This article was downloaded by [Karolinska Institute] On 7 August 2007 Access Details [subscription number 779857372] Publisher Informa Healthcare Informa Ltd Registered in England and Wales Registere[.]
This article was downloaded by:[Karolinska Institute] On: August 2007 Access Details: [subscription number 779857372] Publisher: Informa Healthcare Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Pharmaceutical Biology Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713721640 Large-scale in Vitro Screening of Egyptian Native and Cultivated Plants for Schistosomicidal Activity Online Publication Date: 01 July 2007 To cite this Article: Yousif, Fouad, Hifnawy, Mohamed S., Soliman, Gamil, Boulos, Loutfy, Labib, Therese, Mahmoud, Soheir, Ramzy, Fatem, Yousif, Miriam, Hassan, Iman, Mahmoud, Khaled, El-Hallouty, Salwa M., El-Gendy, Mohamed, Gohar, Lamiaa, El-Manawaty, May, Fayyad, Walid and El-Menshawi, Bassem S (2007) 'Large-scale in Vitro Screening of Egyptian Native and Cultivated Plants for Schistosomicidal Activity', Pharmaceutical Biology, 45:6, 501 - 510 To link to this article: DOI: 10.1080/13880200701389425 URL: http://dx.doi.org/10.1080/13880200701389425 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article maybe used for research, teaching and private study purposes Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material © Taylor and Francis 2007 Downloaded By: [Karolinska Institute] At: 13:55 August 2007 Pharmaceutical Biology 2007, Vol 45, No 6, pp 501–510 Large-scale in Vitro Screening of Egyptian Native and Cultivated Plants for Schistosomicidal Activity Fouad Yousif1, Mohamed S Hifnawy2, Gamil Soliman3, Loutfy Boulos4, Therese Labib5, Soheir Mahmoud1, Fatem Ramzy1, Miriam Yousif2, Iman Hassan1, Khaled Mahmoud6, Salwa M El-Hallouty6, Mohamed El-Gendy6, Lamiaa Gohar6, May El-Manawaty6, Walid Fayyad6, and Bassem S El-Menshawi6 Theodor Bilharz Research Institute, Cairo, Egypt; 2Faculty of Pharmacy, Cairo University, Cairo, Egypt; Faculty of Science, Cairo University, Cairo, Egypt; 4Faculty of Science, Alexandria University, Alexandria, Egypt; Orman Botanical Garden, Giza, Egypt; 6National Research Centre, Dokki, Giza, Egypt Abstract In vitro bioassay screening of 346 methanol extracts originated from 281 native and cultivated plant species growing in Egypt, and related to 81 families, was carried out for schistosomicidal activity The extracts were bioassayed at 100 mg=mL on viable Schistosoma mansoni mature worms in culture medium Viability of worms was examined after exposure for 24 h, and mortality determined Negative (DMSO) and positive (praziquantel) controls were used Of the tested plant extracts, 72 were found to possess reproducible in vitro antischistosomal activity These active extracts were further subjected to determination of their LC50 and LC90 values Strong antischistosomal activity was found in the extracts of 15 species (possessing LC50 ! 15 mg=mL), viz Agave americana L var marginata Trel (Agavaceae), A lophantha Schiede (Agavaceae), Furcraea selloa C.Koch (Agavaceae), Calotropis procera (Aiton) W.T.Aiton (Asclepiadaceae), Pergularia tomentosa L (Asclepiadaceae), Asclepias sinaica (Boiss.) Muschl (Asclepiadaceae), Alkanna orientalis (L.) Boiss (Boraginaceae), Khaya grandifoliola DC (Meliaceae), Swietenia mahogani (L.) Jacq (Meliaceae), Pimenta racemosa (Mill.) J.W.Moore (Myrtaceae), Pinus canariensis C.Sm (Pinaceae), Verbascum sinaiticum L (Scrophulariaceae), Solanum elaeagnifolium Cav (Solanaceae), S nigrum L (Solanaceae), and Brachychiton rupestris (Lindl.) K.Schum (Sterculiaceae) These 15 species could represent promising bioactive sources that deserve further investigation, with the aim of finding novel antischistosomal agents The current study represents the first report on a systematic screening of schistosomicidal activity utilizing a large number of plant species Keywords: Egyptian plants, in vitro bioassay, Schistosoma mansoni, schistosomicidal effect, screening Introduction Schistosomiasis represents the second most prevalent tropical disease affecting more than 200 million people worldwide It is a grave and debilitating disease of socioeconomic importance and is increasing in incidence despite concerted efforts to control and contain the disease in its endemic regions Although a multipronged method of control using health education, sanitation, and snail control has been used, chemotherapy and chemoprophylaxis play the most important and crucial role in containing=preventing the transmission of the disease Praziquantel is the mainstay of schistosomiasis control programs worldwide, and thus an enormous investment in terms of money and training rests solely on the efficacy of a single compound This extensive reliance on just one drug might represent a serious situation, due to the possible development of drug-resistant parasite (Ismail et al., 1999; Doenhoff et al., 2002) Therefore, there is now an urgent need for developing new antischistosomal drugs (Lambertucci et al., 1980; Souza et al., 1982) Accepted: December 12, 2006 Address correspondence to: Prof Bassem S El-Menshawi, Department of Pharmacognosy, National Research Centre, El-Tahrir Str., Dokki, Giza 12622, Egypt Tel.: þ 20-2-762-1363; Fax: þ 20-2-336-9603; E-mail: menshawi@soficom.com.eg DOI: 10.1080/13880200701389425 # 2007 Informa Healthcare Downloaded By: [Karolinska Institute] At: 13:55 August 2007 502 F Yousif et al Whereas herbal medicine has produced some very effective treatments for malaria, for example, quinine and recently artemisinin (Frederich et al., 2002), few attempts have been made to screen plants against schistosomiasis (Sparg et al., 2000; Molgaard et al., 2001) Accordingly, it would be useful to collect a large number of plant species and screen them for bioactivity against this widespread parasite To fulfill this objective, the current study has utilized a validated in vitro schistosomicidal bioassay to screen Egyptian plants (native and cultivated species) on S mansoni worms, under a collaborative research project, during 1998–2006 (El-Menshawi, 2003) Table Egyptian plants (native and cultivated) screened for antischistosomal activity in vitro S=N 2% 3% 4% 5% 8% 10 Materials and Methods Plant material Plant material used in this study amounts to 281 species that yielded 346 extracts from different plant organs as shown in Table Plant specimens were collected randomly Native species were collected from various areas of Egypt (namely, Mediterranean coastal region, Sinai, Red Sea coastal region, Nile Valley, Eastern and Western deserts, including the oases), and cultivated taxa were obtained from various botanical gardens (namely, Zoological Garden, Orman Garden, and Mansoria Garden, at Giza, and Aswan Botanical Garden, Aswan) Voucher specimens are deposited in the National Research Center (NRC-Plant Drug Discovery Herbarium), Dokki, Giza, Egypt The identification of native plants was carried out by Loutfy Boulos, and the nomenclature is in accordance with Boulos (1999, 2000, 2002, 2005) The identification of cultivated plants was done by Therese Labib, and the nomenclature follows Huxley et al (1992) Preparation of extracts A small quantity of each plant, sufficient to yield about 50 g dry weight, was collected for preliminary bioscreening Routine protection of natural plant constituents from denaturation, or artifact formation, during the extraction and concentration procedures was ensured during the preparation of crude extracts (El-Menshawi, 2003) Whole plant or plant part was dried in a solar oven at 40#C, ground, and extracted with methanol at ambient temperature by percolation Extracts were filtered and methanol was evaporated to dryness under reduced pressure and totally freed from water by freeze-drying and stored under freezing at $20#C until use Parasite material Schistosoma mansoni adult worms were obtained from the Schistosome Biological Supply Center at Theodor 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26% 27 28 29 30% 31 32 33% 34% 35 36% 37 38% 39 40 41 42% Plant name Family: Agavaceae Agave heteracantha Zucc Agave americana L var marginata Trel Agave angustifolia Haw Agave decipiens Bak Agave lophantha Schiede Agave macrantha Salm-Dyck Agave macroacantha Zucc Furcraea selloa C Koch Sansevieria trifasciata Prain Family: Aizoaceae Aptenia cordifolia (L.f.) Schwantes Family: Amaranthaceae Aerva javanica (Burm f.) Juss ex Schult Amaranthus caudatus L Amaranthus graecizans L Amaranthus viridis L Family: Amaryllidaceae Clivia miniata Regel Hippeastrum vittatum (L’ He´r.) Herb Family: Anacardiaceae Schinus terebenthifolius Raddi Family: Annonaceae Annona cherimolia Mill Annona cherimolia Mill Annona squamosa L Annona squamosa L Family: Apocynaceae Acokanthera oblongifolia (Hochst.) Codd Acokanthera oblongifolia (Hochst.) Codd Alstonia scholaris (L.) R Br Alstonia scholaris (L.) R Br Carissa carandus L Nerium oleander L variegata Nerium oleander L variegata Nerium oleander L variegata Thevetia peruviana (Pers.) Schum Thevetia peruviana (Pers.) Schum Family: Araliaceae Schefflera arboricola (Hayata) Hayata Family: Asclepiadaceae Calotropis procera (Aiton) W.T Aiton Calotropis procera (Aiton) W.T Aiton Cryptostegia grandiflora R.Br Pergularia tomentosa L Solenostemma arghel (Delile) Hayne Asclepias sinaica (Boiss.) Muschl Family: Basellaceae Anredera cordifolia (Ten.) Steenis Family: Bignoniaceae Jacaranda mimosifolia D Don Markhamia lutea (Benth.) Schum Family: Boraginaceae Alkanna orientalis (L.) Boiss Part L L L L L L L L L Sh H Br,L H W Sh L W L Br Br L L Br L B Br,L H Br L Br L Br,L H Fr L H H H Br B L H (Continued) Downloaded By: [Karolinska Institute] At: 13:55 August 2007 Schistosomicidal bioassay of Egyptian plants Table Continued S=N 43 44 45 46 47 48 49% 50% 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67% 68 69 70% 71 72 73 74 75 76 77 78 79 80 81 82 503 Table Continued Plant name Anchusa aegyptiaca (L.) A DC Cordia dentata Poir Echiochilon fruticosum Desf Heliotropium digynum (Forssk.) C Chr Moltkiopsis ciliata (Forssk.) I M Johnst Trichodesma africanum (L.) R Br Family: Burseraceae Commiphora myrrha (Nees) Engl Family: Cactaceae Cereus jamacaru DC Opuntia leucotricha DC Opuntia tomentosa Salm-Dyck Pereskia aculeata Mill Pereskia aculeata Mill Family: Cannaceae Canna indica L Family: Capparaceae Capparis sinaica Veill Family: Caprifoliaceae Lonicera japonica Thunb Sambucus nigra L Sambucus nigra L Family: Caricaceae Carica papaya L Family: Caryophyllaceae Herniaria fontanesii J Gay Herniaria hemistemon J Gay Paronychia arabica (L.) DC Polycarpaea repens (Forssk.) Asch & Schweinf Silene fruticosa L Silene rubella L Silene succulenta Forssk Spergularia marina (L.) Bessler Stellaria pallida (Dumort.) Murb Family: Celastraceae Euonymus japonica Thunb Family: Chenopodiaceae Anabasis articulata (Forssk.) Moq Atriplex holocarpa F Muell Atriplex portulacoides L Atriplex lindleyi Moq subsp inflata (F Muell.) P.G Wilson Cornulaca monacantha Delile Bassia indica (Wight) A.J Scott Salsola villosa Schult Family: Chenopodiaceae Suaeda aegyptiaca (Hasselq.) Zohary Family: Cleomaceae Cleome amblyocarpa Barratte & Murb Cleome chrysantha Decne Cleome droserifolia (Forssk.) Delile Family: Commelinaceae Tradescantia pallida (Rose) D Hunt Family: Compositae Part H Br H H H H S=N 83 84 85 86 87% 88 89 Resin Br,L St Br L,Br Br,L 90 91 92% 93 94 95 96 Sh 97 H 98 Br,L Br L Fr,S H H H H H W H H W Br H H H H H H H H H H H Br,L (Continued) 99 100 101% 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122% 123% 124 125 Plant name Part Achillea fragrantissima (Forssk.) Sch Bip H Achillea santolina L H Artemisia monosperma Delile H Symphyotrichum squamatum (Spreng.) Nesom H Calendula arvensis L H Centaurea pallescens Delile H Pseudognaphalium luteoalbum (L.) W Hilliard & B.L.Burtt Iphiona scabra DC H Launaea spinosa (Forssk.) Sch Bip ex Kuntze H Nauplius graveolens (Forssk.) Wiklund H Pluchea dioscoridis (L.) DC H Senecio cineraria DC L Klenia fulgens Hook.f Br,L Chiliadenus candicans (Delile) Brullo H Family: Convolvulaceae Convolvulus althaeoides L H Family: Cruciferae Cakile maritima Scop subsp aegyptiaca W (Willd.) Nyman Eruca sativa Mill W Farsetia aegyptia Turra H Family: Cupressaceae Chamaecyparis lawsoniana (Murray) Parl B Cupressus lusitanica Mill B Family: Cycadaceae Cycas revoluta Thunb L Family: Cyperaceae Cyperus alopecuroides Rottb H Cyperus alternifolius L subsp Sh flabelliformis (Rottb.) Kuăk Cyperus alternifolius L subsp Br flabelliformis (Rottb.) Kuăk Cyperus rotundus L H Family: Ebenaceae Diospyros kaki L.f L Diospyros kaki L.f Br Family: Ephedraceae Ephedra alata Decne H Family: Euphorbiaceae Codiaeum variegatum (L.) Blume Br Euphorbia paralias L H Euphorbia dendroides L H Euphorbia hirta L H Euphorbia lactea Haw Br,L Euphorbia neriifolia L Br,L Euphorbia nubica N.E.Br St Euphorbia paralias L H Euphorbia peplis L W Euphorbia pseudocactus A Berger Sh Euphorbia retusa Forssk H Euphorbia tirucalli L Br,L Joannesia principes Vell B Mercurialis annua L H Pedilanthus tithymaloides (L.) Poit Br,L (Continued) Downloaded By: [Karolinska Institute] At: 13:55 August 2007 504 F Yousif et al Table Continued S=N 126 127 128% 129 130% 131% 132 133 134 135 136 137% 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162% 163 164% Table Continued Plant name Ricinus communis L Ricinus communis L Synadenium grantii Hook.f Synadenium grantii Hook.f Family: Flacourtiaceae Dovyalis caffra Warb Family: Frankeniaceae Frankenia hirsuta L Family: Geraniaceae Erodium laciniatum (Cav.) Willd Pelargonium zonale (L.) L’ He´r Pelargonium zonale (L.) L’ He´r Erodium cicutarium (L.) L’ He´r Family: Gingkoaceae Gingko biloba L Gingko biloba L Family: Gramineae Avena sterilis L Dendrocalamus giganteus Munro Panicum turgidum Forssk Paspalum dilatatum Poir Phragmites australis (Cav.) Trin ex Steud Poa annua L Stipagrostis plumosa (L.) Munro ex T Anderson Family: Hydrophyllaceae Wigandia caracasana HBK Family: Juglandaceae Carya illinoensis (Wagnenh.) C.Koch Carya illinoensis (Wagnenh.) C.Koch Family: Labiatae Lamium amplexicaule L Lavandula coronopifolia Poir Marrubium alysson L Origanum syriacum L subsp sinaicum (Boiss.) Greuter & Burdet Phlomis aurea Decne Prasium majus L Stachys aegyptiaca Pers Teucrium leucocladum Boiss Thymus capitatus (L.) Link Micromeria nervosa (Desf.) Benth Family: Lauraceae Cinnamomum glanduliferum (Wallich) Meissn Cinnamomum glanduliferum (Wallich) Meissn Laurus nobilis L Laurus nobilis L Family: Leguminosae Acacia nilotica (L) Delile subsp tomentosa (Benth.) Brenan Acacia nilotica (L.) Delile subsp.Nilotica Acacia saligna (Labill.) H Wendl Part S=N Br,Fl,Fr L L,Br H 165% 166 L,Br H H L Br H L Br H Sh H Sh H W W L L Br W H H H 167 168 169% 170 171 172% 173 174 175 176 177 178 179 180 181% 182 183 184 185 186 187 188 189 190 191 192 193 194% 195 196 197 Br H H H H H 198 199 200 201 202 Br 203% L 204 L Br 205 Br,L Br,L,Fr L (Continued) 206 207 208 209 210% Plant name Acacia saligna (Labill.) H Wendl Acacia tortilis (Forssk.) Hayne subsp Tortilis Albizia lebbek (L.) Benth Albizia lebbek (L.) Benth Amorpha fruticosa L Astragalus fruticosus Forssk Caesalpinia gilliesii (Wallich ex Hook.) Benth Caesalpinia spinosa (Molina) Kuntze Cassia fistula L Cassia renigera Wallich ex Benth Dalbergia sissoo Roxb ex DC Dalbergia sissoo Roxb ex DC Delonix regia (Bojer) Raf Delonix regia (Bojer) Raf Delonix regia (Bojer) Raf Dichrostachys cineria (L.) Wight & Arn Ebenus armitagei Schweinf & Taub Erythrina corallodendrum L Erythrina poeppigiana (Walp.) Cook Gleditsia caspica Desf Gleditsia triacanthos L Haematoxylum campechianum L Leucaena leucocephala (Lam.) De Wit Lonchocarpus speciosa L Ononis vaginalis Vahl Parkinsonia aculeata L Saraca cauliflora Baker Senna bicapsularis (L.) Roxb Sophora japonica L Tephrosia purpurea (L.) Pers subsp leptostachya (DC.) Brummitt var pubescens Baker Trifolium alexandrinum L Trifolium resupinatum L Vicia sativa L Family: Liliaceae, s.l Aloe vera (L.) Burm.f Aloe ciliaris Haw Asparagus stipularis Forssk Asphodelus aestivus Brot Asphodelus aestivus Brot Family: Loganiaceae Buddleja x hybrida Farq Family: Lythraceae Lagerstroemia indica L Family: Malvaceae Gossypium barbadense L Family: Meliaceae Aphanamixis polystachya (Wallich) Parker Toona ciliata M Roemer Toona ciliata M Roemer Khaya grandifoliola DC Khaya grandifoliola DC Part Br H L Br Br,L W Br,Fl,L Br Br L Br Br,Fr,L H Br Fl,L Br H B B L Pods,S Br,L Br,L B H Br,Fr,L L Sh L H H W W L L H R Sh L L Br L L,Fr Br L Br (Continued) Downloaded By: [Karolinska Institute] At: 13:55 August 2007 Schistosomicidal bioassay of Egyptian plants Table Continued S=N 211% 212% 213 214 215% 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230% 231 232% 233 234% 235 236% 237 238 239% 240% 241% 242 243 244 245 246 247 248% 249 250 251% 252 253% 254 505 Table Continued Plant name Swietenia mahogani (L.) Jacq Swietenia mahogani (L.) Jacq Family: Moraceae Ficus benjamina L Ficus elastica Roxb ex Hornem ‘‘decora’’ Ficus elastica Roxb ex Hornem ‘‘decora’’ Ficus elastica Roxb ex Hornem ‘‘decora’’ Ficus saussureans DC Ficus saussureans DC Ficus hispida L Ficus hispida L Ficus lyrata Warb Ficus macrophylla Desf ex Pers Ficus macrophylla Desf ex Pers Ficus macrophylla Desf ex Pers Ficus drupacea Thunb Ficus obliqua Forst Ficus platyphylla Delile Ficus palmata Forssk Ficus religiosa L Ficus sperguana L Ficus sperguana L Morus alba L Family: Moringaceae Moringa peregrina (Forssk.) Fiori Family: Myrtaceae Syzygium jambos (L.) Alston Syzygium jambos (L.) Alston Syzygium jambos (L.) Alston Eugenia uniflora L Eugenia uniflora L Pimenta racemosa (Mill.) J.W Moore Pimenta racemosa (Mill.) J.W Moore Psidium littorale Raddi var longipes (O.Berg) McVaugh Psidium guajava L Psidium guajava L Family: Neuradaceae Neurada procumbens L Family: Nyctaginaceae Bougainvillaea glabra Choisy Bougainvillaea spectabilis Willd Bougainvillaea spectabilis Willd Family: Oleaceae Fraxinus velutina Torr Family: Orobanchaceae Cistanche phelypaea (L.) Cout Family: Palmae Calamus rotang L Chrysalidocarpus lutescens (Bory) H.A Wendl Phoenix dactylifera L Calamus indica Family: Peganaceae Peganum harmala L Part L Br Br L B Br Br L L Br L Br,L Br L L L Br Br B L Br L,Br Br,Fr,L L,Br Br L L Br L B L,Br L Br S=N 255 256 257% 258 259 260% 261 262 263 264 265 266 267 268% 269 270 271 272 273 274 275% 276 277 278 279 280% H Br L Br 281 282 283% 284 285% L,Br H L L L L,Br H (Continued) 286% 287 288 289 290 291 292 293 Plant name Family: Pinaceae Pinus canariensis C Sm Pinus canariensis C Sm Pinus canariensis C Sm Pinus halepensis Mill Pinus halepensis Mill Pinus halepensis Mill Pinus roxburghii Sarg Pinus roxburghii Sarg Family: Plantaginaceae Plantago notata Lag Family: Platanaceae Platanus orientalis L Platanus orientalis L Platycladus orientalis (L.f.) Franco Family: Plumbaginaceae Limonium lobatum (L f.) Chaz Limonium pruinosum (L.) Chaz Plumbago aurticulata Lam Plumbago aurticulata Lam Family: Podocarpaceae Podocarpus gracilior Pilg Family: Polygonaceae Calligonum polygonoides L subsp comosum (L’He´r.) Soskov (locality: Burg El-Arab) Calligonum polygonoides L subsp comosum (L’He´r.) Soskov (locality: Sharm El-Sheikh) Coccoloba pubescens L Polygonum equisetiforme Sm Ruprechtia polystachya Grisab Ruprechtia polystachya Grisab Ruprechtia salicifolia C.A.Mey Ruprechtia salicifolia C.A.Mey Family: Primulaceae Primula boveana Decne ex Duby Family: Rhamnaceae Hovenia dulcis Thunb Hovenia dulcis Thunb Ziziphus spina-christi (L.) Desf Ziziphus spina-christi (L.) Desf Ziziphus spina-christ (L.) Desf Family: Rosaceae Cydonia oblonga Mill Eriobotrya japonica (Thunb.) Lindl Eriobotrya japonica (Thunb.) Lindl Rosa bankesia Aiton Family: Rutaceae Citrus sinensis (L.) Osbeck Severinia buxifolia (Poir.) Ten Family: Salicaceae Salix mucronata Thunb Salix tetrasperma Roxb Part Br L Fr Br L B Fr L H Br L Br,L H H L Br Br H H Br,L H L Br Br L H L Br W L Br Br,L L B Br L Br Br Br (Continued) Downloaded By: [Karolinska Institute] At: 13:55 August 2007 506 F Yousif et al Table Continued S=N 294% 295% 296 297 298 299% 300 301% 302 303 304 305 306 307 308% 309% 310 311 312 313 314 315 316 317 318 319 320% 321% 322 323 324% 325% 326 327 328% 329% 330% 331 332 Table Continued Plant name Family: Sapindaceae Dimocarpus longan Lour Dimocarpus longan Lour Harpullia pendula Planch ex F Muell Harpullia pendula Planch ex F Muell Koelreuteria paniculata Laxm Nephelium tomentosum F Muell Family: Sapotaceae Mimusops elengi L Mimusops elengi L Family: Scrophulariaceae Antirrhinum majus L Lindenbergia indica (L.) Valke Paulownia tomentosa (Thunb.) Steud Paulownia tomentosa (Thunb.) Steud Verbascum fruticulosum Post Verbascum letourneuxii Asch & Schweinf Verbascum sinuatum L Family: Solanaceae Cestrum diurnum L Cestrum diurnum L Cestrum parqui L’He´r Cestrum parqui L’He´r Cestrum parqui L’ He´r Cestrum parqui L’He´r Datura stramonium L Hyoscyamus boveanus (Dunal) Asch & Schweinf Hyoscyamus desertorum (Asch ex Boiss.) Taăckh Petunia x hybrida hort Solanum elaeagnifolium Cav Solanum elaeagnifolium Cav Solanum nigrum L Family: Sterculiaceae Brachychiton australis (Schott & Endl.) A Terracc Brachychiton australis (Schott & Endl.) A Terracc Brachychiton australis (Schott & Endl.) A Terracc Brachychiton australis (Schott & Endl.) A Terracc Brachychiton rupestris (Lindl.) K Schum Brachychiton rupestris (Lindl.) K Schum Dombeya tiliacea (Endl.) Planch Family: Sterlitziaceae Strelitzia nicolai Regel & Koărn Family: Taxodiaceae Taxodium distichum (L.) Rich Family: Thymeleae Thymelaea hirsuta (L.) Endl Family: Ulmaceae Ulmus parvifolia Jacq Part S=N 333 Br L Br L L L 334 335 336 337 338 L Br Sh H L Br H H Fr Br L Br Br,L Fl,L Br,Fr,L Fr,S H H Br,L Br Fr H L 339 340 341% 342% 343 344 345% 346% Plant name Ulmus pumila L Family: Umbelliferae Ammi majus L Deverra tortuosa (Desf.) DC Eryngium creticum Lam Pseudorlaya pumila (L.) Grande Family: Urticaceae Urtica urens L Family: Verbenaceae Citharexylum spinosum L Duranta erecta L Lantana camara L Verbena bipinnatifida Nutt Family: Violaceae Viola odorata L Viola odorata L Family: Zygophyllaceae Fagonia cretica L Fagonia mollis Delile Part Br H H H H W B H Br,Fl,L Br,Fl,L L Br H H % This species proved to possess a confirmed antischistosomal activity in this screening B, bark; Br, branches; Fl, flower; Fr, fruit; L, leaves; R, roots; S, seeds; St, stem; H, herb; W, weed; Sh, shoot system S=N, Serial=number Bilharz Research Institute Laboratory-bred hamsters weighing 80–100 g, maintained on standard diet (24% protein), were infected percutaneously with 350–400 cercariae=hamster After 6–7s weeks, worms were cleared from hamsters’ blood by perfusion technique using phosphate buffer through 20-mm mesh sieves and rapidly placed in culture medium RPMI 1640 containing 300 mg streptomycin, 300 units penicillin, and 160 mg gentamicin=100 mL medium Br B Br,L Br L Br Br,L Br,L H L (Continued) Schistosomicidal bioassay A stock solution (10 mg=mL) of each plant extract was prepared in DMSO, diluted with RPMI to produce mL test solution of 100 mg=mL final concentration in a 10-mL vial for the screening Three replicates were used for each extract, and three pairs of worms, males and females equally represented, were placed in each vial using sterilized tissue forceps Incubation was maintained at 37#C Positive (praziquantel, at 0.1 mg=mL) and negative (DMSO) controls were similarly used Examination for worm viability was done after 24 h using a stereomicroscope Worms showing no signs of motility for were considered dead The activity of the extract was measured by calculating the number of dead worms relative to the total number of worms Downloaded By: [Karolinska Institute] At: 13:55 August 2007 Schistosomicidal bioassay of Egyptian plants Determination of LC50 and LC90 Active extracts resulted from the bioassay screening were similarly bioassayed at descending concentrations (e.g., 50, 30, 10, 7, and mg=mL) and the mortality of worms was recorded The results were used to calculate the LC50 and LC90 of the extract using probit analysis and utilizing the SPSS computer program (SPSS for Windows version 9=1989; SPSS Inc., Chicago, IL, USA) Determination of bioassay method validation The method was examined for reproducibility and accuracy, using the reference drug praziquantel Five different experiments at five different dates using five concentrations, each in triplicate, were done to calculate different validation parameters Statistical analysis of the resulting LC50 and LC90 of praziquantel (average 0.08 and 0.12, respectively) using one-way ANOVA on SPSS computer program version revealed that there is no significant day-to-day variation (p > 0.01), which ensures reproducibility Also, the calculated % coefficient of variation of the LC values lies within the accepted range, thus proving the precision and accuracy Results and Discussion The bioscreening results revealed that 72 extracts possessed reproducible and confirmed in vitro antischistosomal activity The plant species producing these active extracts are marked with an asterisk (% ) in Table The LC50 and LC90 values of the above active extracts were determined and are recorded in Table 2, where 15 extracts out of these were found to possess strong antischistosomal activity (LC50 values equal to or less than 15 mg=mL) The plant species that produced these most effective extracts are listed as follows [after the serial number of each]: [2] Agave americana, [5] A lophantha, [8] Furcraea selloa, [33] Calotropis procera, [36] Pergularia tomentosa, [38] Asclepias sinaica, [42] Alkanna orientalis, [210] Khaya grandifoliola, [212] Swietenia mahogany, [240] Pimenta racemosa, [257] Pinus canariensis, [308] Verbascum sinaiticum, [320] Solanum elaeagnifolium, [321] S nigrum, and [325] Brachychiton rupestris These 15 most effective plant species deserve further investigation, with the aim to isolate and characterize their active constituents The antischistosomal effects of these species should be confirmed by investigation in an infected animal model to determine their therapeutic value and toxicity Studies are currently ongoing and will be reported in a future publication Several articles had reported on different biological activities of the above species, namely, anti-inflammatory and molluscicidal properties of Agave americana (Shoeb et al., 1984; Peana et al., 1997), molluscicidal activity of 507 Agave lophantha (El-Sayed et al., 1991), anti-inflammatory activity of Calotropis procera (Kumar & Basu, 1994), hypoglycemic activity of Pergularia tomentosa (Shabana et al., 1990), antiviral activity of Alkanna orientalis (El-Sohly et al., 1997), antibacterial, antinociceptive, and antiinflammatory effects of Pimenta racemosa (Garcia et al., 2004; Saenz et al., 2004), antimicrobial activities of Verbascum sinaiticum (Tadeg et al., 2005), molluscicidal properties of Solanum elaeagnifolium (Bekkouche et al., 2000), and molluscicidal and cercaricidal activities of Solanum nigrum (Ahmed & Ramzy, 1997) Few investigators have conducted in vitro bioassay screening of plants for antischistosomal activity Molgaard et al (2001) screened extracts of 23 plant species from Zimbabwe and found that the stem and root extracts from Abrus precatorius (Fabaceae) and stem bark extracts from Elephantorrhiza goetzei (Mimosaceae) have a good activity against schistosomules Sparg et al (2000) screened 21 species from South Africa against the schistosomula of S haematobium, where Berkheya speciosa (Asteraceae), Euclea natalensis (Ebenaceae), and Trichilia emetica (Meliaceae) were found lethal A 90–100% mortality of Schistosoma worms was found affected in vitro by mg=mL goyazensolide, a component extracted from Eremanthus goyazensis (Barth et al., 1997), and 200 mg l $1 of an ethyl acetate extract of ginger, Zingiber officinale (Sanderson et al., 2002) In vitro antischistosomal activity was possessed by robustic acid and an isoflavone compound isolated from the seeds of the tree Millettia thonningii (Lyddiard et al., 2002), as well as extracts of Scilla natalensis and Ledebouria ovatifolia (Sparg et al., 2002) Concerning in vivo antischistosomal activity of natural products, Utzinger et al (2001) reported that artemether, the methyl ether derivative of artemisinin, which is a Chinese active antimalarial principle in the leaves of Artemisia annua, exhibited antischistosomal properties by oral doses of mg=kg in randomized controlled clinical trials Koko et al (2005) determined the efficacy of oral therapy with Balanites aegyptiaca fruit mesocarp in a dose of 200 mg=kg body weight of mice infected with Sudanese strain of S mansoni, and found a significant reduction in egg count per gram of feces Ramadan et al (2004) studied the therapeutic effect of Ferula assafoetida on S mansoni in experimentally infected mice Massoud et al (2001) and Abo-Madyan et al (2004) reported that Commiphora molmol extract (Myrrh capsules) given to patients at a dose of 10 mg=kg of body weight per day for or consecutive days induced a curative rate higher than 90% However, contrary to these results, Botros et al (2004) and Barakat et al (2005) did not recommend the use of this plant drug in treating human cases of schistosomiasis based on negative results from several experiments performed on patients and infected animals Downloaded By: [Karolinska Institute] At: 13:55 August 2007 508 F Yousif et al Table In vitro antischistosomal effect (LC50 and LC90) of the bioactive extracts S=Na Plant name 2% 5% 8% 26 30 33% 34 36% 38% 42% 49 50 67 70 87 92 101 122 123 128 130 131 137 162 164 165 169 172 181 194 Agave americana L var marginata Trel Agave angustifolia Haw Agave decipiens Bak Agave lophantha Schiede Furcraea selloa C Koch Carissa carandus L Thevetia peruviana (Pers.) Schum Calotropis procera (Aiton) W.T Aiton Calotropis procera (Aiton) W T Aiton Pergularia tomentosa L Asclepias sinaica (Boiss.) Muschl Alkanna orientalis (L.) Boiss Commiphora myrrha (Nees) Engl Cereus jamacaru DC Silene succulenta Forssk Euonymus japonica Thunb Calendula arvensis L Nauplius graveolens (Forssk.) Wiklund Chamaecyparis lawsoniana (Murray) Parl Euphorbia tirucalli L Joannesia principes Vell Synadenium grantii Hook f Dovyalis caffra Warb Frankenia hirsuta L Gingko biloba L Acacia nilotica (L) Delile subsp tomentosa (Benth.) Brenan Acacia saligna (Labill.) H Wendl Acacia saligna (Labill.) H Wendl Amorpha fruticosa L Caesalpinia spinosa (Molina) Kuntze Ebenus armitagei Schweinf & Taub Tephrosia purpurea (L.) Pers subsp leptostachya (DC.) Brummitt var pubescens Baker Buddlejaxhybrida Farq Khaya grandifoliolaDC Swietenia mahogani (L.) Jacq Swietenia mahogani (L.) Jacq Ficus elastica Roxb ex Hornem ‘‘decora’’ Ficus sperguana L Morus alba L Syzygium jambos (L.) Alston Syzygium jambos (L.) Alston Pimenta racemosa (Mill.) J.W Moore Pimenta racemosa (Mill.) J.W Moore Psidium littorale Raddi var longipes (O.Berg) McVaugh Fraxinus velutina Torr Chrysalidocarpus lutescens (Bory) H.A Wendl Calamus indica Pinus canariensis C Sm Pinus halepensis Mill Limonium pruinosum (L.) Chaz Polygonum equisetiforme Sm Primula boveana Decne.ex Duby Ziziphus spina-christi (L.) Desf Ziziphus spina-christi (L.) Desf 203 210% 211 212% 215 230 232 234 236 239 240% 241 248 251 253 257% 260 268 275 280 283 285 LC50b (ppm) LC90b (ppm) L L L L L L,Br Br H Fr H H H Resin Br,L H Br H H B L,Br B L,Br L,Br H Br L,Br Br L L,Br Br H H 11.45 29.30 21.50 8.20 7.10 40.40 30.70 11.50 18.70 9.40 11.70 7.00 49.80 45.00 51.10 42.40 26.10 73.60 59.60 48.30 32.60 43.00 24.30 46.80 50.70 57.01 67.10 32.10 31.00 56.40 37.00 17.30 18.40 44.40 53.20 11.70 9.40 62.70 37.20 19.00 28.90 12.00 16.20 13.00 58.60 70.00 70.06 64.40 40.60 85.30 72.20 62.90 51.90 60.30 43.90 68.50 59.70 77.30 80.30 50.29 45.18 67.40 43.80 40.41 L Br L Br B L L,Br L,Br L L B L,Br L,Br L L,Br Fr B H H H W Br 50.50 8.70 26.10 7.40 46.95 21.46 49.70 32.60 57.26 31.30 14.20 27.30 19.20 38.70 17.30 12.80 24.10 41.30 33.10 73.30 18.90 18.90 66.20 24.40 39.05 13.30 65.71 41.82 58.50 51.60 79.80 53.10 23.70 53.60 33.79 56.90 31.90 22.00 38.90 64.13 58.00 58.20 27.50 27.50 Part (Continued) Downloaded By: [Karolinska Institute] At: 13:55 August 2007 Schistosomicidal bioassay of Egyptian plants 509 Table Continued S=Na 286 294 295 299 301 308% 309 320% 321% 324 325% 328 329 330 341 342 345 346 Part LC50b (ppm) LC90b (ppm) L,Br Br L L Br Fr Br Fr H B Br,L Br L,Br L,Br L,Fl,Br L,Fl,Br H H 42.50 47.70 47.70 42.30 41.30 8.00 22.00 6.00 13.30 38.50 11.60 40.20 41.30 57.39 28.20 76.15 22.72 27.26 57.80 56.00 56.09 69.30 64.30 14.20 45.20 8.41 28.80 54.36 14.53 59.76 55.41 84.36 34.90 32.30 33.80 42.72 Plant name Cydonia oblonga Mill Dimocarpus longan Lour Dimocarpus longan Lour Nephelium tomentosa F Muell Mimusops elengi L Verbascum sinuatum L Cestrum diurnum L Solanum elaeagnifolium Cav Solanum nigrum L Brachychiton rupestris (Lindl.) K Schum Brachychiton rupestris (Lindl.) K Schum Dombeya tiliacea (Endl.) Planch Strelitzia nicolai Regel & Koărn Taxodium distichum (L.) Rich Lantana camara L Verbena bipinnatifida Nutt Fagonia cretica L Fagonia mollis Delile % Most effective extract The same serial number (S=N) used in Table b Confidence limit ¼ 95% a The importance of plants as sources of natural product bioactive molecules to medicine lies not only in their pharmacological or chemotherapeutic effects but also in their role as template molecules for the production of new drug substances (Phillipson, 1994) The current study is a trial toward that direction and introduces some new antischistosomal plant sources This is the first paper to report on a systematic screening of schistosomicidal activity that utilizes a large number of plants The bioassay method used proved to furnish accurate and reproducible results and hence could be used to screen larger numbers of plants, with the aim to discover new antischistosomal agents Accordingly, we are continuing this task by subjecting the other available plant species growing in Egypt to this screening bioassay Acknowledgments This work was financed by The Academy of Scientific Research and Technology and The National Research Centre, Egypt, under the project ‘‘The use of biotechnological methods for drug discovery from Egyptian plants: antitumor, cancer chemopreventive, immunomodulatory, antiviral, and schistosomicidal agents’’; and by Program of the National Strategy for Biotechnology, contract agreement no 10 (1998–2007), Principal Investigator B El-Menshawi References Abo-Madyan AA, Morsy TA, Motawea S (2004): Efficacy of myrrh in the treatment of schistosomiasis (haematobium and mansoni) in Ezbet El-Bakly, Tamyia Center, El-Fayoum Governorate Egypt J Egypt Soc Parasitol 34: 423–446 Ahmed AH, Ramzy RM (1997): Laboratory assessment of the molluscicidal and cercaricidal activities of the Egyptian weed, Solanum nigrum L Ann Trop Med Parasitol 9: 931–937 Barakat R, EL-Morshedy H, Fenwick A (2005): Efficacy of myrrh in the treatment of human schistosomiasis mansoni Am J Trop Med Hyg 73: 365–367 Barth LR, Fernandes AP, Ribeiro-Paes JT, Rodrigues V (1997): Effects of goyazensolide during in vitro cultivation of Schistosoma mansoni Mem Inst Oswaldo Cruz 92: 427–429 Bekkouche K, Markouk M, Larhsini M, Jana M, Lazrek HB (2000): Molluscicidal properties of glycoalkaloid extracts from Moroccan Solanum species Phytother Res Aug 14: 366–367 Botros S, William S, Ebeid F, Cioli D, Katz N, Day TA, Bennett JL (2004): Lack of evidence for an antischistosomal activity of myrrh in experimental animals Am J Trop Med Hyg 7: 206–210 Boulos L (1999): Flora of Egypt 1: Azollaceae–Oxalidaceae Cairo, Egypt, Al Hadara Publ., pp 1–419 Boulos L (2000): Flora of Egypt 2: Geraniaceae–Boraginaceae Cairo, Egypt, Al Hadara Publ., pp 1–352 Downloaded By: [Karolinska Institute] At: 13:55 August 2007 510 F Yousif et al Boulos L (2002): Flora of Egypt 3: Verbenaceae–Compositae Cairo, Egypt, Al Hadara Publ., pp 1–373 Boulos L (2005): Flora of Egypt 4: Alismataceae–Orchidaceae Cairo, Egypt, Al Hadara Publ., pp 1–617 Doenhoff MJ, Kusel JR, Coles GC, Cioli D (2002): Resistance of S mansoni to praziquantel: Is there a problem? Trans R Soc Trop Med Hyg 96: 465–469 El-Menshawi B (2003): The Use of Biotechnological Methods for Drug Discovery from Egyptian Plants Technical Report, Phase I (2003) Cairo, Acad Sci., Res., & Technol., pp 1–366 El-Sayed MM, Abdel-Hadi M, el-Nahas HA (1991): Molluscicidal activity and clinico-pathological effect of Agave lophantha Arch Pharm Res 14: 81–86 El-Sohly HN, El-Feraly FS, Joshi AS, Walker LA (1997): Antiviral flavonoids from Alkanna orientalis Planta Med 63: 384 Frederich M, Dogne JM, Angenot L, De Mol P (2002): New trends in anti-malarial agents Curr Med Chem 9: 1435–1456 Garcia MD, Fernandez MA, Alvarez A, Saenz MT (2004): Antinociceptive and anti-inflammatory effect of the aqueous extract from leaves of Pimenta racemosa var ozua (Mirtaceae) J Ethnopharmacol 91: 69–73 Huxley A, Griffiths M, Lexy M (1992): The New Royal Horticultural Society Directory of Gardening, Vols 1, 2, 3, & London, Macmillan Ismail M, Botros S, Metwally A, William S, Farghally A, Tao LF, Day TA, Bennett JL (1999): Resistance to praziquantel: Direct evidence from Schistosoma mansoni isolated from Egyptian villagers Am J Trop Med Hyg 60: 932–935 Koko WS, Abdalla HS, Galal M, Khalid HS (2005): Evaluation of oral therapy on mansonial schistosomiasis using single dose of Balanites aegyptiaca fruits and praziquantel Fitoterapia 76: 30–34 Kumar VL, Basu N (1994): Anti-inflammatory activity of the latex of Calotropis procera J Ethnopharmacol 44: 123–125 Lambertucci JR, Pedroso ERP, Souza DWC, Lim DP, Neves J, Salazar HM, Marinho RP, Rocha MOC, Coelho PMS, Costa MFFL, Greco DB (1980): Therapeutic efficacy of oral oxamniquine in the toxemic form of Schistosomiasis mansoni: Treatment of eleven individuals from two families, and experimental study Am J Trop Med Hyg 29: 50–53 Lyddiard JR, Whitfield PJ, Bartlett A (2002): Antischistosomal bioactivity of isoflavonoids from Millettia thonningii (Leguminosae) J Parasitol 88: 163–170 Massoud A, Sheir Z, Nasr AA, Salama O, Badra GA, El-Shennawy H, Hassan N, Hammad SM (2001): View publication stats A safe, effective, herbal antischistosomal therapy derived from myrrh Am J Trop Med Hyg 65: 700–704 Molgaard P, Nielsen SB, Rasmussen DE, Drummond RB, Makaza N, Andreassen J (2001): Anthelmintic screening of Zimbabwean plants traditionally used against schistosomiasis J Ethnopharmacol 74: 257–264 Peana AT, Moretti MD, Manconi V, Desole G, Pippia P (1997): Anti-inflammatory activity of aqueous extracts and steroidal sapogenins of Agave americana Planta Med 63: 199–202 Phillipson JD (1994): Natural products as drugs Trans R Soc Trop Med Hyg 88: 17–19 Ramadan NI, Abde Same-Ekobo A, Lohoue J, Essono El-Aaty HE, Abdel-Hameed DM, El Deeb HK, Samir NA, Mansy SS, Al Khadrawy FM (2004): Effect of Ferula assafoetida on experimental murine Schistosoma mansoni infection J Egypt Soc Parasitol 34: 1077–1094 Saenz MT, Tornos MP, Alvarez A, Fernandez MA, Garcia MD (2004): Antibacterial activity of essential oils of Pimenta racemosa var terebinthina and Pimenta racemosa var grisea Fitoterapia 75: 599–602 Sanderson L, Bartlett A, Whitfield PJ (2002): In vitro and in vivo studies on the bioactivity of a ginger (Zingiber officinale) extract towards adult schistosomes and their egg production J Helminthol 76: 241–247 Shabana MM, Mirhom YW, Genenah AA, Aboutabl EA, Amer HA (1990): Study into wild Egyptian plants of potential medicinal activity Arch Exp Veterinarmed 44: 389–394 Shoeb HA, Hassan AA, el-Sayed MM, Refahy L (1984): The molluscicidal properties of Agave decepiens and Agave americana (var marginata) J Egypt Soc Parasitol 14: 265–273 Souza LCD, Pedro RJ, Deberaldin ER (1982): Use of praziquantel in patients with schistosomiasis mansoni previously treated with oxamniquine and=or hycanthone: Resistance of Schistosoma mansoni to schistosomicidal agents Trans R Soc Trop Med Hyg 76: 652–659 Sparg SG, Van Staden J, Jager AK (2000): Efficiency of traditionally used South African plants against schistosomiasis J Ethnopharmacol 73: 209–214 Sparg SG, van Staden J, Jager AK (2002): Pharmacological and phytochemical screening of two Hyacinthaceae species: Scilla natalensis and Ledebouria ovatifolia J Ethnopharmacol 80: 95–101 Tadeg H, Mohammed E, Asres K, Gebre-Mariam T (2005): Antimicrobial activities of some selected traditional Ethiopian medicinal plants used in the treatment of skin disorders J Ethnopharmacol 100: 168–175 Utzinger J, Xiao S, N’Goran EK, Bergquist R, Tanner M (2001): The potential of artemether for the control of schistosomiasis Int J Parasitol 31: 1549–1562