPOCRATEA CELASTROIDES. A las plantascitadas, se les atribuye en Mexico propiedades medicinales e insecticidas. Estudios previos handemostrado que las raıces (corteza) de Hippocratea excelsa poseen actividad insecticida contrala plaga de granos almacenados Sithophilus zeamais. Para identificar las substancias activasde las raıces, se obtuvieron diversos extractos (eter de petroleo, CH2Cl2, acetona, metanol yagua) y compuestos; estos fueron ensayados mediante pruebas de alimentacion obligada (1%p/p) con S. zeamais. Todos los extractos de H. excelsa redujeron drasticamente la alimentacione incrementaron moderadamente la mortalidad. De los extractos de hexano y metanol se ais-laron el triterpeno pristimerina y una mezcla de alcaloides sesquiterpenicos, respectivamente;dichas substancias presentaron actividad antialimentaria alta. Otros triterpenoides aislados delextracto hexanico (friedelina, b-sitosterol, canofilol) y el alditol galactitol obtenido del extractoacuoso resultaron inocuos, o bien, su actividad no fue significativa estadısticamente. Los ex-tractos organicos de H. celastroides presentaron actividad antialimentaria moderada, en tantoel extracto acuoso resulto inocuo. De dicho extracto tambien se obtuvo galactitol.

Key Words: Hippocratea excelsa, Hippocratea celastroides, Sitophilus zeamais, insecticidalplants, medicinal plants, botanical insecticides, stored grain pests, alkaloids, triterpenes, alditols,polyols, pristimerin, galactitol, friedelin, canophyllol.

Higher plants synthesize chemical substancesthat can be toxic, repellent or inhibitory to the

1 Received 20 July 2000; accepted 21 November 2002.

growth and feeding of insects. Knowledge ofthese species is ancient and they are used worldwide nowadays, especially by peasants and na-tive ethnic groups, for the control of insect pests(Secoy and Smith 1983). During the first half of

2003] 55REYES-CHILPA ET AL.: NATURAL INSECTICIDES

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 cimarron, izcate blanco, mata piojo, piojoso, quinaHippocratea excelsa cancerina, hierba del piojo, ixcate, izcate rojo, mata piojo

the 20th century, several botanical insecticides,generally used as plant dusts or extracts, wereavailable in the international markets. Thesecomprise pyrethrins from Chrysanthemum ciner-riaefolium Vis. (Asteraceae), rotenone fromLonchocarpus and Derris spp. (Leguminosae),nicotine and anabasine from Nicotiana glaucaGraham and N. tabacum L. (Solanaceae), quas-sin from Quassia amara L. and Aeschrion ex-celsa Kuntze (Simaroubaceae), ryanodine fromRyania speciosa Vahl (Flacourtiaceae), and cev-eratrum alkaloids from Schoenocaulon and Ve-ratrum spp. (Liliaceae) (Benner 1993; Jacobson1982, 1989).

Most botanical insecticides languished after1950, 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 phytochemicalshas surged because these compounds couldserve as models for developing new syntheticinsecticides 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 havebeen proposed as part of the agricultural tech-nology support directed toward poor farmers ofless 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-tınez 1959; Soto Nunez and Sousa 1995; Stand-ley and Steyermark 1949). Hippocratea excelsaroot cortex, known as cancerina, is commonlyfound in popular markets all over the country(Hersch-Martınez 1995, 1997). Experimental ev-idence has demonstrated cancerina antifeedingactivity against four stored-grain insect pests, in-cluding Sitophilus zeamais Mots (Lagunes andRodrıguez 1989), but its active principles havenot yet been determined. Therefore, we decidedto examine the effects of the root extracts fromboth species and several isolated compounds on

the feeding and survival of S. zeamais. A pre-liminary analysis of cancerina water decoctionand capsules containing cancerina is also pre-sented. The botany, ethnobotany, chemistry, andbiological activity of H. excelsa and H. celas-troides are briefly reviewed.

BOTANY

The Hippocrateaceae comprises approximate-ly 115 species distributed pantropically. InAmerica, 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 somecases, 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 andHemiangium, which include two species of in-terest to the present study Pristimera celastro-ides (5 Hippocratea celastroides) and Hemian-gium excelsum (5 Hippocratea excelsa). Theauthor 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 weredistinguished by their growth, size and mor-phology of the leaves, inflorescence, and colorof the flowers. Fonseca (1995), in her floristicand taxonomic study of the Hippocrateaceae ofthe State of Guerrero, Mexico, also consideredboth species in the same genus and distinguishedthem by fruit characters and flower size.

Both species are climbing vines, glabrous orpuberulent with opposite leaves, persistent, el-liptical or oblong, coriaceous. In the case of H.celastroides, the flowers are 5 mm in diameterwith green glabrous sepals and green-yellowpetals. The inflorescences are axillary; the fruitlobules are separated from the base. Regarding

56 [VOL. 57ECONOMIC BOTANY

Fig. 1. Hippocratea excelsa root cortex (canceri-na). Upper right: Roots from a collected plant. Lowerright: Raw cancerina as expended in Mexican markets.Left: Cancerina capsules.

H. excelsa, the flowers are 10 mm in diameterwith pulverulent greenish sepals and green-yel-low petals; the fruit lobules are united until halfof their length. In both species flowering andfruiting are long lasting, but plants both withfruits and flowers are seldom found. The collec-tion of samples with flowers is important in thedelimitation of these species.

ETHNOBOTANY AND ETHNOPHARMACOLOGY

Several Hippocratea species are known inMexico for their presumed medicinal and insec-ticide properties. Among the Maya people of theState of Yucatan, H. celastroides has been usedas a sedative (Smith 1940; Standley and Stey-ermark 1949; Dıaz 1976) and as a remedyagainst dysentery (Sanabria-Diago 1986). In thisstate, one of its vernacular names is matapiojo(lice killer—Table 1) because a paste made ofthe ground seeds or the whole fruit is applied tokill head lice (Standley and Steyermark 1949).Insecticide application is also widespread inCentral America (Standley and Steyermark1949) and extends to other species and regionsof Mexico. For instance, peasants of the statesof Michoacan and Guerrero apply a paste madeof the grounded seeds of H. excelsa, H. acapul-censis (5 H. celastroides), or H. uniflora as aremedy against lice and other insect and miteskin parasites (Soto-Nunez and Sousa 1995).Hippocratea excelsa is also known as matapiojoin 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 (INI1994). It is also purported to be useful in thetreatment of gynecological ailments, cancer,wounds, and sores (Legorreta 1989). Cough isalso mitigated with an infusion of the leaves(Dıaz 1976; Martınez 1959).

In Mexico, H. excelsa is the most importantspecies of the genus from an economic point ofview. The root cortex is popularly known as can-cerina. It has a characteristic pink-orange colorwith yellow strips and is somewhat elastic, mak-ing it easy to recognize (Fig. 1). In folk medi-cine, cancerina is prepared as a water decoctionwhich is used for the treatment of gastritis, gas-tric ulcers, and as an anti-inflammatory and cic-atrizant agent (INI 1994). Recent reports alsopoint out the application of cancerina in thetreatment 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-Lopezand Basurto-Dorantes 1985) and ethanol extracts(Perez et al. 1995) exhibit anti-inflammatory ac-tivity using animals models of experimental ede-ma. The petroleum ether extract has also shownhigh cytotoxicity against KB, UISO-SQC-1, andHCT carcinoma cell lines (Popoca et al. 1998).

Hippocratea excelsa root cortex, but not thewhole roots or other organs, is collected in thewild and sold in markets and herbal stores inrural and urban centers in Mexico (Hersch-Mar-tınez 1995, 1997). New releases, such as pack-aged brands and capsules presumably containingcancerina (Fig. 1) have appeared, but theclaimed botanical origin of these products hasnot been verified. The medicinal applicationsand mode of preparation have been provided bythe sellers in the markets, but several brandsnow include this information on the labels. Forexample, the label on the commercially preparedcancerina (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 itcool to room temperature, and drink it duringthe day instead of water’’ (agua de tiempo). Onthe other hand, H. celastroides is not an objectof 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-


tigated by a Mexican entomological researchgroup during 1985–1990. This group tested 387plant species under laboratory conditions againstfour stored-grain pests: Acanthocelides obtectusSay, Prostephanus truncatus Horn, Zabrotes su-bfasciatus Boh., and Sitophilus zeamais Mots.(Lagunes and Rodrıguez 1989). A dozen prom-ising active plants against each pest were iden-tified; interestingly cancerina was effectiveagainst 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 tocontrol stored-grain pests in rustic granaries ofpoor rural areas (Lagunes and Rodrıguez 1989).Cancerina recommended dose in granaries was1 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 ofseed (Lagunes and Rodrıguez 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 ofS. frugiperda first instar larvae (Lagunes andRodrıguez 1990).

CHEMISTRY

Hippocratea excelsa root cortex chemistryhas been thoroughly studied. The petroleumether extract contains several triterpenoids, suchas friedelin (I), canophyllol (II), canophyllal andcanophyllic acid as well as the methylene-qui-nones pristimerin (III), celastrol (IV), tingenone,and excelsin (Calzada et al. 1991) (Fig. 2). Thechloroform 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 asource of natural rubber (Palacios et al. 1989).The methanol extract contains five evoninoatesesquiterpene alkaloids: hippocrateine I, II andIII (VI–VIII), as well as emarginatine A andmayteine (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 isolatedfrom the methylene chloride extract (Jimenez-Estrada et al. 2000). Celastroidine A is presum-ably produced by a fusion of a triterpene andditerpene, whereas celastroidine B is a diterpene

dimer. The leaves of H. celastroides are knownto contain triterpenes of the friedelin and lupanetypes, such as: friedelin (I), friedelan-3b-ol (epi-friedelinol), lup-20-en-3b,30-diol, and 3-oxo-lup-20-en-30-ol (Gonzalez et al. 1989).

METHODS AND MATERIALS

PLANT MATERIALS

Cancerina samples were bought during 1996at the Mercado de Sonora located in MexicoCity, and at the Central de Abastos of IgualaCity, State of Guerrero, Mexico. Both sampleswere positively identified as Hippocratea excel-sa root cortex by comparison with an authenticspecimen collected at Chamela Biological Sta-tion, State of Jalisco, Mexico (voucher MEXU830,828). 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 (Fonseca1995; Standley and Steyermark 1949). Capsulescontaining a pink powder and labeled ‘‘Cancer-ina’’ (brand PROSA) also were purchased at theCentral 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 threetimes, and the extracts were pooled and thenconcentrated in a rotary evaporator. Extractionwith water (24 hours) was done twice; the ex-tract was concentrated by evaporation in a vaporbath. The hexane, acetone, methanol, and waterextract yield was 7.3, 9.4, 41.9, and 74.8 g, re-spectively. The methylene chloride extract yieldwas not determined; it appeared as a gummy liq-uid that polymerized into an amorphous brownsolid, which was soluble in metacresol.

The petroleum ether extract (7.3 g) was dis-solved in CH2Cl2, and then treated with coldmethanol. The precipitated long chain hydrocar-bons were removed by vacuum filtration. Theremaining extract was concentrated (6.38 g) andsubjected to column chromatography (CC) overSilica Gel 60 (Merck 180 g). Elution was carriedout with mixtures of petroleum ether-ethyl ace-tate in order of increasing polarity. Fractionswere first analyzed by thin layer chromatogra-


Fig. 2. Compounds isolated from Hippocratea excelsa.

phy (TLC) using Silica Gel plates (Merck, 0.25mm) with different elution systems. DevelopedTLC plates were observed under UV light; andafterwards sprayed with a reagent (Cerium IVtetrahydrate 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 (857mg), 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 acetate8:2), 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


CH2Cl2, acetone, and mixtures of these solventsin 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 ofalkaloids, whereas fractions 155 to 171 elutedwith a solvent mixture CH2Cl2-acetone (8:2) re-sulted positively. These fractions showed a sim-ilar TLC profile and were pooled obtaining apale brown powder (m.p.112–1208C). The con-centrated water extract was a syrup, which aftertreating 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 capsulescontained H. excelsa root cortex, the pink pow-der was extracted with petroleum ether, and theextract was then analyzed by TLC (Silica Gel0.25 mm; petroleum ether-ethyl acetate 8:2).Several triterpenoids isolated from an authenticH. excelsa sample (see above), were used asstandards: canophyllol (I) (Rf 5 0.48), pristi-merin (III) (Rf 5 0.33, red spot without sprayreagent). Finally, the chemical composition of atraditional 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 previouslydescribed for the water extract, obtaining againgalactitol (V) (1.43 g). The remaining decoctionwas 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) wereground and extracted sequentially at room tem-perature with petroleum ether, methylene chlo-ride, acetone, methanol, and water. The extractswere prepared and concentrated as previouslydescribed. The petroleum ether, methylene chlo-ride, acetone, and methanol yields were 6.1814.1, 4.1 and 21.6 g, respectively. The methy-lene chloride and methanol extracts afforded awhite precipitate after treatment with acetone.The acetone extract was dissolved with metha-nol and also yielded a precipitate. Finally, thewater 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 mortalityof the plant extracts, fractions, or compoundswere evaluated in a force feeding test with Sith-

ophilus zeamais as described by Villavicencioand Perez-Escandon (1993). The chemical in so-lution was mixed with commercial maize flour(Brand Maizena) and water. The paste was cutinto tablets (9 mm diameter, 2 mm height) anddried (608C, 10 min.) in an oven. Control tabletswere treated only with the solvents used for dis-solving the samples. Positive control tabletswere also prepared using rotenone (ICN). Thefinal concentration of all the extracts, fractions,or compounds in the tablets was 1% (w/w). Eachtablet was deposited in a petri dish along with10 adult insects. These were taken from coloniesraised on corn grains and kept in glass flasks.Ten replicates (tablets) per treatment were runsimultaneously. Mortality and feeding (as indi-cated by number of excreta) were evaluated afterfive days. Results were expressed as AntifeedingActivity Index (AAI) and Corrected Mortality(CM). Where: AAI 5 100 2 (number of excretain treatment/number of excreta in control)100,and CM 5 (Y 2 X/100 2 X)100. Y 5 treatmentmortality, X 5 control mortality.

RESULTS

Hippocratea excelsa root cortex (cancerina)was easily found in the Mercado de Sonora, thebiggest herbal market of Mexico City, and in themain market of Iguala City, Mexico. In bothmarkets, the average price for cancerina was US$9.0/kg during 1996. This species could not becollected in the wild in nearby locations withinthe State of Morelos, where, according to her-barium labels, it was present several years ago.This is in agreement with the warnings thatoverexploitation and inadequate gathering prac-tices are leading to H. excelsa extinction insoutheastern Morelos and in the neighboringsouthwestern area of the State of Puebla(Hersch-Martınez 1995, 1997). The hypothesisthat excessive demand is threatening H. excelsasurvival in those regions is also supported by thefact that large populations of H. celastroides (aspecies without commercial value) are foundwithin the State of Morelos, even next to urbandevelopments.

CHEMICAL ANALYSIS AND

INSECTICIDE TESTS

All the extracts from the root cortex of H.excelsa tested at 1% reduced the survival andfeeding of Sitophilus zeamais (Table 2). The or-ganic extracts were the best, inhibiting the feed-


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

ControlRotenone (positive control)Petroleum etherFriedelin (F 10-17) 9.5:0.5

0.088.6 6 0.3**83.8 6 0.8**30.0 6 4.0

0.022.6 6 0.1*68.0 6 0.4**

3.0 6 0.22b-sitosterol (F 26-33) 9.5:0.5Canofilol (F 37-39) 9.5:0.5Pristimerin (F 49-53) 9.5:0.5F 65-71 9.5:0.5

22.0 6 5.10.0 6 6.7

89.2 6 0.5**44.2 6 1.59*

2.0 6 0.220.0 6 0.02

16.0 6 0.317.9 6 0.2

F 72-79 9:1F 80-87 8.5:1.5F 88-97 8.5:1.5F 98-106 8:2F 107-115 7:3F 116-124 6:4

21.2 6 2.634.3 6 2.274.2 6 1.5**76.6 6 1.6**81.5 6 1.6**88.2 6 0.7**

5.3 6 0.213.8 6 0.435.8 6 0.2**40.0 6 0.5**83.5 6 0.7**73.6 6 0.7**

F 125-130 5:5F 131-140 4:6Methylene chlorideAcetone

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*

MethanolAlkaloids (F 155-171)WaterGalactitol

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 waterextract 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 remainingextracts were less than 25.5%.

The petroleum ether extract was subjected toCC, and afforded several fractions and purecompounds, which were in turn examined usingS. zeamais (Table 2). Four triterpenoids wereisolated: 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 thatexhibited by the reference insecticide rotenone.Although friedelin (I) and b-sitosterol exhibitedmild antifeedant activity (22–30% inhibition),these figures were not statistically significant.Canophyllol (II) was completely harmless. Themost polar fractions (F 107–140) obtained byCC exhibited high antifeedant activity (.80%)and increased (.46%) insect mortality (Table 2).The chemical composition of these fractions iscurrently 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 andpooled. Its 1HNMR (200 MHZ) spectrum clearlyindicated a mixture of sesquiterpene evoninoatealkaloids, identified as: hippocrateine I, II, andIII (VI–VIII), as well as emarginatine (Calzadaand Mata 1995; Mata et al. 1970). The intensityof a singlet at 9.00 ppm assigned to H-20 of thenicotinic residue of hippocrateine III, indicatedthis compound was the most abundant in themixture. No attempt was done to further purifythe individual components. The alkaloid mixturetested with the insects exhibited high antifeedingactivity (93.8%) and increased the mortality ofS. zeamais 64%.

The water extract yielded an alditol that wasidentified as galactitol (V) (Voelter et al. 1973).This compound was not toxic to the insects, butwas 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).


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

ControlHexaneMethylene chlorideAcetone (soluble part)

0.067.8 6 3.7**70.3 6 3.3**72.3 6 2.7**

0.07.3 6 0.49.4 6 0.8

12.5 6 0.4PrecipitateMethanol (soluble part)PrecipitateWater (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 0.16.2 6 0.25.2 6 0.20.0 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 leadto 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 withan extract prepared from an authentic sample ofH. excelsa root cortex, and clearly showed thepresence of canophyllol (II) and pristimerin (III).A preliminary analysis of the cancerina tradi-tional water decoction indicated it contains highamounts of galactitol (V), but also the low po-larity compounds friedelin and pristimerin weredetected in the organic phase by TLC.

In the case of Hippocratea celastroides, onlythe 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 mortalityrate. (Table 3). The highest antifeedant activitywas elicited by the precipitate obtained from theacetone extract, this was closely followed by theacetone extract (soluble part), and then by themethylene chloride and hexane extracts. Themethanol extract (both the soluble part and theprecipitate) 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 wereisolated from the hexane and methanol extracts,respectively, and were found in part responsiblefor the antifeeding and toxic properties of Hip-pocratea excelsa root cortex against the insectpest 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 alsoactive. On the other hand, only the organic ex-tracts of H. celastroides exhibited mild antifee-dant activity (Table 3), indicating that activecompounds may be circumscribed to the lowand 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 alkaloidmixture was estimated to be 2.8 times more tox-ic than rotenone. This is noteworthy, consideringthat rotenone has been reported as the best nat-ural antifeedant compound so far tested againstseveral insect storage pests, including Sitophilusgranarius L. (Nawrot et al. 1989; Nawrot andHarmatha 1994). Interestingly, the insecticideactivity 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


Haller 1950; Beroza 1951). To date, approxi-mately 10 sesquiterpene alkaloids have been iso-lated from this species (Ya, Strunz, and Calhoun1990), but further evaluation of their insecticideproperties is needed.

To our best knowledge, pristimerin (III) pre-viously has not been reported as a stored-grainpest antifeedant. Celastrol, known also as trip-terine-(IV), a methylenequinone closely relatedto pristimerin, was once proposed as the activeprinciple of T. wilfordii Hook (Schechter andHaller 1942), but afterwards it was found to beinsecticide inert (Acree and Haller 1950). Struc-ture activity relationship may be keen, becausepristimerin differs from celastrol by only an es-ter instead of a carboxyl functional group.

Galactitol (V) is an abundant component ofH. excelsa and H. celastroides root water ex-tracts. This compound did not show insecticideor antifeeding activity (Table 2). Nevertheless, itcould play a physiological role as an organic os-molyte, such as has been described for galactitolin Bostrychia tenella, a epiphytic red algae ofmangrove trees, which is subjected to long pe-riods of desiccation (Karsten et al. 1996). Ga-lactitol (V) has not been reported previously asa constituent of H. excelsa and H. celastroides.However, galactitol has been found commonlyin the Hippocrataceae (Pristimera, Salacia, andTontelea) 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 foundin species of the Lauraceae, Saxifragaceae, andScrophulariaceae (Plouvier 1963).

Hippocratea excelsa water decoction, whichis currently consumed for medicinal purposes inMexico, contains high concentrations of galac-titol (V), but also low polarity compounds, suchas canophyllol (II) and pristimerin (III). Thepresence of these compounds should be takeninto account in the evaluation of its medicinaland/or potential toxic properties. For example,pristimerin (III) is known to exhibit antibacterialactivity (Bhatnagar and Divekar 1951). Thisproperty could be relevant, because the bacteriaHelicobacter pylori is implicated as a cause ofchronic gastritis, peptic ulcer, and probably inthe 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 slowlyabsorbed in rats fed with high oral doses of thispolyol (Makinen and Hamalainen 1985). Inthese experiments, galactitol also elicited someinteresting metabolic effects, such as retardedgrowth 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 wouldbe interesting to investigate if galactitol metab-olism in humans is similar to that reported forrats, to assess any risk from the H. excelsa de-coction.

ACKNOWLEDGMENTSThis 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 Ramırez,and Dagoberto Alavez for field and laboratory assistance. Thanks to Wil-ber Matus, Luis Velasco, Javier Perez, and Rocio Patino for recordingthe spectra, and to Mazahiro Tanikawa for photographic work.

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