Extratos clorofórmicos ou diclorometânicos de 357 espécies vegetais de ocorrência no sul do Brasil foram testados quanto à presença de peróxidos, através de cromatografia em camada delgada, utilizando o reagente de detecção de Huber & Fröhlke. Das espécies testadas, 71 (20%) apresentaram resultado positivo, a maioria delas (56%) pertencentes à família Asteraceae. A maioria das espécies testadas pertencia a Asteraceae, mas outras 55 famílias foram também testadas, abrangendo um total de 77 gêneros.Chloroform or dichloromethane extracts of 357 southern Brazilian plant species were tested for the presence of peroxides by thinlayer chromatography, using the spray reagent from Huber & Fröhlke. From the species tested, 71 (20%) showed positive results and most of them (56%) are Asteraceae species. The species tested were mainly from Asteraceae, but 55 more families were screened, in a total of 77 genera surveyed

Bordignon, Sérgio A.L.

Falkenberg, Miriam B.

Heinzmann, Berta M.

Manns, Detlef

Matzenbacher, Nelson I.

Mentz, Lilian A.

Rücker, Gerhard

Schenkel, Eloir Pedro

Sobral, Marcos

English

Chloroform or dichloromethane extracts of 357 southern Brazilian plant species were tested for the presence of peroxides by thinlayer chromatography, using the spray reagent from Huber & Fröhlke. From the species tested, 71 (20%) showed positive results and most of them (56%) are Asteraceae species. The species tested were mainly from Asteraceae, but 55 more families were screened, in a total of 77 genera surveyed.Extratos clorofórmicos ou diclorometânicos de 357 espécies vegetais de ocorrência no sul do Brasil foram testados quanto à presença de peróxidos, através de cromatografia em camada delgada, utilizando o reagente de detecção de Huber & Fröhlke. Das espécies testadas, 71 (20%) apresentaram resultado positivo, a maioria delas (56%) pertencentes à família Asteraceae. A maioria das espécies testadas pertencia a Asteraceae, mas outras 55 famílias foram também testadas, abrangendo um total de 77 gêneros

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Revista Brasileira de Ciências FarmacêuticasBrazilian Journal of Pharmaceutical Sciencesvol. 38, n. 2, abr./jun., 2002Screening of Brazilian plants for the presence of peroxidesEloir Pedro Schenkel1*, Gerhard Rücker2, Detlef Manns2, Miriam B. Falkenberg3,Nelson I. Matzenbacher4, Marcos Sobral1, Lilian A. Mentz4 , Sérgio A.L. Bordignon5,Berta M. Heinzmann61Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, 2Pharmazeutisches Institut, Universität Bonn,3Departamento de Ciências Farmacêuticas,Universidade Federal de Santa Catarina, 4Departamento de Botânica,Universidade Federal do Rio Grande do Sul, 5Curso de Ciências Biológicas, Universidade Luterana do Brasil,6Departamento de Farmácia Industrial, Universidade Federal de Santa MariaChloroform or dichloromethane extracts of 357 southern Brazilianplant species were tested for the presence of peroxides by thin-layer chromatography, using the spray reagent from Huber &Fröhlke. From the species tested, 71 (20%) showed positive resultsand most of them (56%) are Asteraceae species. The species testedwere mainly from Asteraceae, but 55 more families were screened,in a total of 77 genera surveyed.*Correspondence:E.P. SchenkelDepartamento de CiênciasFarmacêuticasCentro de Ciências da SaúdeUniversidade Federal de SantaCatarinaCampus Universitário /Trindade88.040-900 – Florianópolis-SC, BrasilE-mail: schenkel@ccs.ufsc.brUniterms:• Peroxides• Antimalarial activity• AsteraceaeINTRODUCTIONThe occurrence of natural products which contain aperoxide group has been known since 1911, whenascaridole was isolated from the essential oil ofChenopodium ambrosioides (Chenopodiaceae). This oilwas utilized as anthelmintic and ascaridole was identifiedas responsible for its activity (Gessner, 1953; Hope, 1958).In the following decades, compounds with a similarstructure were not isolated; peroxides were considered tobe too unstable and ascaridole was thought to be anexception.In the meantime, as much as 320 peroxides havebeen isolated from natural material (Casteel, 1992). Theyoccur mainly as terpene derivatives, although cumarine,fatty acids and alkaloids peroxides derivatives have alsobeen isolated. Beside plants, many species of fungi andmarine organisms produce metabolites containing aperoxide group. The peroxide function has been detectedin different classes of natural compounds that arementioned in Table I, based on the review published byCasteel (1992). The occurrence in vascular plant familiesaccording the literature published until 1999 issummarized by Table II.Many biological activities were described for theperoxides. They show ichthyotoxic and antimicrobialactivity, fungicide, anthelmintic and cytotoxic activity. Atthe moment, the most important activity exhibited byperoxides is the antimalarial activity (Vennerstrom,Easton, 1988; Rücker, 1994). The prevention and cure ofmalaria depends on a limited number of drugs. Resistanceof the malaria parasites Plasmodium ssp. to drugs such asquinine (and more lately chloroquine) occurs withincreasing frequency and emphasize the necessity todevelop new agents for malaria chemotherapy (Kirby,1996). Quinghaosu (artemisinin), a sesquiterpene isolatedfrom Artemisia annua, is the best known plant derivativeas an antimalarial drug after quinine (Klayman, 1985).Another sesquiterpene peroxide, named yingzhaosu, wasisolated from Artabotrys uncinatus (Annonaceae) and alsopossesses a significant antimalarial activity. Bothcompounds contain an endoperoxid group, which isresponsible for their activity (Klayman, 1985) and haveopened a new chapter in the chemotherapy of malaria. Themajority of drugs active against malaria are in fact derivedfrom natural products or from structures suggested bynatural products prototypes, but, as a source of noveldrugs, plants remain largely understudied (Kirby, 1996).E. P. Schenkel, G. Rücker, D. Manns, M. B. Falkenberg et al.192Herewith we report the screening for detection ofperoxides in extracts from higher plants from the states ofRio Grande do Sul and Santa Catarina, Brazil, based onHuber & Fröhlke’s chromatographic test. The screeningwas developed to select promising plants for detailedinvestigation, aiming the isolation and structureelucidation of new peroxides.MATERIAL AND METHODSPlants were collected from several localities in theaforementioned States, between December 1989 andFebruary 1990, and identified by the authors N.Matzenbacher, M. Sobral, L. A. Mentz, and Sérgio A.L. orby the botanist Daniel Falkenberg (Universidade Federalde Santa Catarina). Voucher specimens were deposited atthe ICN- Herbarium of the Instituto de Biociências daUFRGS, Porto Alegre, RS, Brazil.Fresh plant material (10-20 g) was crushed andextracted with CH2Cl2 or CHCl3 (10-20 mL) previouslytested for peroxides. The extraction procedures wereperformed at the same day of the plant collection; in the caseof distant places, the macerates were prepared at the fieldand concentrated at the laboratory. After maceration for 4-10 h, the extracts were filtered and concentrated underreduced pressure until 1 mL, at temperatures below 40 °C.The concentrated extracts were then chromato-graphed on silicagel F254 TLC plates, with CH2Cl2. In thecase that peroxide-like spots appeared at the front, theTLC analysis was repeated using petroleum ether: AcOEt(9:1) as eluting solvent. After elution, the TLC wasobserved under UV light at 254 and 366 nm. Afterwardsthe peroxides were detected by a specific reagentdescribed by Huber and Fröhlke (1972). With this reagent,hydroperoxides develop blue spots immediately, and forendoperoxides the blue color appears after a few minutes.RESULTS AND DISCUSSIONA total of 500 samples of 357 species werecollected, including male and female individuals (someBaccharis species), and samples from different regions.The main target was Asteraceae, considering previousoccurrence reports of peroxide compounds (Table II), andthus any found species was collected for the screening.Concomitantly, ca. the same number of species from otherTABLE I - Peroxide structures described in the literatureconsidering the different classes of natural productsClass Number of compoundsMarine metabolitesFatty acid derivatives 121,2-dioxancarboxilates 39Others 11Terrestrial sourcesSmall molecules, hemiterpenes, 22      and monoterpenesSesquiterpenes 149Diterpenes 14Triterpenes 5Steroids 23Others 9TABLE II - Number of species of the different plantfamilies for which peroxides were reported (literature until1999)Family Number os speciesAnnonaceae 2Apiaceae 4Araceae 4Aristolochiaceae 3Asclepiadaceae 3Asteraceae 214Celastraceae 1Chenopodiaceae 4Clusiaceae 1Cyperaceae 1Ericaceae 2Euphorbiaceae 3Fagaceae 1Hypericaceae 3Hippocastanaceae 1Hydrocharitaceae 1Lamiaceae 9Lauraceae 1Magnoliaceae 3Myrtaceae 3Pinaceae 5Pteridophyta 2Rosaceae 2Rutaceae 2Scrophulariaceae 6Solanaceae 1Valerianaceae 2Verbenaceae 2Zingiberaceae 16Screening of Brazilian plants for the presence of peroxides 193plant families were included to test the hypothesis of arestrictive distribution of peroxides. These samples ofother families were collected randomly, and in the case ofa positive result, more species from the same family weretested. Apart from the Asteraceae species, we tested 182plants from 55 families, ca.1-3 species for each family, butin some cases a larger number were tested, as in the caseof Myrtaceae (24 species), Euphorbiaceae (14),Verbenaceae (12) and Lamiaceae (11). Table III presentsthe 71 species tested which gave positive results and theplant material used for the extraction. Among the 175species of Asteraceae tested for the presence of peroxidesin leaves and blossoms, 40 (30%) showed positive results.In the 182 plants classified in other families, 26 (14%)showed positive results. These results indicated a higheroccurrence of peroxides in the Asteraceae family andconfirmed the literature data (Table II), but suggested thatthey can also be found frequently in other plant families,such as Myrtaceae and Verbenaceae.Within the Asteraceae a higher rate of positiveresults were observed in genera belonging to the tribesAnthemideae and Senecioneae. Otherwise, in some tribesthere were no samples with positive reaction, as in the caseof Vernonieae (13 species tested), Cichorieae (3),Cynareae (2) , Helenieae (2) (results not shown), or witha low rate, as in the case of tribes Inuleae (19 speciestested, 1 positive result). The species which gave negativereaction with the peroxide reagent are not presented. Thelist of these plants can be obtained from the author towhom correspondence should be addressed.From the plants listed on Table III, several wereselected for detailed investigation, observing also factorsas the abundance of the species and the foliar mass. Fromthese works (Falkenberg, 1991; Marek, 1994; Heinzmann,1996; Rücker et al., 1996) some new peroxides wereisolated. Some of the isolated peroxides were also testedfor antimalarial activity, semi-synthesized and derivatized(Rücker, 1994). The compound with most promisingactivity was 1 (bisabolene-1,4-endoperoxide=1,4-epidioxy-bisabolan-2,12-diene) (Figure 1), for which invitro an IC50 0.05 was observed, in comparison with thevalue 0.005 for artemisinine as reference compound(Heinzmann, 1996; Rücker et al., 1997). Since thecompound 1  was isolated from Heterothalamuspsiadioides, H. alienus, Senecio selloi and Eupatoriumrufescens (Heinzmann, 1996; Rücker et al., 1996; Rückeret al., 1996) it seems to have a wide occurrence, being firstreported from Rudbeckia lasciniata (Bohlmann et al.,1978), and also from Ligularia speciosa (Bohlmann, Fritz,1980), Chamaemelum fuscatum (Pascual et al. 1983) andSenecio desfontnei (Metwally, Dawidar, 1986). Twoothers bisabolan-type sesquiterpenes, zingiberene-3,6-a-and zingiberene-3,6-b-endoperoxides (3 and 4) wereisolated from S. selloi and E. rufescens and also showedantimalarial activity in vitro, but this activity was weakerthan the activity of bisabolene-1,4-endoperoxide. S. selloialso contains two hydroperoxides (5 and 6) , which wereidentified indirectly by isolation, identification and poste-rior photooxidation of α-curcumene, their precursor in theplant (Figure 1). The detailed investigation of the CH2Cl2extract of S. selloi also showed a greyish blue spot with theHuber’s peroxide reagent. After isolation, this compoundwas identified as a triterpene ozonide (7), which containsa new 9-epi-cucurbitan skeleton (8) (Rücker et al., 1999).The possible occurrence of peroxides in species ofMyrtaceae, as suggested by our screening, was a subjectof a detailed investigation. The substances giving positivereaction in the extracts from Eugenia hyemalis andParamyrciaria glazioviana were isolated; the structureelucidation revealed these compounds to be the quinones9, 10 (Figure 1) (Falkenberg, 1996). The high oxidationpotential of this class is pointed as the reason for thepositive reactions with the Huber’s peroxide reagent(Kiefer, 1994).ACKNOWLEDGMENTSThis work was supported by the German InstitutionStifterverband für die Deutsche Wissenschaft (SV), theBrazilian agencies Fundação de Amparo a Pesquisa doEstado do Rio Grande do Sul (FAPERGS) and ConselhoNacional de Desenvolvimento Científico e Tecnológico(CNPq).RESUMOInvestigação de plantas brasileiras quanto à presen-ça de peróxidosExtratos clorofórmicos ou diclorometânicos de 357 es-pécies vegetais de ocorrência no sul do Brasil foram tes-tados quanto à presença de peróxidos, através decromatografia em camada delgada, utilizando o reagentede detecção de Huber & Fröhlke. Das espécies testadas,71 (20%) apresentaram resultado positivo, a maioria de-las (56%) pertencentes à família Asteraceae. A maioriadas espécies testadas pertencia a Asteraceae, mas outras55 famílias foram também testadas, abrangendo um totalde 77 gêneros.UNITERMOS: Peróxidos. Atividade antimalárica.Asteraceae.E. P. Schenkel, G. Rücker, D. Manns, M. B. Falkenberg et al.194Tribe Inuleae (19 species tested)Pterocaulon sp. 1 +Tribe Mutisieae (13 species tested)Gochnatia cordata +~Gochnatia orbiculata +~Mutisia sp. +++Trichocline macrocephala +~Tribe Senecioneae (9 species tested)Erechthites hieraciifolia ++Erechthites valerianifolia ++Senecio bonariensis +~Senecio crassiflorus +Senecio heterotrichus +Senecio heterotrichus +~Senecio selloi +++EUPHORBIACEAE (14 species tested)Croton gnaphalii +Euphorbia hirta +LAMIACEAE (10 species tested)Hyptis mutabilis + MYRSINACEAE (3 species tested)Myrsine umbellate +~MYRTACEAE (24 species tested)Blepharocalyx salicifolius +Eucalyptus calophylla ++Eucalyptus camaldulensis +Eucalyptus citriodora ++Eucalyptus globulus new leaves ++Eucalyptus sideroxylon old leaves +new leaves +Eucalyptus viminalis old leaves +new leaves+Eugenia hyemalis ++Eugenia hyemalis +Gomidesia palustris ++Myrciaria cuspidata +Plinia trunciflora +RUBIACEAE (11 species tested)Cephalanthus glabratus +VERBENACEAE (12 species tested)Aloysia gratissima +Aloysia sp. (Uruguaiana) ++Verbena cf. bonariensis +Verbena cf. litoralis +ANACARDIACEAE (8 species tested)Lithraea molleoides +Astronium balansae +Schinus lentiscifolius +Schinus molle +BORAGINACEAE (1 species tested)Cordia curassavica +ASTERACEAE (175 species tested)Tribe Anthemideae (6 species tested)Artemisia alba ++Artemisia cf. annua +Artemisia verlotorum +Chrysanthemum vulgare ++Tribe Astereae (33 species tested)Baccharis anomala (Guaiba) +~Baccharis anomala (Porto Alegre) +~Baccharis cf. artemisioides +Baccharis cylindrica (male; flowers) +~Baccharis cylindrica (male; leaves) +~Baccharis dracunculifolia ++Baccharis ochracea +Baccharis pseudotenuifolia ++Baccharis spicata +~Grindelia buphtalmoides ++Grindelia cf. pulchella +~Heterothalamus alienus +Heterothalamus psiadioides +Hysterionica pinifolia +~Tribe Eupatorieae (31 species tested)Eupatorium ascendens ++Eupatorium commersonii ++Eupatorium intermedium ++Eupatorium pedunculosum +Eupatorium serrulatum ++Eupatorium spathulatum +~Eupatorium tanacetifolium ++Mikania laevigata +~Mikania ternifolia ++Symphyopappus casarettoi +++Symphyopappus casarettoi (flowers) +Symphyopappus casarettoi (leaves) +Tribe Heliantheae (19 species tested)Eclipta megapotamica +Galinsoga parviflora +Helianthus annuus ++Verbesina subcordata +TABLE III - Extracts tested for the presence of peroxides which gave a positive reaction with the reagent described byHuber and Frölke, 1972. The signals +~ (weak positive reaction), +, ++ and +++ were assigned considering the colorintensityScreening of Brazilian plants for the presence of peroxides 195FIGURE 1 - Compounds isolated from Heterothalamus alienus (1, 2, 3 and 4), H. psiadioides (1), Eupatorium rufescens(1,3 and 4), Eugenia hyemalis and Paramyrciaria glazioviana.E. P. Schenkel, G. Rücker, D. Manns, M. B. Falkenberg et al.196REFERENCESBOHLMAN, F., FRITZ, U. Isofukinone, an eremophilanefrom Ligularia speciosa. Phytochemistry, v.19, p. 2471-2472, 1980.BOHLMAN, F., JAKUPOVIC, J., ZDERO, C. NeueNorsesquiterpene aus Rudbeckia lasciniata und Seneciopaludoaffinis. Phytochemistry, v.17, p. 2034-2436, 1978.CASTEEL, D. A. Peroxy natural products. Natural ProductReports, v. 24, p. 289-312, 1992.FALKENBERG, M. B. Chinone und andere Inhaltsstoffe ausEugenia hiemalys Camb. und Paramyrciaria glazioviana(Kiaerskou) Sobral. Boon, 1996. 84p. [PhD Thesis.Universität Bonn].GESSNER, O. 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Investigação de plantas brasileiras quanto à presença de peróxidos

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Screening of brazilian plants for the presence of peroxides

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