Forest Plot As a Tool to Demonstrate the Pharmaceutical Potential of Plants in a Tropical Forest of Panama Author(S): Angela I

Forest Plot as a Tool to Demonstrate the Pharmaceutical Potential of Plants in a Tropical Forest of Panama Author(s): Angela I. Calderon, Cindy K. Angerhofer, John M. Pezzuto, Norman R. Farnsworth, Robin Foster, Richard Condit, Mahabir P. Gupta and Djaja D. Soejarto Reviewed work(s): Source: Economic Botany, Vol. 54, No. 3 (Jul. - Sep., 2000), pp. 278-294 Published by: Springer on behalf of New York Botanical Garden Press Stable URL: http://www.jstor.org/stable/4256322 . Accessed: 10/12/2012 14:47

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This content downloaded by the authorized user from 192.168.52.67 on Mon, 10 Dec 2012 14:47:28 PM All use subject to JSTOR Terms and Conditions FOREST PLOT AS A TOOL TO DEMONSTRATE THE PHARMACEUTICALPOTENTIAL OF PLANTS IN A TROPICAL FOREST OF PANAMA1,2

ANGELA I. CALDERON, CINDY K. ANGERHOFER, JOHN M. PEZZUTO, NORMAN R. FARNSWORTH, ROBIN FOSTER, RICHARD CONDIT, MAHABIR P. GUPTA, AND DJAJA D. SOEJARTO

Calderon, Angela I. and Mahabir P. Gupta (Centerfor PharmacognosticResearch on Pan- amanian Flora-CIFLORPAN,Apartado 10767, College of Pharmacy,University of Panama, Republic of Panama), Robin B. Foster (Environmentaland ConservationPrograms, Botany Department,Field Museum,Roosevelt Road at Lake Shore Drive, Chicago, IL 60612, USA, and SmithsonianTropical Research Institute,Balboa, Republicof Panama), Richard Condit (SmithsonianTropical Research Institute,Apartado 2072, Balboa, Republicof Panama), and Cindy K. Angerhofer, John M. Pezzuto, Norman R. Farnsworth, and Djaja Doel Soejarto (Programfor CollaborativeResearch in the PharmaceuticalSciences, College of Pharmacy, Universityof Illinois at Chicago, Chicago, IL 60612, USA). FOREST PLOT AS A TOOLTO DEM- ONSTRATETHE PHARMACEUTICALPOTENTIAL OF PLANTS IN A TROPICALFOREST OF PANAMA. Eco- nomic Botany 54(3):278-294, 2000. Based on literatureanalysis of 308 angiospermspecies inventoriedfrom a 50-hectareforest plot on Barro ColoradoIsland, Panama,40 species were selected and 80 samples (two samplesfor every species; leaf + twig and stembarksamples) were collectedfor investigationof their medicinal/pharmaceuticalpotential. Extractsof these 80 samples were tested in bioassay systems to assess cancer chemoprevention(eight assays), antiplasmodial,cytotoxicity, and anti-HIVactivities. Details of bioassay techniquesare described. Of the 40 species, 12 (30%) showed activityin one or more of the 11 bioassay systems used. These active species are Bombacopsis (= Pachira)quinata, Calophyllum longifolium, Caseariacommersoniana, Lozania pittieri, Maclura tinctoria, Mouriri myrtilloides, Olmedia aspera (= Trophiscaucana), Pseudobombax septenatum, Spondias radlkoferi, Stylogyne standleyi, Turpiniaoccidentalis, and Vochysia ferruginea.Because literaturedata on the chemistryof Bombacopsis (= Pachira)quinata, Lozania pittieri, Mouririmyrtilloides, Olmedia aspera (= Trophiscaucana), Pseudobombax septenatum, and Stylogyne standleyi,are lacking,and similar data on the other six species are deficient,further fractionation and isolation work on these active species potentially may yield novel, biologically active structures.This study demon- strates that a plot-based selection of plant species for evaluationof their potential medicinal/ pharmaceuticalvalue has merit in achievingsuch a goal, and should be used in a programon plant drug discovery.

PARCELADE BOSQUE COMOUN DISENO PARADEMOSTRAR EL POTENCIALMEDICINAL/FARMACEUTICO DE PLANTAS DE UN BOSQUE TROPICALDE PANAMA. A base de andlisis de 308 especies de an- giospermas inventariadasde una parcela de 50 hectareas de un bosque en la Isla de Barro Colorado,Panama, 40 especiesfueron seleccionadasy 80 muestras(hojas +ramitas y corteza de tallo, de cada especie) fueron coleccionadaspara la investigacionhacia su potencial medicinal/farmacetitico.Los extractos de estas 80 muestrasfueron suministradosa bioensayos para detectar sus actividades en la prevenci6n de cancer (8 tipos de ensayos), antipaluidica, citotoxicidad,y anti-HIV.Los detalles de los metodos de bioensayos se presentan.De las 40 especies, 12 (30%) demostraronactividad en uno o mds de los 11 bioensayosempleados. Estas especies son Bombacopsis (= Pachira)quinata, Calophyllum longifolium, Caseariacommer- soniana, Lozania pittieri, Macluratinctoria, Mouriri myrtilloides, Olmedia aspera (= Trophis caucana),Pseudobombax septenatum, Spondias radlkoferi, Stylogyne standleyi,Turpinia occidentalis, y Vochysia ferruginea.En vista de que Bombacopsis (= Pachira)quinata, Lozania pittieri,Mouriri myrtilloides, Olmedia aspera (= Trophiscaucana), Pseudobombax septenatum, y Stylogyne standleyi,carecen de datos quimicosde la literatura,mientras que datos quzmicos sobre las otras seis especies son deficientes,un trabajode fraccionamientoy aislamientosobre estas especies potencialmentenos pueda dar compuestosnovedosos, biologicamenteactivos. Este estudio demuestraque el metodo de selecci6n de especies de plantas a partir de especies

EconomicBotany 54(3) pp. 278-294. 2000 ?) 2000 by The New York BotanicalGarden Press, Bronx, NY 10458-5126 U.S.A.

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inventariadas de una parcela de bosque, para someterlas a pruebas biol6gicas, tiene sus mer- itos para empleo mds amplio. Key Words: Panama;plot-based plant selection;pharmaceutical potential; biological activity; bioassays;cancer chemoprevention;antiplasmodial; cytotoxicity; anti-HIV.

In view of the increasingneed for investigat- is among the 25 most plant-richcountries in the ing plants of the tropical rain forests for their world (World ConservationMonitoring Centre nontimber economic potential, the present re- 1992:84). For the presentresearch, a list of iden- search was undertaken.In the present study, the tified angiospermspecies as a basis for selection potential economic value of plants from a trop- was obtainedfrom the 1990 census data of a 50- ical rain forest of Panama was assessed, focus- hectareforest plot on the BarroColorado Island ing on medicinal and pharmaceuticalvalue, by (BCI) tropical rain forest (Foster, Condit, and carrying out biological evaluation of samples Hubbell 1991). Similar plot-basedmethodology collected, utilizing a biodiversity-basedplant se- for the purposeof biological evaluationhas been lection approach. Specifically, plants were se- described and used previously (BohLke 1997; lected and collected from a large plot that had Bohlke et al. 1996; Horgen 1997; Horgen et al. been already established and inventoried. Al- 1997; Soejartoet al. 1996). though a numberof selection approaches(Soe- Barro Colorado is an island in the Panama jarto 1996) may be utilized in a program to Canal Zone, lying midway between the Atlantic screen plants for biological activities, a plot- and Pacific Oceans. At about 15.6 kM2, BCI is based selection approachwas utilized based on the largest island in Gatun Lake, which was the following rationale:(1) The results of such formedbetween 1911 and 1914 by the damming an approach may provide semiquantitativere- of the ChagresRiver to the Canal. BCI was set sults (percentof active species) on the potential aside as a biological preserve in 1923 and is pharmaceuticalvalue of plants found within the currentlysupervised by the SmithsonianTropi- plot, and hence, in a forest tract;(2) Recollection cal ResearchInstitute, which operatesa modem of active species, even from sterile plants, may field station at the LaboratoryClearing, on the be performed using the and still-standing northeast shore (Leigh, Rand, and Windsor marked plants in the plot as a living reference 1982). The vegetation on Barro Colorado has (Soejarto 1991). mature(> 500 years old) semievergreentropical In the present study, the choice of a Pana- forest (Fosterand Brokaw 1982). Thereare 1207 maniantropical rain forest was based on the fol- species of vascularplants in BCI, including409 lowing criteria:(a) high plant diversity in these tree and shrub species (Hubbell forests (D'Arcy 1987; Gupta 1995); (b) avail- and Foster 1992). A previous field ability of a large forest plot and census data study by a team of ecol- ogists at the (Foster and Hubbell 1990; Foster, Condit, and SmithsonianInstitution's Tropical Hubbell 1991); (c) availability of botanist col- Research Station on this island in the early laboratorsin Panama; (d) familiarity with the 1980s (Hubbell and Foster 1983) showed that country.Proof of a high biodiversityis given by 108 species of trees with diametersof 20 cm and the total numberof vascularplant species in this above were found in a 50-hectareplot. A census country,estimated between 8000 and 10 000, of in 1990 of free-standingwoody plants of 1 cm which 17.3% are considered endemic (Gupta diameter at breast height (dbh) from the same 1995). Among the nonvascularplants are 350 50-hectareplot yielded 308 species. Such an as- mosses, plus an unknownnumber of hepaticand sortmentof plant species was consideredan ap- hornwortspecies (D'Arcy 1987). Thus, Panama propriateamount of diversity in searching for compoundswith biological activities that could be of potentialpharmaceutical value. ' Received 5 March 1999; accepted 22 November To match this biodiversity-basedapproach in 1999. the selection of plantsto study,a broadspectrum 2 Part of a M.Sc. thesis in Pharmacognosy,Depart- ment of Medicinal Chemistry and Pharmacognosy, of biological activities was chosen for screening College of Pharmacy,University of Illinois at Chicago, 80 plant extracts.The rationalefor the selection Chicago. of the bioassays was to find new leads for dis-

This content downloaded by the authorized user from 192.168.52.67 on Mon, 10 Dec 2012 14:47:28 PM All use subject to JSTOR Terms and Conditions 280 ECONOMICBOTANY [VOL. 54 covering medicinal agents for diseases that have TABLE 1. NINETY-ONE FIRST-PRIORITY SPECIES shown high demographicincidence around the SELECTED FOR FURTHER STUDY FROM A POOL OF world, but for which no appropiatedrugs are 303 FREE-STANDING WOODY SPECIES FOUND IN 1 availablefor currenttherapies. The details of the HECTARE FOREST PLOT IN BARRO COLORADO IS- biological assays selected are discussed under LAND. the Materialsand Methods section. Speciesa (family) MATERIALS AND METHODS Adelia trilobab (Euphorbiaceae) LITERATURESEARCH AND PRIORITIZATIONOF Allophylus psilospermusc (Sapindaceae) PLANTSFOR COLLECTION Amaioua corymbosab (Rubiaceae) Literaturedata from previous biological stud- Anaxagorea panamensisc (Annonaceae) ies on every plant species in the 1990 census of Bertiera guianensisc (Rubiaceae) Calophyllum longifoliumc (Guttiferae) the 50-hectareplot of the tropicalrain forest on Capparis frondosac (Capparidaceae) the BarroColorado Island were queriedfrom the Casearia commersonianac (Flacourtiaceae) NAPRALERTdatabase (which summarizesthe Cassipourea ellipticac (Rhizophoraceae) world literature on medicinal uses, chemistry, Cavanillesia platanifoliab (Bombacaceae) and bioactivity of plants; Loub et al. 1985). Chimarrhis parvifiorab (Rubiaceae) Based on the amountof data availablefor a par- Chrysochlamys eclipesc (Guttiferae) ticularspecies and on data from previous studies Clidemia septuplinerviac (Melastomataceae) of different species of the same genus, certain Conostegia bracteatab (Melastomataceae) species were excluded and otherswere classified Conostegia cinnamomeab (Melastomataceae) into various priorities for collection. To avoid Cordia lasiocalyxc (Boraginaceae) Coussarea curvigemmiac missing crucial informationthat had been pub- (Rubiaceae) Cyphomandra hartwegiid (Solanaceae) lished in the literature,scientific synonyms for Dendropanax stenodontus (= arboreus)c (Araliaceae) each species were verified, and each synonym Desmopsis panamensisb (Annonaceae) was also queried from the NAPRALERTdata- Diospyros artanthifoliac (Ebenaceae) base. Subsequently,a list of plants was created Drypetes standleyic (Euphorbiaceae) according to three priorities depending on the Eugenia nesioticac (Myrtaceae) amount of informationavailable for each spe- Faramea talamancarumc (Rubiaceae) cies. These were: (a) plants that appearnever to Garcinia intermediac [Syn.: Rheedia edulis] have been studied chemically and/or pharma- (Guttiferae) cologically (91 species, Table 1); (b) those that Guapira (= Pisonia) standleyanac (Nyctaginaceae) Guatteria have been studied chemically and/or pharma- dumetorumc (Annonaceae) Gustavia superbac (Lecythidaceae) cologically, but need furtherstudies (24 species, Hampea appendiculatac (Malvaceae) Table 2); and (c) those that have been exten- Heisteria concinnac (Olacaceae) sively studied both chemically and pharmaco- Koanophyllon wetmoreic (Compositae) logically, to the extent that no additionalstudies Lacmellea panamensisc (Apocynaceae) appearto be warranted(four species, Table 3). Lafoensia punicifoliac (Lythraceae) Of the 308 species of trees with a diameterof Leandra dichotomac (Melastomataceae) 1 cm at breastheight (dbh), which represent183 Licania platypusd (Chrysobalanaceae) genera from the 50-hectare forest plot, 119 Lindackeria laurinac (Flacourtiaceae) (38%) species were found to be of interest for Malpighia romeroanac (Malpighiaceae) Marila laxiflorac (Guttiferae) biological evaluation,based on available ethno- Miconia hondurensisc (Melastomataceae) medical, chemical, and pharmacologicaldata. Miconia impetiolarisc (Melastomataceae) The criteriaused for exclusion of the remaining Mouriri myrtilloidesb (Melastomataceae) species were the amountof pharmacologicaland Myrospermum frutescensc (Leguminosae) chemical data, and the time and budget consid- Nectandra cissiforac (Lauraceae) erations for plant collection and biological test- Neea amplifoliac (Nyctaginaceae) ing. Ochroma pyramidalec (Bombacaceae) Ocotea oblongac (Lauraceae) PLANT COLLECTIONSTRATEGY Olmedia aspera (= Trophis caucana)b (Moraceae) The list of 91 plants of priorityone (Table 1) Pavonia (= Lopimia) dasypetalac (Malvaceae) was sent to the SmithsonianTropical Research Pentagonia macrophyllac (Rubiaceae)

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TABLE 1. CONTINUED. TABLE 2. SECOND PRIORITY SPECIES FROM A POOL OF 303 FREE-STANDING WOODY SPECIES FOUND IN Speciesa(family) 1 HECTARE FOREST PLOT IN BARRO COLORADO IS- Perebea xanthochymac (Moraceae) LAND. Picramnia latifoliac (Simaroubaceae) Piper perlasensec (Piperaceae) Speciesa(family) Pithecellobium (= Abarema) macradeniumc Aegiphila panamensisb (Verbenaceae) (Leguminosae) Alibertia edulisc (Rubiaceae) Platymiscium pinnatumc (Leguminosae) Alseis blackianal (Rubiaceae) Platypodium elegansb (Leguminosae) Annona hayesiic (Annonaceae) Pochota (= Bombacopsis; Pachira) quinatab Annona spragueic (Annonaceae) (Bombacaceae) Apeiba tibourboub (Tiliaceae) Pochota (= Bombacopsis; Pachira) sessilisb Brosimum alicastrume (Moraceae) (Bombacaceae) Casearia guianensisb (Flacourtiaceae) Poulsenia arnatab (Moraceae) Cespedesia macrophyllab (Ochnaceae) Pourouma bicolorc (Moraceae) Cupania rufescensb (Sapindaceae) Prioria copaiferab (Leguminosae) Guarea guidoniad (Meliaceae) Protium panamensec (Burseraceae) Hamelia axillarisb (Rubiaceae) Pseudobombax septenatumb (Bombacaceae) Hasseltia floribundac (Flacourtiaceae) Quararibea asterolepisc (Bombacaceae) Herrania purpureac (Sterculiaceae) Quiina schippiic (Quiinaceae) Lacistema aggregatumb (Flacourtiaceae) Randia armatac (Rubiaceae) Ormosia amazonicac (Leguminosae) Rinorea sylvaticac (Violaceae) Ouratea lucensb (Ochnaceae) Schefflera morototonic (Araliaceae) Phoebe (= Cinnamomum) cinnamomifoliac Sloanea terniflorac (Elaeocarpaceae) (Lauraceae) Sorocea affinisc (Moraceae) Posoqueria latifoliac (Rubiaceae) Spachea membranaceab (Malpighiaceae) Schizolobium parahybumc (Leguminosae) Spondias radlkoferic (Anacardiaceae) Stemmadenia grandifoliae (Apocynaceae) Stylogyne standleyib (Myrsinaceae) Triplaris cumingianab (Polygonaceae) Tachigali versicolor (Leguminosae) Zanthoxylum belizense (= ekmanii)c (Rutaceae) Talisia princepsc (Sapindaceae) Zuelania guidoniae (Flacourtiaceae) Ternstroemia tepezapotec (Theaceae) Tetrathylacium johanseniib (Flacourtiaceae) a Nomenclaturefollows the Floraof Panama. I Tocoyena pittieric (Rubiaceae) Speciesis in NAPRALERT,with eitherbiological or chemicaldata. c Species is in NAPRALERT,chemical data only. Trattinnickia aspera (Burseraceae) d Species is in NAPRALERT,with biologicaland ethnodata. Trichanthera gigantead (Acanthaceae) e Species is in NAPRALERT,chemical and ethnomedicaldata. Trophis racemosad (Moraceae) Turpinia occidentalisc (Staphyleaceae) Unonopsis pittieric (Annonaceae) Vismia billbergianac (Guttiferae) Vochysia ferrugineac (Vochysiaceae) Xylopia macranthac (Annonaceae) Xylosma oligandrumc (Flacourtiaceae) TABLE 3. THIRD PRIORITY SPECIES FROM A POOL a Nomenclaturefollows the Floraof Panama,checklist and index. (Vol OF 303 FREE-STANDING WOODY SPECIES FOUND IN 2, 1987). Genusis not in NAPRALERT. 1 HECTARE FOREST PLOT IN BARRO COLORADO IS- Species is not in NAPRALERT,but genus is. LAND. d Species is in NAPRALERT,with ethnomedicaldata only. Speciesa(family)

Aphelandra sinclairianab (Acanthaceae) Institute (STRI) for review, to determine the Jacaranda copaiac (Bignoniaceae) ease of their collection in adequatequantities for Swartzia simplexc (Leguminosae) biological testing (300-500 g dry weight per Symphonia globuliferab (Guttiferae) sample). Forty species were determined to be a Nomenclaturefollows the Floraof Panama. relatively easy to collect in testing quantities. b Species is in NAPRALERT,with biological,chemical and ethnomed- ical data. From a biodiversitypoint-of-view, these 40 spe- c Species is in NAPRALERT,with both biologicaland chemicaldata, cies are taxonomicallydiverse, consisting of 39 but withoutethnomedical data.

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TABLE 4. PLANTS COLLECTED IN PANAMA IN 1995, SELECTED FROM NINETY-ONE FIRST-PRIORITY SPECIES, BASED ON LOGISTICAL AND SPECIES ABUNDANCE CONSIDERATIONS.a

Species (Family) (voucherspecimen) Place, collectors,and date of collection Adelia triloba (Muell.-Arg.)Hemsl. Districtof Panama,Soberania Park, 100 m from the entranceof Euphorbiaceae the Plantation trail, 9?04' N. lat. and 79039' W. long. FLORPAN#2105 S. Aguilar and A. Castillo 05/26/95 Allophyluspsilospermus Raldk. Districtof Panama,Gamboa, 5 km from entranceof Oleoducto Sapindaceae trail, 9?08' N. lat. and 79?44' W. long. FLORPAN#2120 S. Aguilar and A. Castillo 06/02/95 Amaioua corymbosaHBK. Districtof Panama,Pelado hill (Gamboa)9?07' N. lat. and Rubiaceae 79043' W. long. FLORPAN#2117 S. Aguilar and A. Castillo 06/01/95 Annona spraguei Saff. Districtof Panama,Plantation trail, SoberaniaNational Park, Annonaceae approximately 9?04' N. lat. and 79?39' W long. FLORPAN#2091 S. Aguilar,A. Hernandezand A. Calderon05/17/95 Bombacopsis(= Pachira) quinata (Jacq.) Districtof Panama,Soberania National Park, 100 m of the en- Dugand trance of the Plantation trail, 9?04' N. lat. and 79039' W. Bombacaceae long. FLORPAN#2115 S. Aguilar and A. Castillo 06/01/95 Bombacopsis(= Pachira) sessilis (Benth.) Districtof Panama,between Paraisoand summit,9?03' N. lat. Pitt. and 79?38' W. long. Bombacaceae S. Aguilar and A. Castillo 05/26/95 FLORPAN#2103 Calophyllumlongifolium Willd. Districtof Panama,300 m from the entranceof Cruces trail, Guttiferae 9007' N. lat. and 79038' W. long. FLORPAN#2107 S. Aguilar and A. Castillo 05/29/95 Casearia commersonianaCamb. Districtof Chorrera,beside the fence of the BarroColorado Flacourtiaceae monument,9?06' N. lat. and 79?52' W. long. FLORPAN#2124 S. Aguilar and A. Castillo 06/09/95 Cavanillesia platanifolia HBK. District of Arraijan, Cocoli, 8?58' N. lat. and 79037' W. long. Bombacaceae S. Aguilar and A. Castillo 05/31/95 FLORPAN#21 10 Coussarea curvigemmia Dwyer District of Arraijan, Cocoli, 8?58' N. lat. and 79037' W. long. Rubiaceae S. Aguilar,A. Hernandezand A. Calderon05/22/95 FLORPAN#2093 Desmopsispanamensis (Rob.) Saff. Districtof Chorrera,beside the fence of the BarroColorado Annonaceae monument,9?06' N. lat. and 79?52' W. long. FLORPAN#2122 S. Aguilar and A. Castillo 06/05/95 Gustaviasuperba (HBK.) Berg. Districtof Panama,Soberania National Park, 9004' N. lat. and Lecythidaceae 79039' W. long. FLORPAN#2101 S. Aguilar and A. Castillo 05/26/95 Heisteria concinna Standl. District of Arraijan, Cocoli 8058' N. lat. and 79037' W. long. Olacaceae S. Aguilar and A. Castillo 05/23/95 FLORPAN#2095 Lacmelleapanamensis (Woods.) Markgr. Districtof Chorrera,beside fence of the BarroColorado monu- Apocynaceae ment, 9?06' N. lat. and 79?52' W. long. FLORPAN#2121 S. Aguilarand A. Castillo 06/05/95 Lindackerialaurina Presl Districtof Panama,Gamboa, entrance to Oleoductotrail, 9?05' Flacourtiaceae N. lat. and 79039' W. long. FLORPAN#2100 S. Aguilarand A. Castillo 05/25/95 Lozaniapittieri (Blake) L.B. Sm. Districtof Capira,Campana hill beside trail to formerforest Flacourtiaceae guardhouse, 8?41' N. lat. and 79?56' W. long. FLORPAN#2128 S. Aguilar and A. Castillo 06/12/96

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TABLE4. CONTINUED.

Species (Family) (voucherspecimen) Place, collectors,and date of collection Luehea seemanniiTr. et P1. Districtof Arraijan,Cocoli 8?58' N. lat. and 79?37' W. long. Tiliaceae S. Aguilarand A. Castillo 05/31/95 FLORPAN#2109 Maclura tinctoira (L.) D. Don ex Steud. District of Arraijan, Cocoli 8?58' N. lat. and 79037' W. long. Moraceae S. Aguilarand A. Castillo 05/31/95 FLORPAN#2113 Miconia impetiolaris(Sw.) D. Don Districtof Panama,Plantation trail, SoberaniaNational Park, Melastomataceae approximately9?04' N. lat. and 79039' W. long. FLORPAN#2090 S. Aguilar,A. Hernandezand A. Calderon05/17/95 Mouririmyrtilloides (Sw.) Poir. Districtof Chorrera,beside fence of the BarroColorado Monu- Melastomataceae ment, 9006' N. lat. and 79052' W. long. FLORPAN#2126 S. Aguilarand A. Castillo 06/09/95 Ochroma pyramidale (Cav. ex Lam.) Ur- District of Arraijan, Cocoli 8058' N. lat. and 79037' W. long. ban S. Aguilarand A. Castillo 05/31/95 Bombacaceae FLORPAN#2112 Olmediaaspera (= Trophiscaucana) Districtof Panama,entrance to the Cruces trail 9?06' N. lat. R. et P. and 79037' W. long. Moraceae S. Aguilar and A. Castillo 05/29/95 FLORPAN#2106 Pentagonia macrophyllaBenth. Districtof Panama,Gamboa, 1 km from Oleductotrail, 9?05' Rubiaceae N. lat. and 79039' W. long. FLORPAN#2097 S. Aguilar and A. Castillo 05/25/95 Perebea xanthochymaKarst. Districtof the Chorrera,beside fence of the BarroColorado Moraceae monument, 9006' N. lat. and 79052' W. long. FLORPAN#2125 S. Aguilar and A. Castillo 06/09/95 Picramnia latifolia Tul. District of Arraijan, Cocoli, 8?58' N. lat. and 79037' W. long. Simaroubaceae S. Aguilar and A. Castillo 05/31/95 FLORPAN#21 14 Poulsenia armata (Miq.) Standl. Districtof Panama,Gamboa, 1 km from the Oleductotrail, Moraceae 9?05' N. lat. and 79039' W. long. FLORPAN#2098 S. Aguilarand A. Castillo 05/25/95 Prioria copaifera Griseb. Districtof the Chorrera,beside fence of the BarroColorado Leguminosae Monument,9?06' N. lat. and 79?52' W. long. FLORPAN#2127 S. Aguilar and A. Castillo 06/09/95 Protiumpanamense (Rose) I. M. Johnst. Districtof Panama,Gamboa, 1 km from the Oleductotrail, Burseraceae 9?05' N. lat. and 79039' W. long. FLORPAN#2099 S. Aguilar and A. Castillo 05/25/95 Pseudobombaxseptenatum (Jacq.) Dugand Districtof Panama,between Paraisoand summitof 9?03' N. Bombacaceae lat. and 79?38' W. long. FLORPAN#2102 S. Aguilar and A. Castillo 05/26/95 Garcinia (= Rheedia) edulis (Seem.) Districtof Chorrera,beside fence of the BarroColorado monu- P1. et Tr. ment, 9?06' N. lat. and 79?52' W. long. Guttiferae S. Aguilar and A. Castillo 06/09/95 FLORPAN#2123 Rinorea sylvatica (Seem.) Kuntze District of Panama, the Cruces trail, 9?06' N. lat. and 79037' W. Violaceae long. FLORPAN#2108 S. Aguilarand A. Castillo 05/29/95 Schefflera morototoni (Aubl.) Maguire, District of Arraijan, Cocoli, 8?58' N. lat. and 79037' W. long. Stey. et Frod. S. Aguilar,A. Hernandezand A. Calderon05/22/95 Araliaceae FLORPAN#2094

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TABLE 4. CONTINUED.

S(pecies (amily) (voucherspecimen) Place,collectors, and date of collection Sorocea affinis Hemsl. Districtof Arraijan,Cocoli, 8?58' N. lat. and 79?37' W. long. Moraceae S. Aguilar,A. Hernandezand A. Calderon05/23/95 FLORPAN#2096 Spondias radlkoferi J.D. Sm. District of Arraijan, Cocoli, 8?58' N. lat. and 79037' W. long. Anacardiaceae S. Aguilar and A. Castillo 05/31/95 FLORPAN#21 11 Stylogynestandleyi Lund. Districtof Panama,Soberania National Park, 100 m of the en- Myrsinaceae trance of the Plantation trail, 9004' N. lat. and 79039' W. FLORPAN#2116 long. S. Aguilarand A. Castillo 06/01/95 Tachigali versicolorStandl. et L.O. Wms. Districtof Panama,Pelado hill (Gamboa)9?07' N. lat. and Leguinosae 79043' W. long. FLORPAN#2118 S. Aguilar and A. Castillo 06/01/95 Tetrathylaciumjohansenii Standl. Districtof Arraijan,Cocoli, 8?58' N. lat. and 79037' W. long. Flacourtiaceae S. Aguilar,A. Hernandezand A. Calderon05/22/95 FLORPAN#2092 Turpiniaoccidentalis (Sw.) G. Don Districtof Capira,Campana hill beside the trail to the former Staphyleaceae forest guardhouse. 8?41' N. lat. and 79?56' W. long. FLORPAN#2129 S. Aguilarand A. Castillo 06/12/95 Vismiabillbergiana (Buerl.) Districtof Panama,Pelado hill (Gamboa),9?07' N. lat. and Guttiferae 79043' W. long. FLORPAN#2119 S. Aguilarand A. Castillo 06/01/95 Vochysia ferruginea Mart. District of Panama, highway to Gamboa, 9003' N. lat. and Vochysiaceae 79038' W. long. FLORPAN#2104 S. Aguilar and A. Castillo 05/26/95 aTwAosamples were collectedfrom each species:LP = leaf + twig sample,and SB = stembarksample. genera in 22 families (Table 5). After a collec- prevent sample molding and deterioration,due tion budget was established,collection work and to the high humidityin the Panamaniantropical identification of the plants collected were un- rain forest. For every tree species collected, four dertakenby staff botanists of STRI. sets of voucher herbariumspecimens, fully labeled, were preparedto documentthe plant sam- PLANT COLLECTION ples. One set of duplicate specimens remainsat Samples of the 40 species were collected in STRI (SCZ), one set was sent to the herbarium an area outside of the plot within a 50-mile ra- of the Field Museumin Chicago (F), one set was dius (to reduce the of probability chemical var- kept at the CIFLORPANHerbarium in Panama iability) on Barro Colorado Island (Table 4). City, and one set was sent to the National Her- This sampling method was used because Barro barium of the Smithsonian Institution (US), ColoradoIsland is a NatureMonument, and col- Washington,DC. lection in an experimentalplot was not allowed. This restriction attempts to strengthen conservation efforts in that experimentalarea, in the CHEMICAL EXTRACTION extractionof plant resources. For each species, two different samples were collected, namely, a The processing and extractionof plant mate- leaf + twig sample (1 sample) and a stembark rial (consisting of 80 samples, representing40 sample (1 sample); each sample was labeled us- species) were carriedout in the ResearchCenter ing the collector'scollection numberand the part for Pharmacognostic Studies on Panamanian of the plant collected. Samples were dried in a Flora (CIFLORPAN)at the University of Pana- herbariumdryer at 30-50?C with electric fans ma. Methanolextraction of 20 g of each sample for air circulation.This measurewas intendedto was performedovernight.

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TABLE 5. TAXONOMICDIVERSITY OF PLANT SAM- inductionof phase II enzymes that detoxify car- PLES COLLECTEDAND INVESTIGATED. cinogens, based on the direct assay of the activity of quinone reductase.The induction of qui- Number of Number of Family genera species none reductasewas based on the procedurede- scribed by Prochaska, Santamaria,and Talaly Anacardiaceae 1 1 (1992). In brief, the extractswere evaluatedfor Annonaceae 2 2 the induction of reductase in Apocynaceae 1 1 quinone Hepa Araliaceae 1 1 lclc7 cells, and their cytotoxicity was assessed Bombacaceae 4 5 throughthe determinationof proteinwith crystal Burseraceae 1 1 violet staining. Euphorbiaceae 1 1 Inhibition of TPA-InducedOrnithine Decar- Flacourtiaceae 4 4 boxylase Activity. The aim of this assay is to Guttiferae 3 3 look for potentialplant extracts that could inhibit Lecythidaceae 1 1 phorbol ester-induced ornithine decarboxylase Leguminosae 2 2 (ODC) activity in cell culture. Both ODC en- 2 2 Melastomataceae zyme activity and the resulting polyamines are Moraceae 5 5 Myrsinaceae 1 1 essential for cellular proliferation of normal Olacaceae 1 1 mammalian cells; however, they are overex- Rubiaceae 3 3 pressed in various cancer cells (Auvinen et al. Sapindaceae 1 1 1992). Simaroubaceae 1 1 The ornithinedecarboxylase activity was as- Staphyleaceae 1 1 sayed directlyin 24-well plates using mouse epi- Tiliaceae 1 1 dermal cells, line 308, derived from adult Balb/ Violaceae 1 1 c mouse skin by measuring the release of Vochysiaceae 1 1 [14C]CO2 from L-[1-'4C]ornithineas described TOTAL 22 39 40 by Lichti and Gottesman(1982), and the cytotoxicity of the extractstowards the cell line was measuredby proteindetermination. AntioxidantActivity. This assay detects poten- SELECTIONOF BIOLOGICALASSAYS tial antioxidantactivity of plant extractsthrough Bioassays were selected to represent diseases their scavenging activity of the stable 1,l-diphe- with a high incidence of occurrence in the nyl-2-picrylhydrazyl(DPPH) free radicals in- world. These include cancer chemopreventive, dependentlyof any enzymaticactivity (Smith et antiplasmodial, cytotoxicity, and anti-HIV re- al. 1987). verse transcriptase assays. Bioassays were per- This assay was performed according to the formed in the Bioassay Research Facility of the method carriedout by Smith (1982; Smith et al. PCRPS, College of Pharmacy, University of Il- 1987). The reaction mixtures containing plant linois at Chicago. extractsand DPPH ethanolic solution in 96-well microtiterplates were incubatedat 37?C for 30 CANCERCHEMOPREVENTIVE ASSAYS min. The absorbancewas measuredat 515 nm Antimutagenicity Assay. This assay is based (Blois 1958). on the ability of mutated bacteria (Salmonella Inhibition of Cyclooxygenase Activity. Oxi- typhimurium strain TM677) to form colonies in dants have an importantrole in the promotion the presence of the purine analog 8-azaguanine of carcinogenesis(Pezzuto 1995). They activate (8-AG). The assay was carried out in Petri dish- phospholipaseA2, leading to productionof ar- es as described previously (Skopek et al. achidonicacid. Arachidonicacid, in turn,serves 1978a,b). A decrease in the mutantfraction due as a substratefor cyclooxygenase, whose func- to the presence of a test substance, compared to tion is to catalyze the productionof prostaglan- the mutagen alone, indicates antimutagenic ac- dins. Cyclooxygenase activity is considered to tivity. be relatedto tumorpromotion as exemplifiedby Induction of Quinone Reductase with Cul- the fact that the inhibitionof TPA-inducedODC tured Hepa ICIC7 Cells. This is a simple sys- activity by indomethacinis overcome by con- tem for rapid detection and measurementof the currentapplication of PGE2(Verma, Ashendew,

This content downloaded by the authorized user from 192.168.52.67 on Mon, 10 Dec 2012 14:47:28 PM All use subject to JSTOR Terms and Conditions 286 ECONOMICBOTANY [VOL. 54 and Boutwell 1980). For instance, prostaglan- tion in Mammary Organ Culture. The develop- dins contribute to the growth and possible ment of preneoplasticalveolar lesions in mam- spreadof tumorsin humancolon carcinogenesis mary gland organ cultureis an attractivemodel (Marnett1992). by which to evaluate the efficacy of potential Cyclooxygenase activity was determinedby chemopreventiveagents prior to an in vivo car- monitoringthe co-oxidation of a peroxidaseco- cinogenesis study (Mehta and Moon 1986). substrate as a prescreen of samples.The con- The extracts,at a concentrationof 10 [ug/mL, sumptionof oxygen was used to confirmthe in- were tested in mammaryglands from four-week hibition of cyclooxygenase activity by active old balbc/cmice in organculture (McCormick and samples. The procedureadopted was described Moon 1986) with some hormones.The percentage by Kulmacz and Lands (1987). of inhibitionof mammarylesions producedby Induction of Differentiation with HL-60 Cells. DMBA (7,12-dimethylbenz(a)anthracene)was as- This assay is used to search for agents that in- sessed. duce terminaldifferentiation in HL-60 cells. The induction of granulocytes, monocytes, eosino- ANTIPLASMODIAL ASSAY phils, or macrophage-likecells, withouttoxicity, This microdilutionmethod was developed for is a unique responsenot detectedin other assays measuringthe activity of potential antimalarial in this panel (Pezzuto 1995). drugs against culturedintraerythrocytic asexual The plant extracts were evaluated for induc- forms of the humanmalaria parasite (Desjardins tion of HL-60 cell differentiationthrough the et al. 1979). The quantitativemeasurements of following assays: the reductionof nitrobluetet- the antiplasmodialactivity in this assay is based razolium (NBT) (indicative of monocytes and on the inhibitionof uptakeof a radiolabelednu- granulocytes), which shows the ability to pro- cleic acid precursorby the parasiteduring short- duce superoxide when challenged with phorbol term culturein microtiterplates. ester. The method was described by Ostrem et The antiplasmodialactivity of extractswas as- al. (1987) and by Sokoloski et al. (1993). The sessed with an in vitro radioisotope-incorpora- nonspecific/specificesterase assay is indicative tion method.Concentrations of both extractsand of monocytes and macrophage-like cells and standardsthat inhibited parasite-specificincor- monocytes and other cell types, respectively,de- poration of [3H]hypoxanthineby 50% (IC50) scribed by Zhou et al. (1989) and Sokoloski et were determinedby nonlinearregression analy- al. (1993). The inhibition of [3H] thymidinein- sis. Zero drug controls indicate 100% incorpo- corporationwas intendedto evaluatethe level of ration (Desjardinset al. 1979). HL-60 cell proliferation,through the determination of the [3H]thymidineincorporation into CYTOTOXICITY ASSAY the DNA (Collins et al. 1978). This assay is a rapidand sensitive methodthat Estrogenic and Antiestrogenic Activity with was developed for in vitro anticancerscreening Ishikawa Cells. This test exploits the hormone- by measuringthe cellular proteincontent of hu- dependent characteristicof human breast can- man epidermoidcarcinoma (KB) culturesin 96- cers. This is evaluatedby measuringagents that well microtiter plates (Likhitwitayawuidet al. are capable of displacing estrogen, which may 1993; Skehanet al. 1990). The KB cell line was demonstrateeither antagonisticor agonistic ac- purchasedfrom the AmericanType CultureCol- tivity (Pezzuto 1995). lection (Rockville, MD). This bioassay utilized the Ishikawahuman en- dometrialadenocarcinoma cell line grown in 96- INHIBITION OF HIV-1 REVERSE well plates. The estrogenic activity was mea- TRANSCRIPTASE ASSAY sured by alkaline phosphatase,which is mark- The evaluationfor the inhibitionof HIV-1 re- edly stimulatedby estrogens in these cells, so verse transcriptaseis a useful approachto find- that the enzyme can be quantifiedin situ using ing new naturalproducts that could be specific a chromogenicsubstrate. Antiestrogenic activity inhibitors.Attention is focused on this enzyme was determinedby blockage of the action of es- because it is specific for the replicationand in- tradiol.The protocol followed was describedby fectivity of retrovirusesincluding HIV. The viral Pisha and Pezzuto (1997). RT has no counterpartin the human cell, con- Inhibition of DMBA-Induced Lesion Forma- sequently,it is unique to the virus.

TABLE 6. SUMMARYOF CHEMOPREVENTIVEACTIVITIES OF PROMISINGPANAMANIAN PLANTS.

HL-60 Anti- Cox-Ia cell mutg Part (% Anti-oxb QRc activity' (% MMOChb Species Family tested inhib) IC50 CDd/IC50 Cje ED50 inhib) (% inhib) Bombacopsis (= Pachira) quinata (Jacq.)Dugand Bombacaceae SB 18.9 Calophyllumlongifolium Willd. Guttiferae LP 17.6 23 Casearia commersoniana Camb. Flacourtiaceae SB 20.0 Lozaniapittieri (Blake) L.B. Sm. Flacourtiaceae SB 79 Maclura tinctoria (L.) D. Don ex Steud. Moraceae LP 3.8/19.1 = 5 29 42.8 Mouririmyrtilloides (Sw.) Poir Melastomataceae SB 17.7 42.8 Pseudobombaxseptenatum (Jacq.)Dugand Bombacaceae LP 12.6 Spondias radlkoferiJ.D. Sm. Anacardiaceae LP 77 7.14 37.5 Vochysia ferruginea Mart. Vochysiaceae LP 72

IC50and ED50values are expressedin pug/mL: a Inhibitionof cyclooxygenaseusing polarographicmethod. b DPPHfree radicalscavenging activity. c Inductionof quinonereductase. dConc. of 2-fold of quinonereductase. e Chemopreventiveindex. f Inductionof HL-60cell differentiation. O Antimutagenicactivity. I Inhibitionof DMBA-inducedlesion formationin mammaryorgan culture.

This assay was conductedas describedby Tan tracts were sent to the PCRPS in Chicago. A et al. (1991). For screening, all plant extracts complete list of the voucher herbariumspeci- initially were tested for enzyme inhibitionwith mens and of the localities of the 40 species is homodimer p66 enzyme. Extracts with inhibi- presentedin Table 4. For each species, data on tion of >50% were then tested for the presence the specific name, the family, the type of sam- of polyphenolic compounds (tannins) utilizing ples collected, the voucher herbariumspecimen FeCl3reagent. Tannins were removedfrom plant number (FLORPAN),their exact collecting lo- extracts using insoluble polyvinylpyrrolidone calities, the habitat,and the collector's name are (PVP). Then, extracts were tested for enzyme given. inhibition of the dimer p66/pSi. Calculationof IC50 values for active extracts with or without EVALUATION OF PANAMANIAN PLANTS IN tannins was performed. CANCER CHEMOPREVENTIONASSAYS Bioassay results are presented in Table 6. RESULTS Types of bioassays are indicated in the column Based on the methodology described above, heading and abbreviatedas follows: COX 1 = 80 plant samples from 40 different species of cyclooxygenase assay with polarigraphicoxygen floweringplants, distributedin 39 generaand 22 electrode; ANT-OX = DPPH free radical scav- families were selected from the 50-hectareforest enging activity (antioxidantactivity); QR = in- plot. Collection of samples (Table 4) was per- ductionof quinonereductase with culturedHepa formed during 17 May through 12 June, 1995, lclc7 cells; HL-60 = induction of HL-60 cell each sample in the amount of 300-500 g dry differentiation;ANTMUT = antimutagenicac- weight. Half (40) of the samples collected con- tivity; MMOC = inhibition of DMBA-induced sisted of leaf + twig and the other half of stem- lesion formationin mammaryorgan culture. bark. These samples were extractedand the ex- Inhibition of Cyclooxygenase Activity (COX

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1). The 80 extracts were tested for their ability of four days. The criterionof activity used was to inhibitcyclooxygenase activity,using the col- a response rate of >40%. When the concentra- orimetricmethod. Fifty-nine extracts were active tion was doubled (40 pRg/mL),leaf + twig of in the co-substrateassay with IC50 values < 25 Calophyllum longifolium exhibited an ED50 val- jig/mL and tested for their ability to inhibit cy- ue of 17.6 ,ug/mL and leaf + twig of Pseudo- clooxygenase activity using the polarographic bombax septenatum exhibited an ED50 value of oxygen electrode method. The extractsactive in 12.6 sg/mL (Table 6). the oxygen consumption assay at 70 ,ug/mL Inhibition of DMBA-Induced Lesion Forma- were: stembarkof Lozaniapittieri (79% inhibition in Mammary Organ Culture (MMOC). Nine tion), leaf + twig of Spondias radlkoferi (77% extracts that were active in at least one of the inhibition), and leaf + twig of Vochysia ferru- previous assays were tested in this assay with ginea (72% inhibition) (Table 6). the objective of findingpromising chemopreven- Antioxidant Activity (ANTI-OX). Several sam- tive activity. Maclura tinctoria (leaf + twig) and ples exhibitedhigh antioxidantactivity with IC50 Mouriri myrtilloides (stembark) exhibited mod- values of less than 20 ,ug/mLout of 80 samples erate activity, each with inhibition of 42.8% in tested. These samples were: leaf + twig of Spon- the DMBA-inducedlesions in mammaryorgan dias radlkoferi,active at 7.14 g/mL; stembarkof culture, based on a cut-off point for activity of Mouriri myrtilloides, at 17.7 ,ug/mL; and stem- 55% (Table 6). bark of Bombacopsis (= Pachira) quinata, at Based on the results presentedabove, as sum- 18.9 ,ug/mL(Table 6). marizedin Table 6, and on considerationof lit- Inhibition of TPA-Induced Ornithine Decar- eraturedata, two species, Maclura tinctoria(leaf boxylase Activity (ODC). In the TPA-induced + twig) and Mouriri myrtilloides (stembark), ornithine decarboxylase activity assay, the cri- may be considered first priority candidates as < terion for activity is defined as IC50values of potential chemopreventive agents, due to the 4 the ,ug/mL.Consequently, none of 80 extracts good correlationthat exists between the induc- exhibited significantactivity (Table 6). tion of quinonereductase activity, antimutagenic Induction of Quinone Reductase with Cul- activity, and the inhibition of DMBA-induced tured Hepa IC] C7 Cells (QR). From 80 extracts lesion formation in mammary organ culture. tested, the leaf + twig sample of Maclura tinc- Furthermore,in the case of M. myrtilloides, toria exhibitedtwo-fold inductionof quinonere- based on the present study, a relationshiphas ductase activity at 3.8 pg/mL and IC50of 19-1 been observed between antioxidant and ,ug/mL, with a correspondingchemopreventive activity index of 5 (Table 6). inhibitionof DMBA-inducedlesion formationin a Antimutagenicity (ANTIMUT). Ten extracts mammaryorgan culture. showing activity in antioxidant,cyclooxygenase The second priority comprises two species inhibition, cell differentiation,and quinone re- that have been shown to be active in at least two ductase induction assays were tested in the an- assays, other than the above. The first species is timutagenicityassay. The criterion for activity Calophyllum longifolium (leaf + twig), which was given by >25% inhibitionof mutagenicity. exhibited significant activity in the HL-60 cell Eight out of 10 extracts did not show activity, differentiationassay and a weak activity in the whereas leaf + twig extract of Calophyllum lon- antimutagenicityassay, and the second, Spon- gifolium (23%) and leaf + twig extract of Ma- dias radlkoferi (leaf + twig), showed antioxi- clura tinctoria (29%) showed a weak inhibitory dant activity as well as inhibition of cyclooxy- activity (Table 6). genase. Estrogenic and Antiestrogenic Activity with The thirdpriority comprises species that were Ishikawa Cells (ISHIK). None of the extracts active in only one of the assays. These include showed activity in the displacementof estrogen Bombacopsis (= Pachira) quinata (stembark) binding to the estrogen receptorin the Ishikawa and Casearia commersoniana (stembark), each cell assay. of which was active in the antioxidantactivity Induction of HL-60 Cell Differentiation (HL- assay, and Lozania pittieri (stembark) and Voch- 60). Of 80 extractstested, initially, two samples ysia ferruginea (leaf + twig) were active in the showed weak activity in this assay, at a concen- inhibition of cyclooxygenase activity. The last tration of 20 ,ug/mL with an incubationperiod two species, by virtue of their cyclooxygenase

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TABLE 7. ANTIPLASMODIAL AND CYTOTOXIC ACTIVITIES OF EXTRACTS OF SELECTED PANAMANIAN PLANTS.

Part KB Clone D6 Clone W2 Species (family) tested ED,,b IC5C IC50C Annona spraguei Saff. (Annonaceae) SB >20 000 5100 4480 Calophyllumlongifolium Willd. (Guttiferae) LP >20 000 2760 2150 Calophyllumlongifolium Willd. (Guttiferae) SB >20 000 3840 3600 Cavanillesia platanifolia HBK. (Bombacaceae) SB >20 000 4780 5430 Lindackerialaurina Presl (Flacourtiaceae) LP >20 000 4720 3550 Lozaniapittieri (Blake) L.B. Smith (Flacourtiaceae) LP >20 000 4160 5030 Maclura tinctoria(L.) D. Don ex Steud. (Moraceae) SB >20 000 2680 2930 Olmedia aspera R. et P. (= Trophis caucana) (Moraceae) LP >20 000 2490 2030 Turpiniaoccidentalis (Sw.) G. Don (Staphyleaceae) SB >20 000 6350 8600 Antiplasmodialstandard drugs: Chloroquine 11 200 3.97 0.01 84.18 0.54 Quinine 7700 10.49 0.97 25.15 3.74 Mefloquine 3000 3.99 0.80 1.2 0.4 Artemisinin >20 000 4.52 0.56 2.72 0.74

a LP = leaf + twig; SB = stembark. bED50 valuesare expressedin ng/mL. c IC5,values are expressedin ng/mL.

inhibition,are possible candidatesas a source of with the homodimer p66 enzyme. All extracts anti-inflammatoryagents. that exhibited >50% inhibitionwere then tested for the presence of polyphenolic compounds EVALUATION OF PANAMANIAN PLANTS IN (tannins),by utilizing FeCl3reagent. All samples ANTIPLASMODIAL AND CYTOTOXICITY that containedtannins were treatedwith insolu- ASSAYS ble polyvinylpyrrolidine(PVP) and tested again. Of the 80 plant extracts examined in this This procedureremoved the tannins so the ac- study, only three exhibited activity, albeit weak, tivity of nontannin,inhibitory compounds could in the antiplasmodialassay (Table 7), namely, be detected. Because tannins and related com- leaf + twig of Calophyllum longifolium, with pounds have been reported as HIV-1 reverse IC50values of 2760 ng/mL for clone D6 and transcriptaseinhibitors (Lee et al. 1992), the 2150 ng/mL for clone W2; stembarkof Maclura PVP treatmentmakes possible the detection of tinctoria, with IC50values of 2680 ng/mL for novel anti-HIV compounds,other than the cur- clone D6 and 2930 ng/mL for clone W2; and rently known class of compounds (Tan et al. + leaf twig of Olmedia aspera (= Trophis cau- 1991). Samples that showed negative reaction cana), with values of 2490 ng/mL for clone IC50 for tannins and those showing positive reaction D6 and 2030 ng/mL for clone W2. The remain- for tannins, but that retainedthe HIV-1 RT in- ing extracts failed to show in vitro antiplasmo- hibitory activity after PVP treatment,were sub- dial activity against either of the Plasmodium jected to IC50 determinations.For falciparum clones tested. All extractswere eval- nontannin- uated for cytotoxicity with human epidermoid containingsamples, the results obtainedshowed carcinoma (KB) cells, and no cytotoxicity was IC50 values of 95 ,ug/mrLfor the leaf + twig observed at the test concentrationof 20 pug/ml sample of Lozania pittieri, 82 ,ug/mL for the (Table 7), which implies that any plant extracts stembarksample of Spondias radlkoferi,and 96 that exhibited activity in the antiplasmodialbio- ,ug/mLfor the leaf + twig sample of Stylogyne assay are relatively selective. standleyi (Table 8). For the active plant extracts that were positive for the presence of tannins, EVALUATION OF PANAMANIAN PLANTS FOR IC50 values were determined with the under- INHIBITION OF HIV-1 REVERSE standingthat the true IC50values would be less TRANSCRIPTASE ASSAY than the values observed. All the extracts evaluated in the HIV-1 RT The following tannin-containingspecies, Cal- assay initially were tested for enzyme inhibition ophyllumlongifolium (leaf + twig), Lozaniapit-

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TABLE 8. HIV- 1 REVERSE TRANSCRIPTASE INHIBITORY ACTIVITY OF EXTRACTS OF SELECTED PANAMANIAN PLANTSUSING P66-PS1 HETERODIMERENZYME.

% HIV-1 RT Species Family Part useda inhibition IC50(pLg/ml) Calophyllum longifolium Willd. Guttiferae LP 61.9 >5.04 Lozania pittieri (Blake) L.B. Sm. Flacourtiaceae LP 55.8b 95 Lozaniapittieri (Blake) L.B. Sm. Flacourtiaceae SB 99.8 >76 Pseudobombax septenatum (Jacq.) Dugand Bombacaceae LP 83.8 >87 Spondias radlkoferiJ.D. Sm. Anacardiaceae LP 98.8 >68 Spondias radlkoferiJ.D. Sm. Anacardiaceae SB 68.1b 82 Stylogyne standleyi Lund. Myrsinaceae LP 63.2b 96 Stylogyne standleyi Lund. Myrsinaceae SB 83.9 >92 Turpiniaoccidentalis (Sw.) G. Don Staphyleaceae LP 91.2 >82

aLP = leaf + twig; SB = stembark. Extracts not treated with PVP. Active extracts have >50% of inhibition.

tieri (stembark), Pseudobombax septenatum soniana, and Spondias radlkoferi. The total (leaf + twig), Spondias radkolferi(leaf + twig) numberrepresents 30% of the species studied. and Turpiniaoccidentalis (leaf + twig), all exhibited interestingIC50 values less than 90 ,ug/ DISCUSSIONAND CONCLUSIONS mL (Table 8). A biodiversity-basedplant selection approach In view of the low IC50value of Calophyllum for the biological evaluation of a pool of taxo- longifolium (Table 8), it may be concluded that nomically diverse tree species found in a Pana- this species (leaf + twig) might be more potent manianforest plot, followed by bioassay of sam- than plants that contain no tannins,thus making ples of these plants in variousbioassay systems, it a high-prioritycandidate for bioassay-guided has successfully led to the identificationof 12 fractionation. of 40 species (30%) that showed activity in a panel of 11 biological assays. SUMMARY OF BIOLOGICAL ACTIVITIES Plot-based plant selection has the following The overall occurrence of biological activity advantages.Firstly, a forest plot provides an ad- in species studied in each of the assays is sum- equately diverse assortmentof species and gen- marized in Tables 9-A and 9-B. As can be seen era in a well defined area, which, in turn, pro- from these tables, in eight of the biological sys- vides an adequatechemical diversityas basis for tems tested, at least one species showed activity. biological screening. The area of a forest plot, Absence of activity was observed in the Ishi- 0.1-1 hectare,is large enough,yet small enough, kawa cell assay, TPA-inducedornithine decar- to allow the performanceof an efficient sam- boxylase assay, and cytotoxicity assay. Some of pling method on the plant species diversity. A the species scored as active were active in more collection of plants from a plot, even if only tree than one test system. When two extracts from species or woody species are targeted,can pro- one species were active, that species was count- vide a wide spectrumof taxonomicdiversity that ed as one. The same criteronwas used to score a general collecting survey does not normally the other three species with lower levels of ac- provide. Plot sampling can captureefficiently a tivity. broader spectrum of chemical diversity repre- The species showing activity in one or more sented within a particulartract of forest, as com- assays were Calophyllumlongifolium, Maclura pared to a general collecting survey. Secondly, tinctoria, Vochysia ferruginea, Pseudobombax a plot-based plant selection provides a semi- septenatum, Mouriri myrtilloides, Stylogyne quantitative result (percent of active species standleyi, Turpinia occidentalis, Bombacopsis from a well-definedunit of area)on the potential (= Pachira) quinata, Lozania pittieri, Olmedia pharmaceuticalvalue of plants found within the aspera (= Trophiscaucana), Casearia commer- plot. An extrapolationof such finding may lead

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TABLE 9-A. RESULTSOF BIOLOGICALEVALUATION OF ANGIOSPERMSPECIES COLLECTED IN PANAMA.

Test results COX-2a ANT-OX, ODCc QRd ISHIKe ANTMUT5 Active 3 3 0 1 0 0 Moderately active 0 19 0 0 0 2 Weakly active 5 2 0 0 0 0 Inactive 32 16 40 39 40 8 Subtotal 40 40 40 40 40 10

a Inhibition of cyclooxygenase with polarographic method. bDPPH free radical scavenging activity. c Inhibition of TPA-induced ornithine decarboxylase activity. d Induction quinone reductase. e Estrogenic and antiestrogenic activities with Ishikawa cells. I Antimutagenic activity. to a sound estimate of the medicinal or phar- ority" species, followed by furtherselection of maceutical potential of plants from the forest species for collection based on (a) ease of field tract. identification,(b) ease of collection, in terms of In a truly random,plot-based approach, using location, and (c) availabilityof material,namely, a small plot size, such as 0.1 hectare, where a size of plant, to allow the collection of 300-500 small numberof species is found, one can per- gramsdry weight. Collectionof these 40 species form bioassays on plant samples collected from was performedoutside the 50-hectare plot be- the pool of species found within this plot, even cause we were not allowed to collect marked without prior taxonomic determination.When and numberedplants/species within the experi- one or more species have been shown to be ac- mentalplot. This "terminal"species selection is tive in a particularbioassay, marked and num- equivalentto using markedand numberedplants bered trees within the plot would facilitate rec- in the plot as a referencefor the recollection of ollection of the active species. active species. In a plot of a 1-hectareor largersize, a larger The plant selection approach used in our numberof species is found (Whitmore1984:5). study could expand the possibility of finding To reduce such a large numberof species into a more than 30% of active species in the screening more manageablepool for biological evaluation, effort, if the number of plant species collected a preliminary"literature screening" to prioritize and screened was increased to include all 91 species following taxonomic determinationof species we consideredto be of "high priority." all species found within the plot should be per- In our study, the NAPRALERTdatabase in- formed using published ethnobotanicaland ex- dicates that of those species in our assays that perimental (chemical and biological/pharmaco- showed activity, Pseudobombax septenatum, logical) data. Such a method was used in our Mouriri myrtilloides,Stylogyne standleyi, Bom- study. We reduced a pool of 308 species found bacopsis (= Pachira) quinata, Lozania pittieri, in a 50-hectare plot to a pool of 91 "high pri- and Olmedia aspera (= Trophiscaucana) have

TABLE 9-B. RESULTS OF BIOLOGICAL EVALUATION OF ANGIOSPERM SPECIES COLLECTED IN PANAMA.

HL-60a KB Antiplasmodial HIV-1 RT Test results activity MMOC5 cytotoxicityc activity inhibitiond Active 1 2 0 0 9 Moderately active 1 1 0 3 0 Weakly active 0 0 0 0 0 Inactive 38 6 40 37 31 Subtotal 40 9 40 40 40

aInduction of HL-60 cell differentiation. bInhibition of DMBA-induced lesion formation in mammary organ culture. c Cytotoxicity assay with KB cells. I Inhibition of HIV-1 reverse transcriptase.

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no recordedphytochemical literature data. This College, Universityof Illinois at Chicago,Chicago, suggests that these species potentiallycould pro- IL, USA, 264 pp. vide novel chemical structuresif fractionation , H. Guinaudeau, C. K. Angerhofer, V. and isolation work were performed.The discov- Wongpanich, D. D. Soejarto, N. R.Farnsworth, G. A. ery of a novel, in vitro, active antimalarialcom- Mora, and L. J. Poveda. 1996. Costaricine, a new antiplasmodialbisbenzylisoquinoline alka- pound, costaricine, a bisbenzylisoquinolineal- loid from Nectandra salicifolia trunk bark. Journal kaloid, from Nectandra salicifolia (Lauraceae) of NaturalProducts 59:576-580. from a Costa Rican forest plot (Bohlke 1997; Collins, S. J., F.W. Ruscetti, R. E. Gallagher, and Bohlke et al. 1996) and of anothernovel cyto- R. C. Gallo. 1978. Terminaldifferentiation of hu- toxic compound, ardisenone,an alkeny phenol, man promyelocyticleukemia cells induced by di- from Ardisia iwahigensis (Myrsinaceae) from a methyl sulfoxide and other polar compounds.Pro- forest plot of Palawan (Philippines) (Horgen ceedings of the National Academy of Sciences 1997; Horgen et al. 1997), two species that had (USA) 75:2458-2462. not been studied chemically, has demonstrated D'Arcy, W. G. 1987. Flora de Panama,Checklist and that plot-basedplant selection has improvedthe Index. Missouri Botanical Garden,St Louis, Vol- ume 2, 670 pp. chances of finding novel compoundsfrom rela- Desjardins, R. E., C. J. Canfield, J. D. Haynes, and tively small, but taxonomically diverse lots of J. D. Chulay. 1979. Quantitativeassessment of an- species. timalarialactivity in vitro by a semiautomatedmi- Our research, and others employing plot- crodilution technique. AntimicrobialAgents and based plant selection method provide an effec- Chemotherapy16:710-718. tive approachin the selection of plants for bio- Foster, R. B., and N. Brokaw. 1982. Structureand logical evaluation. Such an approach may in- history of the vegetationof BarroColorado Island. crease the chances of discoveringnovel, biolog- Pages 67-81 in E. G. Leigh, A. S. Rand, and D. ically active compounds from plants of the M. Windsor,eds., The ecology of a tropicalforest, tropical rain forests, of which only a minuscule SmithsonianPress, Washington,DC. , and S. P. Hubbell. 1990. The floristiccom- percentage(estimated at less than 1%;Balick et position of a 50-ha plot on BarroColorado Island. al. 1996: xi) has been investigatedfor their me- Pages 85-98 in A. Gentry, ed., Four neotropical dicinallpharmaceuticalpotential. forests. Yale UniversityPress, New Haven, CT , R. Condit, and S. P. Hubbell. 1991. Data ACKNOWLEDGMENTS from 1990 Recensus of Barro Colorado(Panama) AIC expresses special gratitude to Fulbright LASPAU for the oppor- 50-Ha ExperimentalPlot. SmithsonianTropical Re- tunity to enrich and improve her scientific knowledge in the area of search Institute,Apartado 2072, Balboa, Republic Pharmacognosy through the award of a scholarship for her Master's de- of Panama. gree studies. Thanks are also expressed to Mary Lou Quinn, Executive Gupta, M. P. 1995. Panamanianflora: Source of bio- Director of NAPRALERT, for providing literature data from NAPRAL- ERT, and to the staff of the UIC Bioassay Research laboratory for per- active compounds.Pages 359-398 in K. Hostett- forming the biological assay procedures described herein. man, A. Marston,M. Mailard,and M. Hamburger, The present study has been supported in part by grant # R29 At34408 eds., Phytochemistryof plants used in traditional awarded by the National Institutes of Allergy and Infectious Diseases, medicine. 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