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Bioactive crude plant seed extracts from the NCAUR oilseed repository.

Summary

Over four-hundred crude extracts from 202 plant species distributed among 131 plant families were evaluated for their bioactivity against brine shrimp (Artemia salina). Activity was determined for both the organic [CH.sub.2] [CI.sub.2]:MeOH) and aqueous extracts against A. salina in a 96 well-plate assay. Of the greater than four-hundred extracts tested, 21 organic and 6 aqueous extracts demonstrated potent cytotoxic activity [LC.sub.50] = <100 [micro]g/ml). Three of these organic extracts (Crateva religiosa, Diospyros dichrophylla, and Olax subscorpioidea) were chosen for chemical investigations due to their high activity and a lack of prior investigations. Chemical analysis of these extracts resulted in the isolation of oleanolic acid (1) and 4-epi-hederagenin (2) from C. religiosa, isodiospyrin (3) from D. dichrophylla, and santalbic acid (4) from O. subscorpioidea.

Key words: cytotoxic, brine shrimp, Artemia salina, extract, plant, seed, Crateva religiosa, Diospyros dichrophylla, Olax subscorpioidea

Introduction

The National Center for Agricultural Utilization Research in Peoria, Illinois is home to a vast and diverse seed repository. This collection contains greater than 15,000 separate seed accessions from more than 130 plant families. It was originally established to identify and quantify the oil composition in a vast array of seeds which may prove economically beneficial. The material remaining after extraction of the oil from various varieties of seeds has often been shown to contain important protein and phytochemical components, such as those found in soy and walnuts.

Our intention is to use this diverse collection of seeds as a source of biologically active natural products with potentially unique structures and mechanisms of action. In general, two different species from every family within this collection were chosen for evaluation, the seeds were extracted with three increasing polarity extractions, and the cytotoxic activity of both the organic ([CH.sub.2][CI.sub.2]:MeOH, 1:1) and aqueous extracts against Arternia Salina was determined. It has been demonstrated that activity against A. sauna correlates well with cytotoxic and antiplasmodial activity (Meyer et al., 1982), as well as other pharmacological activities (Solis et al., 1993). Presented here is the brine shrimp screening data from over 400 crude extracts prepared from 202 plant species. In addition to screening data, the chemical investigation of three bioactive extracts will be discussed.

* Material and Methods

Plant material and extract preparation

Over four hundred different crude seed extracts were prepared from more than two-hundred different plant species collected at various locations throughout the world. The seeds are part of an oilseed repository housed in Peoria, Illinois where each collection has been given a unique identifier and authenticated by USDA taxonomists in Beltsville, Maryland.

Approximately 10 g of seeds from each species were ground in a coffee grinder (Braun, Type 4041). Ground material was placed in a 300 ml separatory funnel (glass wool above the stop-cock for filtration) and soaked in 200 ml of hexane for 18 hours at which time the solvent was drained and the extract concentrated by rotary evaporation. Seed material was allowed to air dry for 6 hours followed by the addition of 200 ml of [CH.sub.2][Cl.sub.2]:MeOH (1:1). After soalting for 18 hours, the solvent was drained and the extract concentrated by rotary evaporation. Lastly, the seed material was allowed to air dry for 6 hours at which time 150 ml of deionized water was added to the separatory funnel and soaked for 18 hours. The aqueous extract was drained, frozen, and placed on a lyophilizer until all water was removed. A portion of both the water and [CH.sub.2][Cl.sub.2]:MeOH extracts were analyzed in the brine shrimp toxicity screen. The hexane extract was not analyzed in this screen due to the insoluble nature of thi s extract in both DMSO and [H.sub.2]O. Prior to chemical investigations of biologically active extracts, approximately 500 g of seeds were extracted using the above procedure with solvent and flask size adjustments made to scale.

Fractionation of Crateva reilgiosa investigation

700 g of Crateva religiosa seeds were extracted as described above to yield hexane (2.0 g), [CH.sub.2][Cl.sub.2] MeOH (6.7 g), and [H.sub.2]O (35 g) extracts. A portion (5 g) of the [CH.sub.2][Cl.sub.2] MeOH, (1:1) extract was dissolved in 50 ml [CH.sub.2][Cl.sub.2] MeOH and applied to a flash chromatography column (5 cm x 50 cm) that had been packed with silica gel (170 g, Merck, 70-230 mesh, 40 A). The column was eluted with mixtures of increasing polarity of [CH.sub.2][Cl.sub.2] MeOH to produce active fraction B ([CH.sub.2][Cl.sub.2] MeOH, 8:2). A portion of fraction B (200 mg) was dissolved in 10 ml of [CH.sub.2][Cl.sub.2] MeOH (1:1) and applied to a flash column (3.5 cm x 100 cm) that had been filled with LH-20 (Sigma, Inc.) and equilibrated in [CH.sub.2][Cl.sub.2]:MeOH (1:1). The column was eluted with 11 of [CH.sub.2][Cl.sub.2]:MeOH (1:1) and passed through a UV-absorbance detector (ISCO V-4) monitoring at 254 nm. Fractions 3 and 4 were the only fractions of the 6 obtained that demonstrated activity ag ainst brine shrimp. Fraction 3 contained 39 mg of oleanolic acid (1). Fraction 4 (12 mg) was further purified by preparative TLC (EM Sciences, Silica Gel, 20 x 20 cm, 0.250 mm thick, [CH.sub.2][Cl.sub.2]:MeOH 9:1) to produce 8 mg of 4-epi-hederagenin (2). Compounds 1 and 2 were identified by comparison of (13) C-NMAR chemical shift data with that which was reported in the literature (Kizu and Tomimori, 1982; Nie et al., 1984; Agrawal and Jam, 1992).

Fractionation of Diospyros dichrophylla investigation

200 g of Diospyros dichrophylla seeds were separated into inner seed and hull. Inner seeds were extracted as described above to yield hexane (2.4 g), [CH.sub.2][Cl.sub.2]:MeOH (1:1, 9.2 g), and [H.sub.2]O (27 g) extracts. A portion (5 g) of the [CH.sub.2][Cl.sub.2]:MeOH, (1:1) extract was dissolved in 50 ml of [CH.sub.2][Cl.sub.2]:MeOH and applied to a flash chromatography column (5 cm x 50 cm) that had been packed with silica gel (170 g, Merck, 70-230 mesh, 40 A). The column was eluted with [CH.sub.2][Cl.sub.2]:MeOH mixtures of increasing polarity to yield active fraction B ([CH.sub.2][Cl.sub.2]:MeOH, 99:1). Fraction B (0.4 g) was further purified using a flash-chromatography column (5 cm x 50 cm) that had been packed with silica gel (140 g, Merck, 70-230 mesh, 40 A) and equilibrated in [CH.sub.2][Cl.sub.2]. The column was eluted with [CH.sub.2][Cl.sub.2] to produce 45 mg of active fraction 2 which was shown to be isodiospyrin (3) Compound 3 was identified by comparison of its (1) HNMR data with that which h ad previously been reported (Lillie and Musgrave, 1977).

Fractionation of Olax subscorpioidea investigation

98 g of Olax subscorpioidea seeds were ground as described above, placed in a Soxhlet extractor containing 11 of [CH.sub.2][Cl.sub.2], and exhaustively extracted for 18 h to yield 1.4 g of crude extract. This procedure was repeated using MeOH as the extracting solvent to give 5.8 g of extract. A portion (3.2 g) of the MeOH extract was dissolved in 20 ml of MeOH and applied to a flash chromatography column (5 cm x 50 cm) that had been packed with reverse phase silica gel (Waters, Inc., preparative C-18, 55-105 [micro]m, 125 A). The column was eluted with decreasing polarity mixtures of MeOH : [H.sub.2]O to produce 40 mg of an active fraction C (100% MeOH). Fraction C was further purified by reverse phase preparative HPLC (ODS-3,250 x 30 mm i.d., 30 ml/min, 5 [micro]m) using isocratic conditions of acetonitrile (0.025% TFA) : [H.sub.2]O (0.025% TFA) (8:2). This led to the purification of santalbic acid (4) (5.7 mg). Compound 4 was identified by mass spectroscopy and various one- and two-dimensional NMR spectros copic techniques.

Brine Shrimp Assay

The procedure described below is a modification of two previously published methods (Meyer et al., 1982; Solis et al., 1993). Brine shrimp eggs (Artemia salina) were obtained online (www.brineshrimpdirect.com) and stored at 10 [degrees]C until used. 400 mg of eggs were added to 11 of deionized water containing 40 g of sea salts (Sigma Chemical Co.). Air was bubbled through the resulting solution for 36 hours at 27 [degrees]C at which time the larvae (nauplii) were collected with a Pasteur pipette by attracting the organisms to one side of the vessel with a light source.

Test compounds, extracts, and fractions were originally made up at a concentration of 1 mg/ml in artificial seawater except for water insoluble compounds, which were dissolved in 50 [micro]1 of DMSO prior to adding enough water to make a 1 mg/ml solution. Serial dilutions were performed in 100 [micro]l of seawater in triplicate in the wells of a 96 well microplate to give appropriate concentrations. Control wells for both water and DMSO were included with each experiment. A 100 [micro]l solution of nauplii (containing 10-30 individuals) was added to each well and the covered plate incubated for 24 hours at 27 [degrees]C. The number of nonmotile and the total number of nauplii were counted in each well using a binocular microscope (x10). [LC.sub.50] values were then calculated by Probit analysis (Finney, 1971).

* Results

The results from screening seed extracts of 202 different plant species against A. salina are shown in Table 1. Both the organic and aqueous extractions from nearly all of the species have been screened and the corresponding [LC.sub.50] values are given. Approximately 28% of the organic extracts demonstrated activity at or below 1000 [micro]g/ml while only 5.6% of the aqueous extracts showed this level of activity. Furthermore, only 9.8% of the organic and 2.8% of the aqueous extracts demonstrated activity low enough ([LC.sub.50] = <100 [micro]/ml) to merit chemical investigation.

These very active ([LC.sub.50] = <100 [micro]g/ml) organic extracts include Anacardium occidentale, Annona reticulate, Garuga pinnata, Crateva religiosa, Cephalotaxus fortunei, Anogeissus pendula, Juniperus osteosperma, Dipterocarpus grandiflorus, Diospyros dichrophylla, Gnetum scandens, Mammea africana, Gonocaryum lobbianum, Strychnos aculeate, Melia azedarach, Mirabilis jalapa, Olax subscorpioidea, Ximenia americana, Pittosporum crassifolium, Plantago aristata, Albizia amara, and Strychnos potatorum. The very active aqueous extracts include Annona reticulate, Juniperus osteosperma, Myrica nagi, Mirabilis jalapa, Ximenia americana, and Gardenia turgida. Certain organic and aqueous extracts were chosen for chemical investigation only if a quick search of the literature suggested that particular species had not been previously investigated or it had been previously investigated but cytotoxic compounds had not been isolated.

In nearly all species extracted, if activity was shown in the aqueous extract, activity was also observed in the organic extract. The exceptions to this observation include Limnanthes douglasii, Myrica nagi, and Camellia japonica var. spontanea. The aqueous extracts from all three of these species were active ([LC.sub.50] = <1000 [micro]g/ml); however, their organic extracts were completely inactive.

Contrary to the above observation, if the organic extract was active, the aqueous extract was usually inactive. In fact, of the sixty organic extracts which demonstrated activity ([LC.sub.50] = <1000 [micro]g/ml), in only nine of these were their corresponding aqueous extracts also active ([LC.sub.50] = <1000 [micro]g/ml). These nine species consist of Acer ginnala, Annona reticulate, Anogeissus pendula, Juniperus osteosperma, Mammea africana, Melia toosendan, Mirabilis jalapa, Ximenia americana, and Gardenia turgida.

* Discussion

Of the 21 organic and 6 aqueous extracts that demonstrated strong activity against A. salina, only three of the organic extracts have been chosen for further investigation. Those three are Crateva religiosa, Diospyros dichrophylla, and Olax subscorpioidea. The organic extracts from all three species demonstrated activity below 100 [micro]g/ml and an initial examination of the literature indicated that no prior chemical investigations had been performed for cytotoxic compounds.

The organic extract of seeds from Crateva religiosa was highly active ([LC.sub.50] = 1.31 [micro]g/ml) against A. salina and a bioassay-guided fractionation was performed in an attempt to isolate the active compound(s). This extract was purified by flash column chromatography on silica gel followed by size-exclusion flash chromatography using LH-20 to yield oleanolic acid (1) and a second impure triterpene. This second compound was further purified by preparative TLC to yield 4-epi-hederagenin (2). Both compounds had been previously isolated from plants of different genera and their [C-NMR.sup.13] data were in full agreement with previously reported values (Kizu and Tomimori, 1982; Nie et al., 1984; Agrawal and Jan, 1992). Their [LC.sub.50] values against A. sauna are reported in Table 2.

Seeds from Diospyros dichrophylla were separated into inner seed and hulls. Both were extracted in an identical manner and it was shown that an organic extract of the inner seed was highly active ([LC.sub.50] = 29 [micro]g/ml) while the hull extracts were completely inactive. This organic extract was twice purified using silica gel flash column chromatography to produce the active compound isodiospyrin (3) ([LC.sub.50] = 0.13 [micro]g/ml). Isodiospyrin (3) was originally isolated from Diospyros mespiliformis and to our knowledge, this is the first report on its isolation from Diospyros dichrophylla. The structure of isodiospyrin (3) was elucidated by various one- and two-dimensional NMR spectroscopy techniques as well as comparison of its [H-NMR.sup.1] data with that which had previously been reported (Lillie and Musgrave, 1977).

Bioassay-guided investigation of the methanol extract from Olax subscorpioidea was performed due to its high activity against A. salina ([LC.sub.50] 44.8 [micro]g/ml). The extract was purified using reverse phase flash column chromatography followed by reverse phase HPLC to produce the active compound santalbic acid (4) ([LC.sub.50] = 26.3 [micro]g/ml). The structure of santalbic acid (4) was determined by mass spectroscopy and one- and two-dimensional NMR spectroscopic techniques. Santalbic acid (4) had been previously isolated from Santalum acuminatum and was shown to possess antibacterial activity (Jones et al., 1995). To our knowledge this is first report on the isolation of santalbic acid from Olax subscorpioidea.
Table 1

Toxicity of crude plant seed extracts against Artemia salina.

Family Species Plant Part



Acanthaceae Justicia simplex D. Don seed
Acanthaceae Monechma debile (Forsk.) Nees seed
Aceraceae Acer campestre L. seed
Aceraceae Acer ginnala Maxim. seed
Alangiaceae Alangium salviifolium (L.F.) seed
 Wangerin
Amaranthaceae Aerva sanguinolenta Blume seed
Amaranthaceae Amaranthus hybridus L. seed
Anacardiaceae Anacardium occidentale L. inner seed
Anacardiaceae Anacardium occidentale L. hull
Anacardiaceae Harpephyllum caifrum Bernh. seed
Annonaceae Annona reticulata L. seed
Annonaceae Monodora myristica Dunal seed
Apiaceae Coriandrum sativum L. seed
Apiaceae Eryngium campestre L. seed
Apocynaceae Thevetia peruviana (Pers.) seed
 K. Schum.
Aquifoliaceae Ilex insignis Hook. F. seed
Aquifoliaceae Ilex purpurea Hassk. seed
Arecaceae Phoenix reclinata Jacq. seed
Aristolochiaceae Aristolochia clematitis L. seed
Asclepiadaceae Asciepias incarnata L. seed
Asclepiadaceae Asciepias syriaca L. seed
Asteraceae Ageratum conyzoides L. seed
Asteraceae Carthamus tinctorius L. seed
Balsaminaceae Impatiens balsamina L. seed
Berberidaceae Podophyllum emodi Wall. ex Hook. F. seed
 and Thorns.
Betulaceae Carpinus betulus L. seed
Betulaceae Carpinus betulus L. seed
Bignoniaceae Campsis grandiflora (Thunb.) seed
 Loisel.
Bignoniaceae Catalpa speciosa Warder seed
Bombacaceae Adansonia digitata L. seed
Bombacaceae Durio pinangianus (Becc.) Ridley seed
Boraginaceae Cerinthe minor L. seed
Boraginaceae Echium plantagineum L. seed
Brassicaceae Brassica compestris L. seed
Brassicaceae Eruca sativa Mill. seed
Burseraceae Garuga pinnata Roxb. seed
Buxaceae Simmondsia chinensis (Link) seed
 C. Schneid.
Cactaceae Pachycereus pecten-aboriginum seed
 (Engelm.) Britt. et Rose
Calycanthaceae Chimonanthus praecox (L.) Link seed
Campanulaceae Lobelia inflata (L.) seed
Campanulaceae Prismatocarpus roelloides Sond. seed
Cannaceae Canna indica L. seed
Capparaceae Capparis spinosa L. seed
Capparaceae Crateva religiosa Forst. F. seed
Caprifoliaceae Sambucus ebulus L. seed
Caprifoliaceae Viburnum opulus L. seed
Caprifoliaceae Viburnum opulus L. seed
Caryocaraceae Caryocar villosum (Aubi.) Pers. inner seed
Caryocaraceae Caryocar villosum (Aubl.) Pers. hull
Caryophyllaceae Gypsophila paniculata L. seed
Caryophyllaceae Vaccaria pyramidata Medic. seed
Celastraceae Cassine crocea (Thunb.) O. Ktze. inner seed
Celastraceae Cassine crocea (Thunb.) O. Ktze. hull
Celastraceae Euonymus europaeus L. seed
Cephalotaxaceae Cephalotaxus fortunei Hook. seed
Chenopodiaceae Atriplex canescens (Pursch.) Nutt. seed
Chenopodiaceae Atriplex lentiformis (Torr.) Wats. seed
Cistaceae Lechea tenuifolia Michx. seed
Clusiaceae Calophyllum pulcherrimum Wall. seed
Clusiaceae Mainmea africana Sabine inner seed
Clusiaceae Manunea africana Sabine hull
Cochlospermaceae Cochlospermum vitifolium (Willd.) Spreng. inner seed
Cochlospermaceae Cochlospermum vitifolium (Willd.) Spreng. hull
Combretaceae Anogeissus pendula Edgew. seed
Combretaceae Terminalia arjuna (Roxb.) Wight and Am. seed
Convolvulaceae Convolvulus Incanus Vahi seed
Convolvulaceae Ipomoea cardiophylla Gray seed
Cornaceae Aucubajaponica Thunb. seed
Comaceae Cornus mas L. seed
Cucurbitaceae Luffa acutaungula Roxb. seed
Cupressaceae Juniperus communis L. seed
Cupressaceae Juniperus osteosperma (Torr.) Little seed
Cycadaceae Macrozamia reidlei (Fisch. ex Gaudich.) seed
Cyperaceae Carex diluta Bieb. seed
Cyperaceae Cyperus esculentus L. seed
Dipterocarpaceae Dipterocarpus grandiflorus Blanco seed
Dipterocarpaceae Shoreafloribunda Kurz seed
Ebenaceae Diospyros dichrophylla (Gand.) DeWint. hull
Ebenaceae Diospyros dichrophylla (Gand.) DeWint. inner seed
Ebenaceae Diospyros mespiliformis Hochst. exA. DC seed
Elaeagnaceae Hippophae rhamnoides L. seed
Ericaceae Arbutus unedo L. seed
Ericaceae Rhododendron ponticurn L. seed
Euphorbiaceae Bridelia retusa (L.) Spreng. seed
Euphorbiaceae Jatropha curcas L. seed
Eupteleaceae Euptelea polyandra Sieb. & Zucc. seed
Fagaceae Lithocarpus garrettianus (Craib) A. Camus seed
Fagaceae Quercus kerrii Craib seed
Flacourtiaceae Flacourtia indica (Burm.F.) Merrill seed
Flaourtiaceae Xylosma ion longifolium Clos. seed
Garryaceae Garryafremontii Torr. seed
Gentianaceae Chironia bacifera L. seed
Gentianaceae Jaeschkea gentianoides Kurz seed
Ginkgoaceae Ginkgo biloba L. seed
Gentaceae Gnetum scandens Roxb. seed
Heamodoraceae Lanaria plumosa Ait. seed
Hamamelidaceae Liquidambar styraciflua L. seed
Hippocastanaceae Aesculus glabra Willd. seed
Hippocastanaceae Aesculus octandra Marsh. seed
Hydrophyllaceae Phacelia integrifolia Torr. seed
Hydrophyllaceae Phacelia neomexicana Thuber ex Torr. seed
Icacinaceae Gonocaryurn lobbianum (Miers) Kurz seed
Iridaceae Alophia amoena (Griseb.) O. Ktze. seed
Iridaceae Watsonia pyramidata (Andr.) Stapf seed
Juglandaceae Carya ovata (Mill.) K. Koch seed
Juglandaceae Pterocarya stenoptera C. DC. seed
Juncaceae Juncus glaucus Ehrh. seed
Juncaceae Luzula spicata (L.) DC. seed
Lamiaceae Leonotis nepetaefolia (L.) R. BR. seed
Lardizabalaceae Akebia quinata Decne. seed
Lardizabalaceae Akebia trifoliata (Thunb.) Koidz. seed
Lauraceae Beilschmiedia tarairi (A. Cunn.) seed
 Benth. + Hook. F. ex Kirk
Lauraceae Litsea salicifolia Hook. F. seed
Lecythidaceae Barringtonia pterocarpa Kurz seed
Lecythidaceae Careya arborea Roxb. seed
Leeaceae Leea indica (Burm. F.) Merrill seed
Leguminosae Albizia amara (Roxb.) Boiv. seed
Leguminosae Bauhinia acuminata L. seed
Liliaceae Colchicum autumnale L. seed
Limnanthaceae Limnanthes douglasii R. BR. seed
Loganiaceae Strychnos aculeata Sol. seed
Loganiaceae Strychnos potatorum L.F. seed
Lythraceae Lagerstroemia parviflora Roxb. inner seed
Lythraceae Lagerstroemia parviflora Roxb. hull
Lythraceae Lagerstroemia tomentosa Presel. seed
Magnoliaceae Michelia cathcartii seed
 Hook. F. + Thoms,
Magnoliaceae Michelia champaca L. seed
Malpighiaceae Bunchosiapalmeri S. Wats. seed
Malpighiaceae Malpighia punicifolia Malpigh. seed
Malvaceae Malva parviflora L. seed
Malvaceae Malvastrum tricuspidatum A. Gray seed
Marantaceae Thaumatococcus daniellii seed
 (Benn.) Benth
Martyniaceae Martynia annua L. seed
Meliaceae Melia azedarach L. seed
Meliaceae Melia dubia Cav. seed
Meliaceae Melia toosendan Sieb. & Succ. seed
Meliaceae Toona sinensis A. Juss. seed
Moraceae Ficus hispida L. F. seed
Moringaceae Moringa oleifera Lam. seed
Musaceae Musa glauca Rozb. seed
Musaceae Musa paradisiaca L. seed
Myricaceae Myrica cordifolia Berg. seed
Myricaceae Myrica nagi Thunb. seed
Myristicaceae Pycnanthus angolensis seed
 (Wewl.) Exell
Myrsinaceae Ardisia japonica (Thunb.) Blume seed
Myrsinaceae Rapanea laetevirens Mez. seed
Nyctaginaceae Mirabilis jalapa L. seed
Nymphaeaceae Nelumbo lutea (Wilid.) Pers. seed
Nymphaeaceae Nuphar luteum L. seed
Olacaceae Olax subscorpioidea Oliv. seed
Olacaceae Ximenia americana L. seed
Oleaceae Fraxinus ornus L. seed
Onagraceae Oenothera strigosa seed
 (Rydb.) Mack & Bush
Orchidaceae Aerides odorata Lour. seed
Pandanaceae Pandanus tectorius seed
 Soland. ex Park.
Papaveraceae Papaver bracteatum Lindl. seed
Phytolaccaceae Phytolacca acinosa Roxb. seed
Phytolaccaceae Phytolacca dioica L. seed
Pinaceae Pinus torreyana Parry ex Carr. seed
Pittosporaceae Pittosporum crassifolium seed
 Banks + Sol. ex A. Cunn
Plantaginaceae Plantago aristata Michx. seed
Plantaginaceae Plantago major L. seed
Plumbaginaceae Limonium tartaricum seed
Plumbaginaceae Statice bahusiensis Fr. seed
Poaceae Aegilops triuncialis L. seed
Podocarpaceae Podocarpus falcatus (Thunb.) R. BR. seed
Podocarpaceae Podocarpus gracilior Pilger seed
Proteaceae Helicia erratica Hook F. seed
Punicaceae Punica granatum L. seed
Ranunculaceae Paeonia brownii Dougl. ex Hook. seed
Ranunculaceae Ranunculus arvensis L. seed
Resedaceae Caylusea abyssinica (Fres.) Fiscgh. seed
 and Mey.
Restionaceae Cannomois virgata (Rottb.) Steud. seed
Restionaceae Chondropetalum macrocarpum Pillans seed
Rhamnaceae Ziziphus mauritiana Lam. seed
Rosaceae Agrimonia eupatoria L. seed
Rosaceae Duchesnea indica (Andr.) Focke seed
Rubiaceae Coffea khasiana Hook. F. seed
Rubiaceae Gardenia turgida Roxb. inner seed
Rubiaceae Gardenia turgida Roxb. hull
Rutaceae Clausena indica Oliv. seed
Rutaceae Murraya koenigii (L.) Spreng. seed
Santalaceae Osyris alba L. seed
Santalaceae Santalum album L. seed
Sapindaceae Blighia sapida Konig seed
Sapindaceae Euphoria longana Lam. seed
Saxifragacene Montina caryophyllacea Thunb. seed
Saxifragaceae Montina caryophyllacea Thunb. hull
Scrophulariaceae Penstemon secundiflorus Benth. seed
Simaroubaceae Ailanthus excelsa Roxb. seed
Simaroubaceae Picramnia nitida Engl. seed
Solanaceae Datura metel L. seed
Solanaceae Datura stramonium L. seed
Staphyleaceae Staphylea bumalda DC. seed
Staphyleacene Staphylea pinnata L. seed
Styracaceae Styrax serrulatum Roxb. seed
Symplocaceae Symplocos theaefolia D. Don seed
Tamaricaceae Tamarix gallica L. seed
Taxaceae Taxus baccata L. seed
Taxaceae Torreya nucifera (L.) Sieb. & Zucc. seed
Taxodiaceae Taxodium distichum L. seed
Theaceae Camellia japonica var. spontanea seed
 L. Nak.
Theaceae Camellia sinensis (L.) O. Kuntze seed
Tiliaceae Triumfetta pilosa Roth. seed
Tropaeolaceae Tropaeolum majus L. seed
Typhaceae Typha angustifolia L. seed
Uapacaceae Uapaca kirkiana Muell. Arg. seed
Ulmaceae Zelkova serrata (Thunb.) Makino seed
Urticaceae Girardinia condensata (Hochst. ex seed
 Steud.) Wedd.
Urticaceae Urtica dioica L. seed
Verbenaceae Tectona grandis L. F. inner seed
Verbenaceae Tectona grandis L. F. hull
Zingiberaceae Amomum subulatum Roxb. seed
Zygophyllaceae Larrea tridentata (DC.) Colville seed
Zygophyllaceae Tribulus terrestris L. seed

Family [LC.sub.50] value [LC.sub.50] value
 ([micro]g/ml) (b) ([micro]g/ml) (b)
 Organic (a) Aqueous

Acanthaceae 200 >1000
Acanthaceae >1000 >1000
Aceraceae 259 >1000
Aceraceae 274 201
Alangiaceae 190.3 >1000

Amaranthaceae >1000 >1000
Amaranthaceae >1000 >1000
Anacardiaceae 90 >1000
Anacardiaceae 1.89 >1000
Anacardiaceae >1000 >1000
Annonaceae 1.15 84
Annonaceae >1000 >1000
Apiaceae >1000 >1000
Apiaceae 869 >1000
Apocynaceae 316 >1000

Aquifoliaceae 818 >1000
Aquifoliaceae >1000 >1000
Arecaceae >1000 >1000
Aristolochiaceae >1000 >1000
Asclepiadaceae >1000 >1000
Asclepiadaceae >1000 >1000
Asteraceae >1000 >1000
Asteraceae >1000 >1000
Balsaminaceae >1000 >1000
Berberidaceae >1000 >1000

Betulaceae >1000 >1000
Betulaceae >1000 >1000
Bignoniaceae >1000 >1000

Bignoniaceae >1000 >1000
Bombacaceae >1000 >1000
Bombacaceae 834 >1000
Boraginaceae >1000 >1000
Boraginaceae >1000 >1000
Brassicaceae >1000 >1000
Brassicaceae >1000 >1000
Burseraceae 51.3 >1000
Buxaceae >1000 >1000

Cactaceae >1000 >1000

Calycanthaceae >1000 >1000
Campanulaceae >1000 >1000
Campanulaceae >1000 >1000
Cannaceae >1000 >1000
Capparaceae >1000 >1000
Capparaceae 1.31 >1000
Caprifoliaceae >1000 >1000
Caprifoliaceae >1000 >1000
Caprifoliaceae >1000 >1000
Caryocaraceae >1000 >1000
Caryocaraceae >1000 >1000
Caryophyllaceae >1000 >1000
Caryophyllaceae >1000 >1000
Celastraceae >1000 >1000
Celastraceae >1000 >1000
Celastraceae 861 >1000
Cephalotaxaceae 58 >1000
Chenopodiaceae 182 >1000
Chenopodiaceae >1000 >1000
Cistaceae >1000 >l000
Clusiaceae 852.27 >1000
Clusiaceae 11.1 960
Clusiaceae 302.6 >1000
Cochlospermaceae >1000 >1000
Cochlospermaceae >1000 >1000
Combretaceae 55 127
Combretaceae 915 >1000
Convolvulaceae 212 >1000
Convolvulaceae >1000 >1000
Cornaceae >1000 >1000
Comaceae >1000 >1000
Cucurbitaceae >1000 >1000
Cupressaceae >1000 >1000
Cupressaceae 90.3 96
Cycadaceae >1000 >1000
Cyperaceae 255 >1000
Cyperaceae >1000 >1000
Dipterocarpaceae 6.64 >1000
Dipterocarpaceae 107.9 >1000
Ebenaceae >1000 >1000
Ebenaceae 29 >1000
Ebenaceae >1000 >1000
Elaeagnaceae >1000 >1000
Ericaceae >1000 >1000
Ericaceae >1000 >1000
Euphorbiaceae >1000 >1000
Euphorbiaceae 230 >1000
Eupteleaceae >1000 >1000
Fagaceae >1000 >1000
Fagaceae >1000 >1000
Flacourtiaceae >1000 >1000
Flaourtiaceae >1000 >1000
Garryaceae >1000 >1000
Gentianaceae 331 >1000
Gentianaceae >1000 >1000
Ginkgoaceae >1000 >1000
Gentaceae 17.1 >1000
Heamodoraceae >1000 >1000
Hamamelidaceae >1000 >1000
Hippocastanaceae 112 >1000
Hippocastanaceae 533 >1000
Hydrophyllaceae >1000 >1000
Hydrophyllaceae 645 >1000
Icacinaceae 78 >1000
Iridaceae >1000 >1000
Iridaceae >1000 >1000
Juglandaceae >1000 >1000
Juglandaceae >1000 >1000
Juncaceae >1000 >1000
Juncaceae >1000 >1000
Lamiaceae 185.23 >1000
Lardizabalaceae >1000 >1000
Lardizabalaceae 136.39 >1000
Lauraceae >1000 >1000

Lauraceae >1000 >1000
Lecythidaceae 259 >1000
Lecythidaceae >1000 >1000
Leeaceae >1000 >1000
Leguminosae 73.39 >1000
Leguminosae >1000 >1000
Liliaceae >1000 >1000
Limnanthaceae >1000 606.2
Loganiaceae 67.8 >1000
Loganiaceae 90.4 >1000
Lythraceae >1000 >1000
Lythraceae >1000 >1000
Lythraceae >1000 >1000
Magnoliaceae >1000 >1000

Magnoliaceae >1000 >1000
Malpighiaceae >1000 >1000
Malpighiaceae >1000 >1000
Malvaceae >1000 >1000
Malvaceae >1000 >1000
Marantaceae >1000 >1000

Martyniaceae 262 >1000
Meliaceae 67 >1000
Meliaceae 366 >1000
Meliaceae 102 731.23
Meliaceae >1000 >1000
Moraceae >1000 >1000
Moringaceae >1000 >1000
Musaceae >1000 >1000
Musaceae >1000 >1000
Myricaceae >1000 >1000
Myricaceae >1000 50.4
Myristicaceae >1000 >1000

Myrsinaceae >1000 >1000
Myrsinaceae >1000 >1000
Nyctaginaceae 27.7 51.7
Nymphaeaceae >1000 >1000
Nymphaeaceae 313 >1000
Olacaceae 44.8 >1000
Olacaceae 67.6 64.7
Oleaceae >1000 >1000
Onagraceae >1000 >1000

Orchidaceae >1000 >1000
Pandanaceae >1000 >1000

Papaveraceae >1000 >1000
Phytolaccaceae >1000 >1000
Phytolaccaceae >1000 >1000
Pinaceae >1000 >1000
Pittosporaceae 86.5 >1000

Plantaginaceae 83.5 >1000
Plantaginaceae >1000 >1000
Plumbaginaceae >1000 >1000
Plumbaginaceae >1000 >1000
Poaceae >1000 >1000
Podocarpaceae >1000 >1000
Podocarpaceae >1000 >1000
Proteaceae >1000 >1000
Punicaceae >1000 >1000
Ranunculaceae >1000 >1000
Ranunculaceae >1000 >1000
Resedaceae >1000 -

Restionaceae >1000 >1000
Restionaceae >1000 >1000
Rhamnaceae >1000 >1000
Rosaceae >1000 >1000
Rosaceae 722 >1000
Rubiaceae >1000 >1000
Rubiaceae 147 75
Rubiaceae >1000 >1000
Rutaceae >1000 >1000
Rutaceae >1000 >1000
Santalaceae >1000 >1000
Santalaceae 130 >1000
Sapindaceae >1000 >1000
Sapindaceae >1000 >1000
Saxifragacene >1000 >1000
Saxifragaceae >1000 >1000
Scrophulariaceae >1000 >1000
Simaroubaceae 866 >1000
Simaroubaceae 734 >1000
Solanaceae >1000 >1000
Solanaceae >1000 >1000
Staphyleaceae >1000 >1000
Staphyleacene 965 >1000
Styracaceae 502 >1000
Symplocaceae 971 >1000
Tamaricaceae >1000 >1000
Taxaceae >1000 >1000
Taxaceae >1000 >1000
Taxodiaceae >1000 >1000
Theaceae >1000 756

Theaceae >1000 >1000
Tiliaceae >1000 >1000
Tropaeolaceae >1000 >1000
Typhaceae >1000 >1000
Uapacaceae >1000 >1000
Ulmaceae >1000 >1000
Urticaceae >1000 >1000

Urticaceae >1000 >1000
Verbenaceae >1000 >1000
Verbenaceae 500 >1000
Zingiberaceae >1000 >1000
Zygophyllaceae 827 >1000
Zygophyllaceae >1000 >1000

(a)[CH.sub.2] [Cl.sub.2] : MeOH (1:1)

(b)Positive control was cycloheximide ([LC.sub.50] = 40 [micro]g/ml)

Table 2

Cytotoxicity of isolated compounds against Artemia salina.

Source Compound [LC.sub.50]
 ([micro]g/ml) (a)

Crateva religiosa oleanolic acid 2.51
Crateva religiosa 4-epi-hederagenin 117
Diospyros dichrophylla isodiospyrin 0.13
Olax subscorpioidea santalbic acid 26.3

(a)Positive control was cycloheximide ([LC.sub.50] = 40 [micro]g/ml)


Acknowledgments

The authors thank Barry Jones, Ray Holloway, and Sejal Patel for technical assistance.

* References

Agrawal PK, Jam DC (1992) 13C NMR Spectroscopy of Oleanane Triterpenoids. Progress in NMR Spectroscopy 24:1-90

Fallas AL, Thomson RH (1968) Ebenaceae extractives. III. Binaphthaquinones from Diospyros species. 3 Chem Soc, C(18): 2279-2282

Finney DJ (1971) Probit analysis. Cambridge [Eng.], University Press

Jones GP, Rao KS, Tucker DJ, Richardson B, Barnes A, Rivett DE (1995) Antimicrobial activity of santalbic acid from the oil of Santalum acuminatum (Quandong). Int J Pharmacogn 33(2): 120-123

Kizu H, Tomimori T (1982) Studies on the constituents of Clematis species. V. On the Saponins of the Root of Clematis chinensis Osbeck. Chem Pharm Bull 30(9):3340-3346

Lillie TJ, Musgrave OC (1977) Ebenaceae extractives. Part 7. Use of Hydroxy-proton Shifts of Juglone Derivatives in Structure Elucidation. J Chem Soc, Perkin Trans 1:355-359

Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL (1982) Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med 45(1):31-34

Nie RL, Morita T, Kasai R, Zhou J, Wu CY, Tanaka O (1984) Saponins from Chinese medicinal plants. (I). Isolation and structures of hemslosides. Planta Med 50(4): 322-327

Solis PN, Wright CW, Anderson MM, Gupta MP, Phillipson JD (1993) A microwell cytotoxicity assay using Artemia salina (brine shrimp). Planta Med 59(3): 250-252

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Title Annotation:National Center for Agricultural Utilization Research, Illinois
Author:Cantrell, C.L.; Berhow, M.A.; Phillips, B.S.; Duval, S.M.; Weisleder, D.; Vaughn, S.F.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:May 1, 2003
Words:4662
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