Abstract
Context: This study is a continuation of our previous work in which a bioassay screening of 346 methanol extracts from 281 Egyptian plant species was carried out for in vitro schistosomicidal activity.
Objective: Another 309 methanol extracts from 278 plant species were subjected to the bioassay screening using the same technique on viable Schistosoma mansoni Sambon (Schistosomatidae) mature worms in specialized culture medium (Roswell Park Memorial Institute medium 1640) in a trial to discover a source for a schistosomiasis drug from Egyptian flora.
Material and methods: The methanol plant extracts were tested in vitro against viable S. mansoni mature worms in culture medium. Viability of worms was examined after exposure to 100 μg/ml of the extract in the medium for 24 h. Negative (dimethyl sulfoxide) and positive (praziquantel) controls were simultaneously used. Extracts showing schistosomicidal activity were further subjected to determination of their (Lethal concentration) LC50 and LC90 values.
Results: Confirmed in vitro antischistosomal activity was found in 42 extracts. Of these, 14 plant species possessed considerably high antischistosomal activity (LC50 ≤ 15 µg/ml), viz. Callistemon viminalis (Soland. Ex Gaertn) Cheel, C. rigidus R.Br., C. speciosus (Sims.) DC, C. citrinus Stapf, Eucalyptus citriodora Hook, E. rostrata Dehnh., Eugenia edulis Vell, E. javanica Lam syn. Syzygium samarangense (Blume) Merril, Melaleuca leucadendron (L.) L., M. stypheloides Sm. (all belong to Myrtaceae), Cryptostegia grandiflora R.Br. (Asclepiadaceae), Zilla spinosa (L.) Prantl (Cruciferae), Ficus trijuja L. (Moraceae) and Fagonia mollis Delile (Zygophylacae).
Discussion and conclusion: These species may represent additional natural sources of bioactive material that deserve further investigation for drug discovery against schistosomiasis.
Introduction
Schistosomiasis is the most prevalent serious parasitic disease in Egypt and several other countries in tropical and sub-tropical regions of Africa, Asia and South America (CitationWHO 2002). Currently, there is only a single effective drug for the treatment and control of this disease, namely, praziquantel (PZQ) (CitationCioli et al., 1995). Moreover, in folk medicine, no plants in the Egyptian region are specifically known to have a curative effect. The present reliance on PZQ represents a serious situation due to the possible development of drug resistant by the parasite (CitationIsmail et al., 1999; CitationDoenhoff et al., 2002). Consequently, there is an urgent need for developing new antischistosomal drugs (CitationLambertucci et al., 1980; CitationSouza et al., 1982; CitationRibeiro-dos-Santos et al., 2006).
Plants represent an important source for drug discovery and have produced some very effective chemotherapeutic treatments for parasites, e.g., antimalarial drugs (CitationFrederich et al., 2002). In Egypt, folk medicine contains several wild plants such as Artemisia judaica L. (Compositae), Balanites aegyptiaca (L.) Del. (Balanitacae), Capparis spinosa L. (Capparaceae), Cyperus rotundus L. (Cyperaceae), and Peganum harmala L. (Zygophyllaceae) which are used as anthelmintics and as parasiticides in veterinary medicine (CitationKarawya et al., 1977; CitationHilal et al., 1978, Citation1979; CitationChaya et al., 1995). Therefore, extensive investigations of Egyptian flora to discover new natural antischistosomal agents are on going. Our preceding publication (CitationYousif et al., 2007) subjected 346 methanol extracts representing 281 Egyptian native and cultivated plants to in vitro screening for schistosomicidal activity on mature Schistosoma mansoni worms, and 15 extracts with strong antischistosomal activity possessing LC50 ≤15 µg/ml) were detected. The present paper represents a second step in the program carried out for screening the Egyptian flora under a collaborative research project (CitationEl-Menshawi, 2003) with an ultimate goal to find a new drug for schistosomiasis.
Materials and methods
Plant material
Plant material used in this study consists of 278 species which yielded 309 extracts from different plant organs as shown in . Plant specimens were collected randomly. Native species collected from various areas of Egypt (namely: Mediterranean coastal region, Sinai, Red Sea coastal region, Nile Valley, Eastern and Western deserts including the oases). Cultivated taxa were also obtained from various botanical gardens (namely: Zoological Garden, Orman Garden, 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 in accordance with CitationBoulos (1999, Citation2000, Citation2002, and Citation2005). The identification of cultivated plants was done by Therese Labib and the nomenclature follows CitationHuxley et al. (1992).
Table 1. Plants extracts (native and cultivated) screened for antischistosomal activity in vitro.
Preparation of extracts
A small quantity of each plant, sufficient to yield about 50 g dry weight, was collected for preliminary bio-screening. Routine protection of natural plant constituents from denaturation or artifact formation during the extraction and concentration procedures was assured during the preparation of crude extracts (CitationEl-Menshawi, 2003). Whole plants or plant parts were 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 untill used.
Parasite material
Clean and schistosome infected hamsters were obtained from the Schistosome Biological Supply Center at Theodor Bilharz Research Institute. The animals are maintained according to the internationally valid guidelines regularly followed in the institute. Hamsters (80–100 g) were infected percutaneously with 350–400 cercariae and worms were cleared from the blood of maturely infected hamsters by perfusion technique using phosphate buffer through 20 µm mesh sieves and rapidly placed in culture medium Roswell Park Memorial Institute medium (RPMI) 1640 containing 300 mg streptomycin, 300 units penicillin and 160 μg gentamycin/100 ml medium.
Schistosomicidal bioassay and determination of LC50 and LC90
The method used in this study is similar to that by CitationYousif et al. (2007) and CitationRamirez et al. (2007). A stock solution (10 mg/ml) of each plant extract was prepared in dimethyl sulfoxide (DMSO) and diluted with RPMI to produce 3 ml test solution of 100 µg/ml final concentration 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 µg/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 one minute, associated with worm deformity such as blackening, twisting, and contracting, etc. were considered dead. The activity of the extract was measured by calculating the number of dead worms relative to the total number of worms. In the case of any doubt about viability of worms, they were allowed to recover in clean medium for 24 h and re-examined.
Active extracts resulted from the above screening were similarly bioassayed at descending concentrations, e.g., 50, 30, 10, 7, 5, 3 and 1 µg/ml and the mortality of worms was recorded at each concentration. 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, statistical analysis software package/version 9/1989 SPSS Inc., Chicago, USA).
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 resulted 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 significant day-to-day variation (p > 0.01) which assures reproducibility. Also, the calculated % coefficient of variation of the LC values lie within the accepted range, thus proving the precision and accuracy.
Results and discussion
The bio-screening results revealed that 42 extracts possessed reproducible and confirmed in vitro antischistosomal activity. The plant species producing these active extracts are marked with (*) in . The LC50 and LC90 values of the above active extracts were determined and recorded in where, 18 extracts out of them were found to possess strong antischistosomal activity (LC50 values equal to or less than 15 µg/ml). The plant species which produced these most effective extracts are listed as follows [after the serial number of each]: [21] Cryptostegia grandiflora R.Br. (Asclepiadaceae), [116] Zilla spinosa (L.) Prantl, (Cruciferae), [219] Ficus trijuja L., (Moraceae), [221, 222] Callistemon viminalis (Soland. Ex Gaertn) Cheel, [223] Callistemon rigidus R. Br., [224] Callistemon speciosus (Sims.) DC, [225 & 226] Callistemon citrinus Stapf, [228 & 229] Eucalyptus citriodora Hook, [230] Eucalyptus rostrata Dehnh., [231] Eugenia edulis Vell, [234] Eugenia javanica syn. Syzygium samarangense (Blume) Merril, [238 & 239] Melaleuca leucadendron (L.) L. and [241] Melaleuca stypheloides Sm (all belong to Myrtaceae), and [302] Fagonia mollis Delile (Zygophyllacae).
Table 2. LC50 and LC90 of the bioactive extracts.
These most effective plant species deserve further investigation, with the aim to isolate and characterize their active constituents, and their antischistosomal effect should be confirmed by in an infected animal model to determine their therapeutic value and toxicity. Studies are currently ongoing and will be reported in a future publication.
While Pimenta racemosa (Mill) J.W. Moore (Myrtaceae) was found active in our first report (CitationYousif et al., 2007), several Myrtaceae species were further detected as active in the present work (), viz., C. viminalis (1 & 2), C. rigidus (3), C. specious (4) C. citrinus (5 & 6), E. citriodora (7 & 8), E. rostrata (9), Euginia edulis (10), E. javanica (11), M. leucadendron (12 & 13) and M. stypheloides (14). Also, while three active species of family Asclepiadaeae were determined in the first report, another active plant was detected here, viz., C. grandiflora (15). It seems that plants of these two families are rich in natural actives which deserve a thorough investigation.
A review of literature finds that several biological activities have been reported by some of the above species. Extracts of C. grandiflora produced significant antibacterial activity against different species of Pseudomonus, Bacillus, Staphylococcus, and Escherichia (CitationMukherjee et al., 1999). CitationSaxena and Gomber (2006) reported that a crude methanol extract of leaves of C. rigidus. R.Br. (Myrtaceae) revealed a potential antibacterial activity against a broad spectrum of multidrug-resistant human pathogens. CitationGuette-Fernandez et al. (2008) reported that the essential oil from the fresh leaves of C. speciosus (Sims) DC. is lethal to Artemia franciscana. E. citriodora Hook extract had shown significant antidiabetic effect in a dose dependent manner (CitationPatra et al., 2009) and their volatile oils used as herbicidal agent specially against the noxious weed Parthenium hysterophorus (CitationSingh et al., 2005). Eucalyptus oil has been traditionally used in ayurveda as an antiseptic and for respiratory tract infections. Moreover, its antibacterial action was reported in CitationTrivedi and Hotchandani (2004). The essential oils of the leaves of Eugenia species showed antibacterial activity, ranging from moderate to strong (CitationMagina et al., 2009). Valdes et al. (2008) found that M. leucadendron extract has selective activity against the parasites Trypanosoma brucei, Trypanosoma cruzi, Leishmania infantum and Plasmodium falciparum. Also, the traditional uses for Melaleuca species in several countries include applications as antiseptic, antihelminthic and skin parasiticide (CitationRoig, 1974; CitationBudhiraja et al., 1999).
The importance of plants to medicine as sources of natural product bioactive molecules lies not only on their pharmacological or chemotherapeutic effects, but also on their role as template molecules for the production of new drug substances (CitationPhillipson, 1994). The present study is a step towards that direction and introduces some new antischistosomal plant sources.
This article is an addition to the systematic work to report on the screening of schistosomicidal activity utilizing 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.
Declaration of interest
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”, Program of the National Strategy for Biotechnology, contract agreement #10(1999–2009), Principal Investigator: B. El-Menshawi.
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