Large scale in vitro screening of egypti

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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

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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

1Theodor Bilharz Research Institute, Cairo, Egypt; 2Faculty of Pharmacy, Cairo University, Cairo, Egypt;

3Faculty of Science, Cairo University, Cairo, Egypt;4Faculty of Science, Alexandria University, Alexandria, Egypt;

5Orman 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 100mg=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

(praziquan-tel) controls were used Of the tested plant extracts,

72 were found to possess reproducible in vitro

antischis-tosomal 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

(Agava-ceae), A lophantha Schiede (Agava(Agava-ceae), 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

Brachychi-ton 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, Schisto-soma 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 socio-economic importance and is increasing in incidence despite concerted efforts to control and contain the dis-ease 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 situ-ation, 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 Pharmaceutical Biology

2007, Vol 45, No 6, pp 501–510

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2007 effective treatments for malaria, for example, quinineWhereas herbal medicine has produced some very

and recently artemisinin (Frederich et al., 2002), few

attempts have been made to screen plants against

schisto-somiasis (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

schisto-somicidal bioassay to screen Egyptian plants (native and

cultivated species) on S mansoni worms, under a

collaborative research project, during 1998–2006

(El-Menshawi, 2003)

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 1 Plant specimens were collected

ran-domly 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

fol-lows 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

bioscreen-ing 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

press-ure 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

Table 1 Egyptian plants (native and cultivated) screened for antischistosomal activity in vitro

Family: Agavaceae

2% Agave americana L var marginata Trel L

Family: Aizoaceae

Family: Amaranthaceae

11 Aerva javanica (Burm f.) Juss ex Schult H

Family: Amaryllidaceae

16 Hippeastrum vittatum (L’ He´r.) Herb L Family: Anacardiaceae

Family: Annonaceae

Family: Apocynaceae

22 Acokanthera oblongifolia (Hochst.) Codd L

23 Acokanthera oblongifolia (Hochst.) Codd Br

Family: Araliaceae

32 Schefflera arboricola (Hayata) Hayata Br,L Family: Asclepiadaceae

33% Calotropis procera (Aiton) W.T Aiton H

34% Calotropis procera (Aiton) W.T Aiton Fr

38% Asclepias sinaica (Boiss.) Muschl H Family: Basellaceae

Family: Bignoniaceae

Family: Boraginaceae

(Continued)

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2007 Table 1 Continued.

46 Heliotropium digynum (Forssk.) C Chr H

47 Moltkiopsis ciliata (Forssk.) I M Johnst H

Family: Burseraceae

Family: Cactaceae

Family: Cannaceae

Family: Capparaceae

Family: Caprifoliaceae

Family: Caricaceae

Family: Caryophyllaceae

64 Polycarpaea repens (Forssk.) Asch

& Schweinf

H

Family: Celastraceae

Family: Chenopodiaceae

74 Atriplex lindleyi Moq subsp inflata

(F Muell.) P.G Wilson

H

Family: Chenopodiaceae

Family: Cleomaceae

79 Cleome amblyocarpa Barratte & Murb H

Family: Commelinaceae

Family: Compositae

(Continued)

Table 1 Continued

83 Achillea fragrantissima (Forssk.) Sch Bip H

86 Symphyotrichum squamatum (Spreng.) Nesom H

89 Pseudognaphalium luteoalbum (L.) Hilliard & B.L.Burtt

W

91 Launaea spinosa (Forssk.) Sch Bip ex Kuntze H

92% Nauplius graveolens (Forssk.) Wiklund H

96 Chiliadenus candicans (Delile) Brullo H Family: Convolvulaceae

Family: Cruciferae

98 Cakile maritima Scop subsp aegyptiaca (Willd.) Nyman

W

Family: Cupressaceae

101% Chamaecyparis lawsoniana (Murray) Parl B

Family: Cycadaceae

Family: Cyperaceae

105 Cyperus alternifolius L subsp

flabelliformis (Rottb.) Ku¨k

Sh

106 Cyperus alternifolius L subsp

flabelliformis (Rottb.) Ku¨k

Br

Family: Ebenaceae

Family: Ephedraceae

Family: Euphorbiaceae

125 Pedilanthus tithymaloides (L.) Poit Br,L

(Continued)

Schistosomicidal bioassay of Egyptian plants 503

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2007 Table 1 Continued.

Family: Flacourtiaceae

Family: Frankeniaceae

Family: Geraniaceae

Family: Gingkoaceae

Family: Gramineae

142 Phragmites australis (Cav.) Trin

ex Steud

H

144 Stipagrostis plumosa (L.) Munro

ex T Anderson

W Family: Hydrophyllaceae

Family: Juglandaceae

Family: Labiatae

151 Origanum syriacum L subsp sinaicum

(Boiss.) Greuter & Burdet

H

Family: Lauraceae

158 Cinnamomum glanduliferum (Wallich)

Meissn

Br

159 Cinnamomum glanduliferum (Wallich)

Meissn

L

Family: Leguminosae

162% Acacia nilotica (L) Delile subsp tomentosa

(Benth.) Brenan

Br,L

163 Acacia nilotica (L.) Delile subsp.Nilotica Br,L,Fr

164% Acacia saligna (Labill.) H Wendl L

(Continued)

Table 1 Continued

165% Acacia saligna (Labill.) H Wendl Br

166 Acacia tortilis (Forssk.) Hayne

subsp Tortilis

H

171 Caesalpinia gilliesii (Wallich ex Hook.)

Benth

Br,Fl,L

172% Caesalpinia spinosa (Molina) Kuntze Br

180 Dichrostachys cineria (L.) Wight & Arn Br

181% Ebenus armitagei Schweinf & Taub H

194% Tephrosia purpurea (L.) Pers subsp

leptostachya (DC.) Brummitt var

pubescens Baker

H

Family: Liliaceae, s.l

Family: Loganiaceae

Family: Lythraceae

Family: Malvaceae

Family: Meliaceae

206 Aphanamixis polystachya (Wallich) Parker L

(Continued)

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2007 Table 1 Continued.

Family: Moraceae

214 Ficus elastica Roxb ex Hornem ‘‘decora’’ L

215% Ficus elastica Roxb ex Hornem ‘‘decora’’ B

216 Ficus elastica Roxb ex Hornem ‘‘decora’’ Br

Family: Moringaceae

Family: Myrtaceae

239% Pimenta racemosa (Mill.) J.W Moore L

240% Pimenta racemosa (Mill.) J.W Moore B

241% Psidium littorale Raddi var longipes

(O.Berg) McVaugh

L,Br

Family: Neuradaceae

Family: Nyctaginaceae

Family: Oleaceae

Family: Orobanchaceae

Family: Palmae

H.A Wendl

Family: Peganaceae

(Continued)

Table 1 Continued

Family: Pinaceae

Family: Plantaginaceae

Family: Platanaceae

266 Platycladus orientalis (L.f.)

Franco

Br,L Family: Plumbaginaceae

Family: Podocarpaceae

Family: Polygonaceae

272 Calligonum polygonoides L subsp

comosum (L’He´r.) Soskov (locality:

Burg El-Arab)

H

273 Calligonum polygonoides L subsp

comosum (L’He´r.) Soskov (locality:

Sharm El-Sheikh)

H

Family: Primulaceae

Family: Rhamnaceae

Family: Rosaceae

Family: Rutaceae

Family: Salicaceae

(Continued)

Schistosomicidal bioassay of Egyptian plants 505

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Bilharz Research Institute Laboratory-bred hamsters weighing 80–100 g, maintained on standard diet (24% protein), were infected percutaneously with 350–400 cer-cariae=hamster After 6–7s weeks, worms were cleared from hamsters’ blood by perfusion technique using phos-phate buffer through 20-mm mesh sieves and rapidly placed in culture medium RPMI 1640 containing

300 mg streptomycin, 300 units penicillin, and 160mg gentamicin=100 mL medium

Schistosomicidal bioassay

A stock solution (10 mg=mL) of each plant extract was prepared in DMSO, diluted with RPMI to produce

3 mL test solution of 100mg=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 0.1mg=mL) and nega-tive (DMSO) controls were similarly used

Examination for worm viability was done after 24 h using a stereomicroscope Worms showing no signs

of motility for 1 min were considered dead The activity of the extract was measured by calculating the number of dead worms relative to the total number of worms

Table 1 Continued

Family: Sapindaceae

296 Harpullia pendula Planch ex F Muell Br

297 Harpullia pendula Planch ex F Muell L

Family: Sapotaceae

Family: Scrophulariaceae

307 Verbascum letourneuxii Asch &

Schweinf

H

Family: Solanaceae

316 Hyoscyamus boveanus (Dunal) Asch

& Schweinf

H

317 Hyoscyamus desertorum (Asch ex Boiss.)

Ta¨ckh

H

Family: Sterculiaceae

322 Brachychiton australis (Schott & Endl.)

A Terracc

L

323 Brachychiton australis (Schott & Endl.)

A Terracc

Br

324% Brachychiton australis (Schott & Endl.)

A Terracc

B

325% Brachychiton australis (Schott & Endl.)

A Terracc

Br,L

326 Brachychiton rupestris (Lindl.) K Schum Br

327 Brachychiton rupestris (Lindl.) K Schum L

Family: Sterlitziaceae

329% Strelitzia nicolai Regel & Ko¨rn Br,L

Family: Taxodiaceae

Family: Thymeleae

Family: Ulmaceae

(Continued)

Table 1 Continued

Family: Umbelliferae

Family: Urticaceae

Family: Verbenaceae

Family: Violaceae

Family: Zygophyllaceae

%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

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2007 Determination of LC50and LC90

Active extracts resulted from the bioassay screening were

similarly bioassayed at descending concentrations (e.g.,

50, 30, 10, 7, 5 and 3mg=mL) and the mortality of worms

was recorded The results were used to calculate the LC50

and LC90of the extract using probit analysis and

utiliz-ing the SPSS computer program (SPSS for Windows

ver-sion 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 9 revealed that there is no

sig-nificant 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

pos-sessed reproducible and confirmed in vitro

antischistoso-mal activity The plant species producing these active

extracts are marked with an asterisk (%) in Table 1

The LC50and LC90values of the above active extracts

were determined and are recorded in Table 2, where 15

extracts out of these were found to possess strong

anti-schistosomal activity (LC50 values equal to or less than

15mg=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]

Pergu-laria tomentosa, [38] Asclepias sinaica, [42] Alkanna

orientalis, [210] Khaya grandifoliola, [212] Swietenia

mahogany, [240] Pimenta racemosa, [257] Pinus

canarien-sis, [308] Verbascum sinaiticum, [320] Solanum

elaeagnifo-lium, [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

Agave lophantha (El-Sayed et al., 1991), anti-inflammatory activity of Calotropis procera (Kumar & Basu, 1994), hypo-glycemic activity of Pergularia tomentosa (Shabana et al., 1990), antiviral activity of Alkanna orientalis (El-Sohly

et al., 1997), antibacterial, antinociceptive, and anti-inflammatory effects of Pimenta racemosa (Garcia et al., 2004; Saenz et al., 2004), antimicrobial activities of Verbas-cum sinaitiVerbas-cum (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 spe-cies 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 spe-ciosa (Asteraceae), Euclea natalensis (Ebenaceae), and Trichilia emetica (Meliaceae) were found lethal

A 90–100% mortality of Schistosoma worms was found affected in vitro by 4mg=mL goyazensolide, a component extracted from Eremanthus goyazensis (Barth

et al., 1997), and 200 mg l$1of 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 ovati-folia (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 6 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 experimen-tally infected mice

Massoud et al (2001) and Abo-Madyan et al (2004) reported that Commiphora molmol extract (Myrrh cap-sules) given to patients at a dose of 10 mg=kg of body weight per day for 3 or 6 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 nega-tive results from several experiments performed on patients and infected animals

Schistosomicidal bioassay of Egyptian plants 507

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2007 Table 2 In vitro antischistosomal effect (LC50and LC90) of the bioactive extracts.

194 Tephrosia purpurea (L.) Pers subsp leptostachya (DC.)

Brummitt var pubescens Baker

(Continued)

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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

screen-ing of schistosomicidal activity that utilizes a large

num-ber 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

Accord-ingly, 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

biotechno-logical methods for drug discovery from Egyptian plants:

antitumor, cancer chemopreventive,

immunomodula-tory, antiviral, and schistosomicidal agents’’; and by

Pro-gram of the National Strategy for Biotechnology,

contract agreement no 10 (1998–2007), Principal

Investi-gator B El-Menshawi

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HB (2000): Molluscicidal properties of glycoalkaloid extracts from Moroccan Solanum species Phytother Res Aug 14: 366–367

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Table 2 Continued

%Most effective extract

aThe same serial number (S=N) used in Table 1

bConfidence limit¼ 95%

Schistosomicidal bioassay of Egyptian plants 509