Journal of Ethnopharmacology 145 (2013) 746–757

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Journal of Ethnopharmacology

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Inhibiton Corr

Chemis

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journal homepage: www.elsevier.com/locate/jep

The traditional medical uses and cytotoxic activities of sixty-one Egyptianplants: Discovery of an active cardiac glycoside from Urginea maritima

Hesham R. El-Seedi a,b,n, Robert Burman a, Ahmed Mansour c, Zaki Turki d, Loutfy Boulos e,Joachim Gullbo f, Ulf Goransson a

a Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Biomedical Centre, Box 574, 751 23 Uppsala, Swedenb Department of Chemistry, Faculty of Science, El-Menoufia University, 32512 Shebin El-Kom, Egyptc Saint Catherine, Sinai, Egyptd Department of Botany, Faculty of Science, El-Menoufia University, 32512 Shebin El-Kom, Egypte Department of Botany, Faculty of Science, Alexandria University, 21561 Alexandria, Egyptf Division of Clinical Pharmacology, Department of Medical Sciences, Uppsala University, 751 85 Uppsala, Sweden

a r t i c l e i n f o

Article history:

Received 20 August 2012

Received in revised form

18 November 2012

Accepted 2 December 2012Available online 8 December 2012

Keywords:

Egyptian medicinal plants

Anticancer screening

Cardiac glycoside

Urginea maritima

FMCA assay

41/$ - see front matter & 2012 Elsevier Irelan

x.doi.org/10.1016/j.jep.2012.12.007

viations: FMCA, Fluorometric Microculture Cy

ate Buffered Saline; FDA, Fluorescein Diacetat

ry Concentration 50%

esponding author at: Division of Pharmacogn

try, Uppsala University, Biomedical Centre,

. Tel.: þ46 18 4714207; fax: þ46 18 509101.

ail address: hesham.el-seedi@fkog.uu.se (H.R.

a b s t r a c t

Ethnopharmacological relevance: Medicinal plants from the Sinai desert are widely used in traditional

Bedouin medicine to treat a range of conditions including, cancers, and may thus be useful sources of

novel anti-tumor compounds. Information on plants used in this way was obtained through

collaboration with Bedouin herbalists.

Aim of the study: To document the traditional uses of 61 species from 29 families of Egyptian medicinal

plants and to investigate their biological activity using a cytotoxicity assay.

Material and methods: MeOH extracts of the 61 plant species investigated were dissolved in 10% DMSO

and their cytotoxic activity was evaluated. The extracts were tested in duplicate on three separate

occasions at three different concentrations (1, 10 and 100 mg/ml) against human lymphoma U-937 GTB.

The most active extract was subjected to bioassay-guided fractionation using HPLC and LC/ESI–MS to

isolate and identify its active components.

Results and discussion: The most potent extracts were those from Asclepias sinaica, Urginea maritima,

Nerium oleander and Catharanthus roseus, followed by those from Cichorium endivia, Pulicaria undulate

and Melia azedarach. Literature reports indicate that several of these plants produce cardiac glycosides.

Bioassay-guided fractionation of alcoholic U. maritima extracts led to the isolation of a bioactive

bufadienolide that was subsequently shown to be proscillaridin A, as determined by 1D and 2D NMR

spectroscopy. This result demonstrates the value of plants used in traditional medicine as sources of

medicinally interesting cytotoxic compounds.

& 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

People have used plants for food and medicinal purposes forthousands of years and have acquired extensive knowledge oftheir properties (Brouwer et al., 2005). Traditional medicinalplants have been used to treat numerous diseases for thousandsof years in various parts of the world (Adebayo and Krettli, 2011)and still play an important role in the cultures and traditions ofmany developing countries (Delgado et al., 2011). There has also

d Ltd. All rights reserved.

totoxicity Assay; PBS,

e; SI, Survival Index; IC50,

osy, Department of Medicinal

Box 574, 751 23 Uppsala,

El-Seedi).

been increasing interest in the use of natural products in the

Western world (Helms, 2004). Naturally occurring compounds,

used either in purified form or as standardized plant extracts,

provide important opportunities for identifying new drug leads

because of their unmatched chemical diversity (Cos et al., 2006).

The World Health Organization (WHO) has reported that around

80% of the world’s population utilize plants as their primary

source of medicinal agents and moreover, traditional medicine

remains the only health resource available to about 60% of the

world’s population, especially those in the vast rural areas of

developing countries (Le Grand and Wondergem, 1989; Cordell,

1995). However, this traditional knowledge is documented only

to a limited extent and is in danger of being lost (Parveen et al.,

2007). This is largely due to social changes within communities,

such as dislocation and the deaths of elders who possess this

knowledge (Smith, 1991). The loss of traditional medical

H.R. El-Seedi et al. / Journal of Ethnopharmacology 145 (2013) 746–757 747

knowledge is a worldwide phenomenon, affecting most indigen-ous societies (Brouwer et al., 2005).

The conservation of medicinal plants and natural resources isof growing importance, and can only be achieved by tapping intothe knowledge of local populations regarding the plants’ uses.Information on the medicinal uses of specific plants is typicallyobtained by interviewing herbalists and traditional healers(Hammond et al., 1998; Uniyal et al., 2006; Al-Qura’n, 2009),making it necessary to establish relationships based on mutualtrust before knowledge can be shared.

Cancer is one of the most important human diseases, and thereis considerable scientific and commercial interest in the discoveryof new anticancer agents from natural sources (Kinghorn et al.,2003). There are many difficulties associated with its treatment,the most common of which include drug resistance, toxicity andthe low specificity of currently available cytotoxic drugs (DeMesquita et al., 2009). These difficulties underpin the importanceof research aimed at the identification and development of novelanticancer compounds.

Medicinal plants have played important roles in the discovery ofanticancer drugs (Balunas and Kinghorn, 2005). Itharata et al. (2004)investigated the in vitro cytotoxic activity of medicinal plants usedtraditionally to treat cancer. The potential of natural products asanticancer agents was recognized in the 1950s by workers at theU.S. National Cancer Institute, who established a large scale-screening program using leukemic mice (Cragg and Newman,2005) and later (1985) set up an in vitro screening panel consistingof more than 60 different human tumor cell lines (Boyd and Paull,1995). Indeed, most of the new clinical applications of plantsecondary metabolites and their derivatives over the last half-century have been in the treatment of cancer (Newman et al., 2000).

There are four main classes of plant-derived anticancer agentsin current clinical use; the Catharanthus alkaloids, the epipodo-phyllotoxins, the taxanes and the camptothecines. The Cathar-

anthus alkaloids and several of their semi-synthetic derivativesinduce metaphase arrest and thereby inhibit mitosis by bindingspecifically to tubulin and causing its depolymerization(Okouneva et al., 2003). Vinblastine and vincristine were isolatedfrom C. roseus (L.) G. Don (Apocynaceae) formerly Vinca rosea L.and have been used clinically for over 40 years (van Der Heijdenet al., 2004). The epipodophyllotoxins bind to tubulin, causingDNA strand breaks during the G2 phase of the cell cycle byirreversibly inhibiting DNA topoisomerase II (Damayanthi andLown, 1998). Podophyllotoxin was isolated from the resin ofPodophyllum peltatum L. (Berberidaceae) (Gordaliza et al., 2004)but was found to be too toxic in mice, so derivatives were made;the first podophyllotoxin-derived drug approved for clinical usewas etoposide (Damayanthi and Lown, 1998).

The prototype taxane paclitaxel was originally isolated fromTaxus brevifolia Nutt. (Taxaceae) and entered the U.S. market inthe early 1990s (Wall and Wani, 1996). The taxanes, whichinclude paclitaxel and its derivatives, act by binding tubulin butdo not induce depolymerization or interfere with tubulin assem-bly (Schiff et al., 1979). Camptothecine was isolated from Camp-

totheca acuminata Decne. (Nyssaceae) but originally causedunacceptable levels of myelosuppression (Wall and Wani, 1996;Cragg and Newman, 2004). Interest in camptothecine was revivedwhen it was found to be a selective inhibitor of topoisomerase I,which is involved in the cleavage and reassembly of DNA (Craggand Newman, 2004). The semi-synthetic taxanes docetaxel(which is used to treat advanced breast cancer and metastaticCRPC) and cabazitaxel (used to treat docetaxel-resistant tumorsand prostate cancer) are two of the most important drugs in thecurrent global anticancer market (Oberlies and Kroll, 2004).

This work describes an ethno-botanical and ethnopharmacologicalstudy conducted in collaboration with herbalists from the Bedouin

tribes, with the intent of protecting traditional aboriginal knowledge.We have documented the medicinal uses of plants collected in thecourse of several trips to the Sinai Peninsula and the Menoufiagovernorate in the Nile Delta. The field trips were undertaken duringdifferent seasons between the years 2006 and 2009. The studyfocused on investigating the chemical components of the activefractions of U. maritima, isolating compounds that exhibit anticanceractivity, and elucidating their structures. In total, the use of 61 plantspecies was documented, and the plants were collected, identifiedand screened against human cancer cells. The current work is a partof our ongoing program of the isolation and structure elucidation ofbioactive chemical compounds from medicinal plants used in folkmedicine (El-Seedi et al., 2010, 2012).

2. Materials and methods

2.1. Instruments

General TLC: silica 60 F254 (Merck; 0.25 mm) with spot detec-tion under UV light (254 and 366 nm) and developed by dippingin vanillin–sulfuric acid and then heating (120 1C). The M.P. wasobtained using a Digital Melting Apparatus (Model 1A 8103,Electrothermal Engineering Ltd., U.K.); uncorrected. The opticalrotation at 251 was obtained with a Perkin–Elmer 241 polari-meter. UV spectra was obtained with a Shimadzu UV-160AUV–vis spectrophotometer. IR spectra was obtained with aPekin–Elmer 1600 FTIR spectrometer. HPLC–ESI–MS chromato-grams and spectra for dereplication were obtained on a FinniganLCQ (San Jose, CA, USA) ion-trap mass spectrometer coupled to anelectrospray interface and an AKTA basic 10 HPLC system with anXTerra MS C18 (2.1�100 mm, Waters, USA) column, eluting withmixtures of acetonitrile and H2O. LC–DAD fractionation wasperformed using a Shimadzu LC-10 system equipped with anSPD-M10AVP Diode Array Detector. UV data were collectedbetween 190 and 600 nm. HPLC was performed using an AKTAbasic system (Amersham Biosciences, Uppsala, Sweden) consist-ing of a P-900 pump, a UV-900 detector, a Frac-900 fractioncollector, a INV-907 injection valve, two PV-908 valves, an M-925mixer, a 50 ml Superloop, and a Unicorn 4.10 work station, withmonitoring at wavelengths of 217, 254, and 335 nm. Elution wasperformed using an analytical and semi-preparative columns(ACE-5C18, 4.6�250 or 10�250 mm, respectively, AdvancedChromatography Technologies, UK) in some cases and with apreparative column (FineLINE pilot 35, 35�160 mm, packed withsource 15RPC from Amersham Biosciences, Uppsala, Sweden) inothers.

HR–FAB–MS was obtained using a Jeol JMS-SX102 spectro-meter in positive-ion mode. Generally, 1H and 13C NMR spectrawere recorded on a Bruker DRX 600 spectrometer at 600.1 and at150.9 MHz, respectively, at 25 1C using CD3OD as the solvent.All chemical shifts are expressed relative to TMS.

2.2. Plant materials

Plant specimens were collected from the North and South Sinaiand Menoufia areas of Egypt between 2006 and 2009. The specieswere identified by two of this. Loutfy Boulos (Department ofBotany, Faculty of Science, Alexandria University, Alexandria,Egypt) and Prof. Zaki Turki (Department of Botany, Faculty ofScience, El-Menoufia University, Shebin El-Kom, Egypt). Voucherspecimens were deposited at the herbarium of the Department ofBotany in the Faculty of Science of El-Menoufia University, Egypt.The vernacular names of species collected in the Sinai area wereprovided by Dr. Ahmed Mansour (co-author). An ethnopharma-cological taxon table was drawn up containing the existing

H.R. El-Seedi et al. / Journal of Ethnopharmacology 145 (2013) 746–757748

scientific data on each collected species and the related nomen-clature, including its accepted scientific name (genius, species,etc), family and known range.

2.3. Extraction and isolation

Plant samples were cut into small pieces and dried at roomtemperature; the drying time varied depending on the nature of theplant parts. The dried material (50–100 g) was ground to a coarsepowder and extracted using methanol, with occasional stirring atroom temperature over four days, three times in succession. Theextracts were filtered, combined and evaporated under reducedpressure.

Dried and ground plant material from U. maritima (0.6 kg) wasextracted three times using a soxhlet apparatus with CH2Cl2:MeOH(2:1) at room temperature. The extract was concentrated in vacuo at30 1C to give 74.5 g of a semisolid material. The crude extract waspre-purified using solid-phase extraction (SPE) cartridges [ISOLUTE-XL C18 (EC), 10 g, International Sorbant Technology Ltd., UK].The SPE-purified solution was then evaporated to dryness, re-dissolved in EtOH (10 mg/ml), and dereplicated by HPLC. Duringthe dereplication process, the EtOH solution was fractionated into 96separate wells (1 ml) and the contents of each well were tested inbioassays and analyzed using LC/ESI–MS. HPLC elution was per-formed with mixtures of MeCN and H2O, starting with an equilibra-tion time of 10 min, followed by a ramp from 0 to 50% MeCN over8 min and then a second ramp from 50 to 100% MeCN over 12 min.A flow of 100% MeCN was then maintained for 10 min. Fractionationwas performed using a flow rate of 1 ml/min with a 500 mg injection,while a flow rate of 0.2 ml/min with a 10 mg injection was used inLC/MS experiments. 400 ml fractions were collected in a plate withdeep wells and then concentrated, re-dissolved in 20% DMSO,transferred to microtiter plates, and used in the bioassay. Afterdereplication, the crude extract (100 mg) was separated on a FineLINEPilot 35 column using a MeCN gradient in dist. H2O 65–100% that wasapplied over 16 column volumes (CV), followed by another 4 CVelution with 100% MeCN. The flow rate was 6.0 ml/min and theeluent was continuously monitored at wavelengths of 254, 217 and335 nm. Fractions were collected automatically as products eluted.Based on their UV spectra, fractions 51–53 appeared to be thosecontaining the active compound and were thus tested using thebioassay and then further purified by semi-preparative HPLC usinggradient elution with an equilibration time of 10 min followed by aramp from 50 to 100% MeCN in water over 50 min, elution with 100%MeCN for 50–70 min, and finally 50% MeCN for 70–75 min. The flowrate was maintained at 4.0 ml/min and the crude material wasinjected in batches of 2.5 mg to yield pure proscillaridin A (4.87 mg).

Proscillaridin A: FAB–MS: NIFAB-MS: m/z 530.3 with molecu-lar formula C30H42O8. 1H- and 13C–NMR spectroscopic data forthis compound are presented in Table 2.

2.4. Human tumor cell line

Cellular bioassays were performed using the human lym-phoma cell line U-937 GTB. The cells were maintained asdescribed previously (Dhar et al., 1996) and were grown inRPMI-1640 (Sigma) supplemented with 10% heat-inactivated fetalcalf serum (Sigma–Aldrich), 2 mM glutamine, 50 mg/ml strepto-mycin, and 60 mg/ml penicillin. Samples to be used in experi-ments were harvested during the exponential growth phase.

2.5. Preparation of microtiter plates

The crude plant extracts were dissolved in 10% DMSO (aq) to aconcentration of 1.0 mg/ml. These crude extracts were furtherdiluted with 10% DMSO to produce stock solutions of 10, 100 and

1000 mg/ml. 20 ml of each stock solution was pipetted intoV-bottomed, 96-well microtiter plates (Nunc, Roskilde, Denmark).Each plate was prepared with six blank wells containing only cell-growth medium, six negative controls containing 20 ml PBS, andsix solvent-control wells containing 20 ml 10% DMSO. All indivi-dual experiments were performed in duplicate, and each pair ofduplicates was tested three times. The prepared plates werestored at �20 1C for at most one month before being used inexperiments.

2.6. Fluorometric microculture cytotoxicity assay

The Fluorometric Microculture Cytotoxicity Assay (FMCA)(Larsson and Nygren, 1989) is based on monitoring fluorescencearising from fluorescein that is produced as a result of FDAhydrolysis by cells with intact cell membranes. Tumor cellssuspended in cell-growth medium were dispensed into theextract-containing microtiter plates. Each well was seeded with180 ml of cell suspension, containing approximately 20,000 cells,to give a total volume of 200 ml/well. The plates were thenincubated for 72 h at 37 1C under an atmosphere containing 5%CO2. Once the incubation time had ended, the plates werecentrifuged at 1000 rpm for 5 min, the medium was removed byaspiration, and the cells were washed with PBS. 100 ml of FDA(10 mg/ml in a physiological buffer) was then added to each well.After 40 min of incubation, the fluorescence at 538 nm in eachwell was measured, with excitation at 485 nm. The fluorescencein each well is proportional to the number of living cells itcontains. The activity of the extracts was reported in terms of asurvival index (SI), which was defined in terms of the fluorescencein the experimental wells, expressed as a percentage of that in thecontrol wells after the fluorescence of the blanks had beensubtracted from both the experimental and the control readings.

3. Results and discussion

The plants used in this study were chosen based on their use intraditional Egyptian medicine and have therefore effectively beenthrough a preliminary screen for biological activity. Most of theplants were collected in the deserts of the northern and southernSinai and in the area around the Menoufia governorate, 70 kmnorth of Cairo. They were collected in collaboration with nativeherbalists, who kindly shared their knowledge of each species’traditional uses.

Worldwide, scientific research has great attention to confirmthe potentials of medicinal herbs in the prevention of differentdiseases (Ung et al., 2007). Even nowadays with the enormouspotential of the high throughput screening methodology of thepharmaceutical industry, it would be a major project to screen allplants in such area (Verpoorte et al., 2005). However, theherbalists of the Sinai’s Bedouin tribes have been using traditionalmedicine for hundreds of years (Friedman et al., 1986), and so wesought to tap into their expertise in order to focus our search onplants that are known to have medicinal uses. Our principalBedouin collaborator was Haj Ahmed Mansour (also known asDr. Ahmed), a respected and well-known traditional herbalist andhealer who keeps a herbal garden in Wadi Itlah to preserve histraditional knowledge for future generations. His collaborationand willingness to share his knowledge was crucial for thisproject.

Some of the plants examined in this work are also used intraditional remedies elsewhere in the world. For example, Bidens

pilosa is used in Brazil to treat fever, diabetes, and inflammation(Pereira et al., 1999). Cichorium endivia, Origanum majorana,Solanum nigrum, and Viola odorata are used for the same purposes

H.R. El-Seedi et al. / Journal of Ethnopharmacology 145 (2013) 746–757 749

in Israel, as shown in Table 1 (Said et al., 2002). Ficus bengalensis isused in India for the treatment of dermatitis and diarrhea, whilePortulaca oleracea is used in China as an antiseptic and diuretic(Mukherjee et al., 1998; Xiang et al., 2005). In addition, some ofthe plants examined have uses elsewhere in the world that differfrom those identified by the Bedouins. For example, Amaranthus

spinosus is used to treat menorrhagia, indigestion, and urinarydisease in Egypt whereas in Burkina Faso it is used as anantimalarial and antimicrobial agent (Hilou et al., 2006). Nigella

sativa is used as a treatment for diabetes in Morocco but is used totreat high blood pressure and heart disease in Egypt. Similarly, C.

roseus is used to treat diabetes and high blood pressure in Egyptwhereas in South Africa it is used for severe gastrointestinalirritation, digitalis-like cardiac effects, and convulsions (Elgorashiet al., 2003; Tahraoui et al., 2007).

In total, 61 species that are used (or have been used) inEgyptian folk medicine were collected. The traditional uses ofeach species related by the Bedouin herbalists, together withthose that have previously been documented in the literature, arelisted in Table 1. This table also lists the scientific and vernacularnames of the species, as well as the plant family to which theybelong and the location at which they were collected. Traditionalherbal remedies are often administered as decoctions or teas,using water as the solvent. However, we used MeOH to extractsoluble species from the plants. MeOH is a polar solventand should extract most substances that are soluble in water.Eom et al. (2008) have reported that MeOH and H2O are equallyefficacious solvents for extracting materials used to make herbalteas. In some cases, we also extracted more hydrophobic speciesfrom the plants using CH2Cl2. For example, this method was usedto remove chlorophyll from Nerium oleander.

After extraction, the solvent was evaporated and the remain-ing crude extract was dissolved in 10% DMSO for the cytotoxicityassay using FMCA. The extracts were screened against thesensitive human lymphoma U-937 GTB cancer cell line.The extracts were tested three times in duplicate at threedifferent concentrations (1, 10 and 100 mg/ml); that is to say, atotal of six tests were performed on each extract at eachconcentration. The final concentration of DMSO in the assayedmaterial never exceeded 1%. The complete list of results obtainedfrom the cytotoxicity analyses is shown in Fig. 1; the results forindividual species are identified using the numbers assigned tothem in Table 1.

The plants whose extracts exhibited the greatest potency wereAsclepias sinaica (species no 4), U. maritima (species no 40), N.

oleander (species no 2), and C. roseus (species no 3), all of whichcaused almost complete cell death at the lowest concentrationevaluated (1 mg/ml). Extracts from C. endivia (species no 15),Pulicaria undulate (species no 11) and Melia azedarach (speciesno 41) also showed high activity: some cell death was observed atthe lowest concentration and they exhibited good activity at thehigher concentrations. Extracts from the other plants were eitherinactive or only moderately active. These results are consistentwith literature reports on the toxicity of certain plant speciesused in traditional medicine, including U. maritima, N. oleander,and C. roseus (Bnouham et al., 2002; Bielawski et al., 2006).

A more detailed investigation was done into the most potentextracts identified in the initial screen, namely that from U.

maritima. The MeOH extract was subjected to bioassay-guidedfractionation in order to isolate chemical species responsible forthe observed biological activity. The most active fractions (51–53)were purified more extensively, leading to the isolation of a purecardiac glycoside. The compound was identified as a bufadieno-lide with m/z at 531, which is consistent with it being proscillar-idin A, whose structure is shown in Fig. 2. The identity of thiscompound by comparing its 1D and 2D NMR spectra to published

data (Krenn et al., 1988; Kopp et al., 1996). Finally, the cytotoxi-city of the purified compound towards the histiocytic lymphomacell line U-937 GTB was tested; its IC50 value was found to be4.271.2 ng/ml (7.9 nM) Fig. 3.

Cardiac glycosides have a range of interesting biologicalactivities and have been used in the clinic to treat heart failurefor many years (Prassas and Diamandis, 2008). They are alsointeresting because of their toxicity (Li et al., 2010). Cardiacglycosides whose aglycone contains or is derived from oleandri-genin exhibit cytotoxic activity and are being studied as potentialcomponents of anticancer drugs (Schmelzer and Gurib-Fakim,2008). It is therefore of interest to examine natural products thatcontain this motif and to investigate their applicability in che-moprevention (Cerella et al., 2010).

Detailed chemical analysis of the active extracts from plantsother than U. maritima was beyond the scope of this study.However, as a preliminary step in this direction, we surveyedthe literature to obtain information on the composition andknown effects of the other active extracts. The second-most activeextract identified in this work was that of A. sinaica (syn.Gomphocarpus sinaicus), which is known to be rich in cardiacglycosides such as calotropin, calactin, and 5,6-dehydrocalotropin(El-Askary et al., 1995a, 1995b; Abdel-Azim, 1998), as well asflavonoid glycosides (Heneidak et al., 2006). It grows in theMediterranean and Saharo–Arabian regions, as well as in theNorth and South Sinai (Boulos, 2000).

The third most active species was N. oleander. It has beenreported to contain cardiac glycosides such as oleander, olean-drin, cardenolide N-1, cardenolide N-4, 3b–O-(b-D-sarmentosyl)-16b-acetoxy-14-hydroxy-5b,14b-card-20-(22)-enolide and 16b-acetoxy,14-dihydroxy-5b,14b-card-20-(22)-enolide (Zhao et al.,2007). The last of these compounds has been demonstrated toexhibit in vitro cytotoxicity against human jurkat leukemia(T-cell), promyelocytic lymphoma (HL-60), cervical carcinoma(HeLa), breast carcinoma (MCF-7), prostate carcinomas (LNCap,DU145, PC3), malignant fibroblast (VA-13) and liver carcinomacells (HepG2) (Raghavendra et al., 2007; Zhao et al., 2007). N.

oleander is found in most of the Mediterranean region, NorthAfrica, Morocco, and the southern parts of China (Tackholm,1974).

The fourth most potent plant extract was that of C. roseus.This species is a rich source of pharmaceutically important alkaloids(Verma et al., 2007), more than 130 of which have been described todate. It originates from Madagascar, but is widely cultivated and hasbeen naturalized in the tropics and subtropics of both hemispheres.It is the sole source of vincristine and vinblastine, which are used inthe clinic against a variety of cancers such as acute leukemia,Hodgkin’s disease, rhabdomyosarcomas, neuroblastoma, and otherlymphomas (Mukherjee et al., 2001; Van der Heijden et al., 2004;Aslam et al., 2009). This is consistent with the high cytotoxicity itsextracts exhibited in this work.

Thus, three of the four most potent species identified in thisstudy are known to be rich in cardiac glycosides, and it is likelythat compounds from this family are responsible for the observedextract cytotoxicity in all three cases (Newman et al., 2008). It hasbeen suggested that compounds from the cardiac glycoside familyaffect the complex mechanisms of cellular signal transduction,resulting in selective inhibition of the proliferation of humantumor cells in mouse xenograft models. As such, they werepreviously regarded as potentially promising starting points forthe development of targeted cancer chemotherapeutics (Balunasand Kinghorn, 2005; Newman et al., 2008). Recently, a moreprecise mechanism for their action has been proposed, involvinginhibition of the Na(þ)/K(þ)-pump and the subsequent blockingof protein synthesis (Perne et al., 2009). The study that led to theproposal of this mechanism also demonstrated that cardiac

Table 1Traditional uses and scientific and vernacular names of the species collected in this study; the table also lists the location at which the plants were collected and the solvents used when extracting them.

Family No Scientific name (Voucher Number) Location Vernacular name Traditional use according to the Bedouins Extraction

solvents

Reference

Amaranthaceae 1 Amaranthus spinosus L. (HRE 100) Met Abo El-Kom,

Menoufia ‘Orf El-Diek

Aerial parts used for menorrhagia, indigestion,

skin diseases, and urinary diseases. b

MeOH Handa et al. (2006)

Apocynaceae 2a Nerium oleander L. (HRE 101) Shebin El-Kom, Menoufia

Defla

Inhalation of the vapors arising from a heated

decoction of the roots is used to treat

headaches and colds. Decoctions of the leaves

are used for skin diseases. Also used against

paralysis and pain in extremities.

CH2Cl2 Said et al., (2002);

Yesilada (2002)

2b MeOH

3 Catharanthus roseus G.Don (HRE 102) Shebien El-Kom,

Menoufia winkle

Used to treat diabetes and high blood pressure. MeOH Pereira et al. (2010)

Asclepiadaceae 4a Asclepias sinaica Boiss. (HRE 103) Saint Catherin, South

Sinai Hargal Barri

Juices are used externally to treat skin

diseases.aCH2Cl2

4b MeOH

Chenopodiaceae 5 Chenopodium ambrosioides L. (HRE 104) Saint Catherin, South

Sinai Zorbeih

Bush tea used against stomach discomfort, and

intestinal worms.aMeOH

6 Anabasis setifera Moq. (HRE 105) Om Shehan, North Sinai

Gelw

Used to treat delayed menstruation.a MeOH

Compositae

(Asteraceae)

7 Artemisia monosperma Delile (HRE 106) Om Shehan, North Sinai

Aader

Used as an antispasmodic, antihelmintic, and

against hypertension.aMeOH

8 Chiliadenus montanus (Vahl) Brullo (HRE 107) Saint Catherin, South

Sinai Heneida

Used for diarrhea, stomach ache, chest

diseases, and as herbal tea for urinary

troubles.a

MeOH

9 Matricaria recutita L. (HRE 108) Shebin El-Kom Menoufia

Babounig

Administered as a sedative and antispasmodic

tea. Also used as antiseptic, mouthwash, and as

eyewash for an infected or inflamed eye.

MeOH Viola et al. (1995)

10 Nauplius graveolens (Forssk.) Less. (HRE 109) Om Shehan, North Sinai

Nogd

Used for blennorrhagia, diabetes, diarrhea,

headache and cold.aMeOH

11 Pulicaria undulate L. (HRE 110) Saint Catherin, South

Sinai Dithdath

Aerial parts used to treat heart disease and as

an antispasmodic.aMeOH

12 Senecio reflexum L. (HRE 111) Om Shehan, North Sinai

Morrar, Umm

Lonein

Used for treating skin diseases.a MeOH

13 Sonchus oleraceus L. (HRE 112) Met Abo El-Kom,

Menoufia Go’edied

Used for treating typhoid.b MeOH Njoroge et al., (2004)

14 Bidens pilosa L. (HRE 113) Met Abo El-Kom,

Menoufia Inyabarasanya

Used to treat fever, angina, diabetes, edema,

infections, inflammation, malaria, and even

tumors.

MeOH Pereira et al. (1999)

15 Cichorium endivia L. (HRE 114) Met Abo El-Kom,

Menoufia Shikoria

Used against bacterial infection, poisoning,

rheumatism, to improve the appetite,

digestion, and as a diuretic. Also used to lower

cholesterol and to treat kidney and liver

problems.b

MeOH Azaizeh et al. (2006);

Said et al. (2002)

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74

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Convolvulaceae 16 Convolvulus arvensis L. (HRE 115) Shebin El-Kom, Menoufia

‘Olleiq

Used as a purgative, to remove dandruff,

against worms, and for skin and stomach

disorders.

MeOH Handa et al. (2006)

17 Ipomoea carica L. (HRE 116) Met Abo El-Kom,

Menoufia Sitt El-Hosn

Used for treating rheumatism, inflammation,

and as a purgative.bMeOH Handa et al. (2006)

Cruciferae 18 Sysimbrium irio L. (HRE 117) Met Abo El-Kom,

Menoufia Figl El-Gamal

Boiled seeds are used as an expectorant and as

a febrifuge.

MeOH Ahmad (2007)

Ephedraceae 19 Ephedra pachyclada Boiss. (HRE 118) Saint Catherin, South

Sinai Alda

Contains tannins for leather.a MeOH

Euphorbiaceae 20 Euphorbia peplus L. (HRE 119) Met Abo El-Kom,

Menoufia Wedeina

Used as a purgative, expectorant, as an

anthelmintic and anti-inflammatory agent.

MeOH Handa et al. (2006)

21 Euphorbia prostrata L. (HRE 120) Shebin El-Kom, Menoufia

Libbein

Used as an anti-inflammatory agent.b MeOH Rene et al. (2007)

22 Euphorbia pulcherrima Willd. ex Klotzsch (HRE

121)

Met Abo El-Kom,

Menoufia Bent El-Quonsol

____ MeOH

23 Ricinus communis L. (HRE 122) Tokh Dalaka, Menoufia

Kherwa’a

Used as anti-swelling agent, a purgative and as

a component in cosmetics.bMeOH Handa et al. (2006)

Globulariaceae 24 Globularia arabica Jaub. & Spach (HRE 123) Om Shehan, North Sinai

Handaqouq

Contains tannins for leather.a MeOH

Gramineae (Poaceae) 25 Avena fatua L. (HRE 124) Met Abo El-Kom,

Menoufia Zommeir

Highly nutritious, promotes the health of the

nervous system, bones, skin, hair and nails.bMeOH

26 Poa annua L. (HRE 125) Met Abo El-Kom,

Menoufia

Sabal Abu El-

Hossein

____ MeOH

27 Polypogon monspeliensis (L.) Desf. (HRE 126) Met Abo El-Kom,

Menoufia Theil El-Qott

Used for treatment of heart palpitations.b MeOH

28 Polypogan semi verticellata L. (HRE 127) Met Abo El-Kom,

Menoufia Theil El-Qott

Leaves used for their antihelminthic and

antimalarial properties.bMeOH

Illecebraceae 29 Pteranthus dichotomus Forssk. (HRE 128) Om Shehan, North Sinai

Na’eema

Leaves used as an ocular antiseptic.a MeOH

Labiatae (Lamiaceae) 30 Ballota undulata (Sieber ex Fresen.) Benth. (HRE

129)

Om Shehan, North Sinai

Ghassa

Used as an antispasmodic, diuretic, antiulcer

agent, and for the treatment of nervous

disorders.a

MeOH

31 Lavandula pubescens Decaisne. (HRE 130) Om Shehan, North Sinai

‘Atan

Used as an antimiocrobial agent, is added to

food to protect it from microbes and for its

pleasant smell.a

MeOH

32 Mentha longifolia L. (HRE 131) Saint Catherin, South

Sinai Habaq El-Barr

A decoction of the leaves in water is used as a

sedative.aMeOH

33 Origanum majorana L. (HRE 132) Shebin El-Kom, Menoufia

Bardaqoush

Used as an anticoagulant, digestive,

mouthwash; for the treatment of respiratory

disorders, migraines; for the control of

menstruation; and for the nervous system.

MeOH Said et al. (2002)

34 Salvia aegyptiaca L. (HRE 133) Om Shehan, North Sinai

Ra’alah

Used to treat diarrhea, gonorrhea, as an

antiseptic, antispasmodic, and stomachic.

MeOH Handa et al. (2006)

35 Stachys aegyptiaca Pers. (HRE 134) Om Shehan, North Sinai

Gortom

Used to treat disorders of the chest and

asthma.aMeOH

36 Teucrium leucocladum Boiss. (HRE 135) Saint Catherin, South

Sinai G’eda

Used to treat disorders of the chest and

asthma.aMeOH

Liliaceae 37 Allium artemisietorum Eig & Feinbrun (HRE 136) Om Shehan, North Sinai

Thoum El-Debaba

Used to treat skin disease and for the scalp.a MeOH

38 Asparagus stipularis Forssk. (HRE 137) Om Shehan, North Sinai

Halaioun

Used to treat urinary tract infections, as well as

for kidneys and gallstones.aMeOH

39a Asphodelus ramosus L. (HRE 138) Om Shehan, North Sinai

Boaraq

Used to treat eczema and cracked skin, and

against ringworm.

MeOH Handa et al. (2006)

H.R

.E

l-Seedi

eta

l./

Jou

rna

lo

fE

thn

op

ha

rma

colo

gy

14

5(2

01

3)

74

6–

75

77

51

Table 1 (continued )

Family No Scientific name (Voucher Number) Location Vernacular name Traditional use according to the Bedouins Extraction

solvents

Reference

39b CH2Cl2

40 Urginea maritima (L.) Baker (HRE 139) Om Shehan, North Sinai

Basal Far’aon

Used as an antibacterial, anthelmintic,

abortifacient, and diuretic. Also used as an

expectorant in bronchitis, chronic catarrh and

pneumonia. Fresh bulbs are applied to wounds

to promote healing. Useful for rheumatism,

edema, gout, slowing down the pulse, and in

the treatment of cancer.a

MeOH Boulos (1983)

Meliaceae 41 Melia azedarach L. (HRE 140) Met Abo El-Kom,

Menoufia Zanzalkht

Leaves used to treat snake bites and skin

infections.

MeOH Handa et al. (2006)

Moraceae 42 Ficus bengalensis L. (HRE 141) Shebin El-Kom, Menoufia

Teen

Used to treat dermatitis and diarrhea.b MeOH Mukherjee et al.

(1998)

Oxalidaceae 43 Oxalis corniculata L. (HRE 142) Met Abo El-Kom,

Menoufia Hommeid

Leaves used to treat bladder liver disorders,

gastrointestinal pain, and vertigo.bMeOH Handa et al. (2006)

Papilionaceae 44 Trifolium resupinatum L. (HRE 143) Met Abo El-Kom,

Menoufia Qort

Used as an expectorant, antiseptic, analgesic

and sedative.

MeOH Sabudak et al. (2008)

45 Sesbania sesban L. (HRE 144) Shebin El-Kom, Menoufia

Saisaban

Used as a hemolytic agent and a molluscicide.b MeOH Reed et al. (2000)

Palmae 46 Hyphaene thebaica L. (HRE 145)

Doom

Used to lower blood pressure.a MeOH

Polygonaceae 47 Rumex dentatus L. (HRE 146) Met Abo El-Kom,

Menoufia Khillala

Used to treat pneumonia, cough abscesses,

stomach ache, smallpox and tumor.

MeOH Fatima et al. (2009);

Midiwo et al. (2002)

Pontederiaceae 48 Eichhornia crassipes (C.Mart.) Solms (HRE 147) Shebin El-Kom, Menoufia

Yasent El-Mayya

The flowers are used for medicating the skin of

horses.bMeOH

Portulacaceae 49 Portulaca oleracea L. (HRE 148) Met Abo El-Kom,

Menoufia Regla

Used as an antiseptic, a diuretic in urinary

disorders, a febrifuge, and in the treatment of

dysentery, carbuncles, snake bite and against

sores.b

MeOH Handa et al. (2006);

Xiang et al. (2005)

Ranunculaceae 50 Nigella sativa L. (HRE 149) Shebin El-Kom, Menoufia

Habea El-braka

The wooden stem is used to treat jaundice.

Seeds are used to treat blood pressure heart

diseases, impotence headaches, nasal

congestion, toothache, and against intestinal

worms.b

MeOH Handa et al. (2006);

Said et al. (2002)

Resedaceae 51 Caylusea hexagyna (Forssk.)M.L.Green (HRE 150) Om Shehan, North Sinai

Danaban

Used with olive oil to treat skin infection.a MeOH

52 Reseda arabica Boiss. (HRE 151) Om Shehan, North Sinai

Theil El-Kharouf

Used to treat pain, infections or disorders of

the vulva and vagina.aMeOH

53 Reseda muricata C.Presl (HRE 152) Om Shehan, North Sinai

Khozama

Used as an antimicrobial agent.a MeOH

Scrophulariaceae 54 Kickxia aegyptiaca [L.] Nabelek (HRE 153) Om Shehan, North Sinai

Megeinina

Used to treat skin infections.a MeOH

55 Scrophularia marilandica L. (HRE 154) Met Abo El-Kom,

Menoufia Sreeda

Used externally on skin disorders, taken

internally to relieve everything from eczema

and psoriasis to mastitis, inflammations of the

mammary gland and chronic lymphatic

stagnation.b

MeOH

Solanaceae 56 Solonum nigrum L. (HRE 155) Met Abo El-Kom,

Menoufia ‘Enab El-Deeb

Used to treat certain human skin carcinomas,

typhoid fever, wounds, and sun burn.bMeOH Azaizeh et al. (2006);

Njoroge et al. (2004);

Said et al. (2002)

57 Ammi majus L. (HRE 156) Shebin El-Kom, Menoufia MeOH

H.R

.E

l-Seedi

eta

l./

Jou

rna

lo

fE

thn

op

ha

rma

colo

gy

14

5(2

01

3)

74

6–

75

77

52

Um

be

llif

era

e

(Ap

iace

ae

)K

hil

la

Use

dto

tre

at

ast

hm

ah

yp

og

lyce

mia

,a

sa

n

an

tisp

asm

od

ic,

diu

reti

c,ca

rmin

ati

ve

ag

en

t,in

the

tre

atm

en

to

fd

ige

stiv

ep

rob

lem

sa

nd

skin

dis

ea

ses

(vit

ilig

oa

nd

pso

ria

sis)

.b

Fab

rica

nt

an

d

Farn

swo

rth

(20

01

)

58

Am

mi

vis

na

ga

L.(H

RE

15

7)

Sh

eb

inE

l-K

om

,M

en

ou

fia

Se

wa

kE

nn

ab

i

Use

dto

tre

at

mil

do

bst

ruct

ion

ao

fth

e

resp

ira

tory

tra

ctin

ast

hm

a,

ve

rtig

o,

dia

be

tes,

kid

ne

yst

on

es,

an

da

sa

diu

reti

c.b

Me

OH

Ba

tan

ou

ny

(19

99

)

59

Dev

erra

trir

ad

iata

Ho

chst

.e

xB

ois

s.(H

RE

15

8)

Om

Sh

eh

an

,N

ort

hS

ina

i

‘Ele

iga

n

Use

dto

tre

at

ast

hm

aa

nd

inte

stin

al

cra

mp

s.a

Me

OH

Vio

lace

ae

60

Vio

lao

do

rata

L.(H

RE

15

9)

Sh

eb

inE

l-K

om

,M

en

ou

fia

Ba

na

ph

seg

Foli

ag

eu

sed

totr

ea

tin

fla

mm

ati

on

of

the

resp

ira

tory

syst

em

,st

om

ach

an

din

test

ine

.b

Me

OH

Sa

ide

ta

l.(2

00

2)

Zy

go

ph

yll

ace

ae

61

Fag

on

iaa

rab

ica

L.(H

RE

16

0)

Om

Sh

eh

an

,N

ort

hS

ina

i

Sh

ob

roq

Use

dto

tre

at

jau

nd

ice

an

dk

idn

ey

dis

ea

ses.

aM

eO

HE

l-D

ari

er

an

d

El-

Mo

ga

spi

(20

09

)

aT

rad

itio

na

lu

ses

as

de

scri

be

db

yM

an

sou

r,A

.b

Tra

dit

ion

al

use

so

bta

ine

db

yco

nsu

ltin

go

the

re

xp

ert

sa

nd

/or

sou

rce

s.

Table 21H and 13C–NMR assignments for Proscillaridin A in CD3OD. d in ppm, J (Hz).

Position 1H 13C

1 – 34.0

2 – 26.8

3 4.35 (m) 73.9

4 5.50 (s) 120.3

5 – 146.9

6 1.70, 1.26 32.0

7 – 28.8

8 1.80 41.9

9 1.07 (td, 12.0, 3.5) 48.0

10 – 37.2

11 1.98, 1.71 20.9

12 1.46, 1.29 40.5

13 – 49.0

14 – 84.0

15 – 31.8

16 – 28.5

17 2.47 (dd, 9.8, 6.6) 50.7

18 0.94 (s) 16.8

19 0.96 (s) 18.4

20 – 123.2

21 7.48 (d, 2.7) 148.6

22 8.23 (dd, 10.0, 2.7) 147.4

23 6.36 (d, 9.7) 114.4

24 – 162.8

1’ 5.50 99.1

2’ 4.49 71.0

3’ 4.53 (dd, 9.0, 2.7) 71.1

4’ 4.40 (t, 9.0) 72.3

5’ 4.24 (dq, 9.0, 6.0) 68.0

6’ 1.72 (d, 6.0) 17.8

H.R. El-Seedi et al. / Journal of Ethnopharmacology 145 (2013) 746–757 753

glycosides induce death in both cancerous and healthy humancells by inhibiting general protein synthesis, casting doubt ontheir utility as anticancer therapeutics. However, several alter-native mechanisms have been suggested, some of which would bemore compatible with therapeutic use. In addition, their biologi-cal activity remains a topic of active research interest, particularlyin terms of their pharmacodynamic interactions with cytotoxicdrugs used in the clinic (Felth et al., 2009).

Communication between scientists and individual healers is anessential component of efforts to preserve the teachings of tradi-tional medicine. By and large, the responsibility for this lies with thetraditional healing community; communication and the free flow ofinformation would be greatly facilitated by the formation of a stronghealers’ association. However, healers often feel exploited in theirinteractions with scientists, and there is a perception that research-ers take information on their secrets without giving anything back inreturn. Many healers therefore do not believe that scientists willactually help them and this (hopefully misguided) sentiment oftenprecludes meaningful cooperation.

Despite these difficulties, the results of this study show thateven if one examines only a small set of the species used intraditional medicine, it is possible to identify plants with inter-esting activity that merit further investigation.

4. Conclusion

An ethno-botanical approach was adopted to identify Egyptianplants for screening against cancer. Despite Egypt’s long history oftraditional medicine, very few of the plants traditionally usedby Egyptian herbalists have had their medicinal properties investi-gated using contemporary scientific methods. In particular, verylittle attention has been paid to the potential utility of compoundsor extracts from plants used in traditional Egyptian medicine as

Fig. 1. Cytotoxicity data for extracts from 61 Egyptian plant species. The extracts’ activity is expressed in terms of the survival index (SI), i.e. as a percentage of the

intensity of fluorescence in the treated well relative to that for the control wells, with the intensity of the blanks subtracted. Each extract was tested at three different

concentrations using the human lymphoma cell line U-937 GTB. The activity shown is the mean value from three separate measurements, each of which was conducted in

duplicate. Error bars show the standard deviation.

H.R. El-Seedi et al. / Journal of Ethnopharmacology 145 (2013) 746–757754

anticancer agents. To address this deficiency, we examined a rangeof plants that are used in this way and identified theirscientific names.

Over 60 species were examined; to the best of our knowledge,this work represents the first time that many of them have been

tested for anticancer activity. Some of the plants studied werepreviously known to exhibit anticancer activity but werere-examined due to their established use in folk medicine. Theplants whose extracts exhibited the strongest cytotoxic activityand thus merited further investigation were A. sinaica, U. maritima,

Fig. 2. Chemical structure of proscillaridin A.

Fig. 3. Concentration–response curve for the cytotoxicity of proscillaridin A

against the lymphoma cell line U-937 GTB using the fluorometric microculture

cytotoxicity assay (FMCA). Each point represents the mean7SEM for three

duplicate measurements.

H.R. El-Seedi et al. / Journal of Ethnopharmacology 145 (2013) 746–757 755

N. oleander and C. roseus followed by C. pumilum and M. azedarach.Bioassay-guided fractionation of extracts from these specieswould facilitate the isolation and identification of the compoundsresponsible for their activity, which could then be evaluatedas potential drug candidates or lead compounds. The specieswhose extracts exhibited the greatest activity in the assay wasU. Maritima.

There have been some previous efforts to document thebiological activity of Urginea species. For example, Naidoo et al.(2004) examined the antibacterial activity of U. sanguine. U. indica

was shown to exhibit activity against the growth of an ascitestumor (Deepak and Salimath, 2006), and U. maritima is used as acardiotonic diuretic (Iizuka et al., 2001). U. maritima syn. Drimia

maritima, Scilla maritima belongs to Liliaceae family. It is culti-vated in the Mediterranean region, where it grows in sandy soils(Kopp et al., 1996) and is commonly known as the white squill. Ithas been used in medicine for many centuries due to its powerfuldigitalis-like cardiac effect (Krenn et al., 2000), and is also used asa diuretic, heart stimulant (Dias et al., 2000), antibacterial,anthelmintic, abortifacient and diuretic in traditional medicine.It has also found uses as an expectorant in the treatment ofbronchitis, chronic catarrh and pneumonia. Fresh bulbs of thisspecies are applied to wounds to promote healing, and it isreported to be useful for treating rheumatism and gout, as wellas in slowing down the pulse and in the treatment of cancer(Boulos, 1983). It has previously been reported to exhibitcytotoxicity against human breast carcinoma cells (MCF-7)in vitro (Bielawski et al., 2006). Other traditional uses confirmedby Bedouins during our trips to collect plants from Sinai includethe treatment of heart diseases, wounds, tumors and edema.

Extracts from this species are known to inhibit the Naþ ,Kþ- adenosine triphosphatase (Naþ , Kþ-ATPase) (Schoenfeldet al., 1985). A number of authors have reported that Urginea speciesare rich in several cardiac glycosides (Krenn et al., 1988, 1994; Koppet al., 1990, 1996; Majinda et al., 1997; Iizuka et al., 2001).

Our ongoing phytochemical investigations into plants used intraditional medicine resulted in the isolation of a bioactivecardiac glycoside from U. maritima. The active compound wasidentified as proscillaridin A based on extensive spectroscopicanalysis including 1D and 2D NMR. Its cytotoxic activity againstthe human tumor cell line (U-937 GTB) was evaluated and foundto be consistent with previous reports. Overall, the resultsobtained demonstrate the value of traditional medical knowledge,and particularly that of Bedouin healers, in identifying naturalproducts with potentially useful biological activity.

Acknowledgment

We are grateful for financial support in the form of a Grant inAid for Scientific Research (2007-6738) awarded to H. R. E. andU. G. under the auspices of the Swedish Research Links programoperated by the Swedish International Development Agency– Middle East and North Africa (SIDA- MENA). U. G. is supportedby the Swedish Foundation for Strategic Research and the Swed-ish Research Council. The assistance of the Bedouins in the Sinai ishighly appreciated; we are particularly grateful to Mr. OmerEl-Gendy, Tawfik Abou-Garad and Salem A. S. Abou-Mosafer fortheir exertions in locating and collecting plants. H. R. E. thanks theChemistry Department of El-Menoufia University for granting himleave to visit Uppsala University on several occasions.

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