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EurAsian Journal of BioSciences EurAsia J BioSci 3, 122-129 (2009) Effect of leaf extracts of Dendrosicyos socotrana
and Jatropha unicostata on the viability of
Echinococcus granulosus protoscoleces
Abdul Kadir Raoof Barzinji1, Ramzi Ahmed Mothana2, Abdul Karim Nasher1*
1Department of Biology, Faculty of Science, Sana'a University, Sana'a, Yemen2Department of Pharmacognosy, Faculty of Pharmacy, Sana'a University, Sana'a, The present work evaluates the effect of aqueous and methanolic extracts of Dendrosicyos socotrana and Jatropha unicostata on the viability of Echinococcus granulosus protoscoleces in vitro, and on the development of secondary hydatid cysts, in vivo. Three different concentrations of each leaf extract were used. Concentrations of 5000 and 1000 μg/mL, for D. socotrana and J. unicostata, respectively, exhibited the highest protoscolicidal activity, significantly reducing and/or stopping protoscolex viability. Oral and intraperitoneal administration of the extracts in white mice invoked noticeable inhibitory effects on the in vivo development of secondary hydatid cysts. These effects were compared with those of albendazole sulfoxide, a commonly used treatment for hydatidosis.
Keywords: Dendrosicyos socotrana, Echinococcus, hydatidosis, Jatropha unicostata, Socotra
Barzinji AKR, Mothana RA, Nasher AK (2009) Effect of leaf extracts of Dendrosicyos socotrana and Jatropha unicostata on the viability of Echinococcus granulosus protoscoleces. EurAsia J BioSci 3, 16, 122-129.
cyst(s) (Shambesh 1997). As with other parasitic diseases, Hydatidosis, or echinococcosis is a disease hydatidosis must be treated. Chemotherapy, caused by the metacestodes of different using benzimidazole carbamate derivatives is species of Echinococcus Rudolphi, 1801. the standard protocol (Liu and Weller 1996).
Tapeworm eggs are passed with the feces of However, chemotherapy is not always infected carnivores and may subsequently effective and may cause serious side effects infect humans who inadvertently ingest them in some patients; therefore, in certain cases (Andersen 1997). Clinical manifestation of surgical intervention becomes mandatory hydatidosis is characterized by tumor-like (Zhou et al. 1988).
growths that occur mostly in the liver and Treatment of a variety of diseases with lungs, with varying degrees of infestation of plant extracts has been practiced for many other organs (Abdel-Hafez and Al-Yaman centuries, with numerous plant species having 1989). These growths are usually filled with a been used (Ye et al. 1990, Al-Saimary and watery fluid known as 'hydatid cyst fluid.' Zeki 1999, Aghwan 1999, Al-Aboody 2001, Hydatidosis is a major world zoonosis Al-Eryani 2002). Extracts of Dendrosicyos affecting humans as well as domestic animals socotrana Balf f. and Jatropha unicostata Balf (Thompson 1995).
f., both of which are endemic to Socotra Because of the slow progression of the Island (Yemen), are used locally for a variety disease, it may initially be asymptomatic or of medical treatments. Extracts of the former show very slight manifestations (Lyagoubi et al. 1997). However, serious clinical symptoms may eventually develop, which Received: June 2009 vary, depending on the extent of infestation, Accepted: November 2009 the site of infection, and the size of the Printed: November 2009 EurAsian Journal of BioSciences, 2009 EurAsian Journal of BioSciences Barzinji et al.
are used by the islanders for the treatment of urinary retention, cystitis symptoms of Sixty five, 6-8 week old white mice (20 diabetes, liver problems, and burns (Miller and males and 45 females), were used in this Morris 2004). Ali et al. (2003) failed to show study. These were descended from eight pairs any antimicrobial effect of D. socotrana and that were originally obtained from Al Azhar only a weak effect for J. unicostata. University Animal House, Cairo, Egypt. However, a methanolic extract of J. Plant collection and extraction
unicostata demonstrated a marked antiviral Fresh leaves of Dendrosicyos socotrana effect against Influenza virus type A and (Cucurbitaceae) and Jatropha unicostata Herpes simplex type 1 (Mothana et al. 2006). (Euphorbiaceae) were collected from Socotra Remarkably, the methanolic extract of D. Island at the end of April 2004. Voucher socotrana exhibited noteworthy cytotoxic specimens of both plants were deposited in activity against human cancer cell lines the herbarium of the Faculty of Science, (Mothana et al. 2007). Sana'a University. The leaves were cleaned According to Miller and Morris (2004), J. and air-dried at room temperature. They were unicostata has various uses. The red sap is then pulverized in a grinder to obtain a fine used as a haemostatic in humans and powder. Seventy grams of powdered leaves livestock, while the colorless sap is used to of each of the two plant species were treat livestock infections. The latter is also extracted with methanol by using a soxhlet used to treat the skin, 'tender' glands, eye extraction apparatus. The residue was dried infections, chest pain, stomach pain, retching, overnight and extracted with water at 60°C.
vomiting, and is used as a laxative and The methanol extracts were evaporated using vermifuge. The present work aimed therefore, a rotary evaporator and the water extracted to evaluate the effect of both plants on the with a freeze-dryer. The dried crude extracts viability of Echinococcus granulosus (Batsch, were kept in dark plastic bags, sealed under 1786) protoscoleces, and on the development vacuum, and kept in a freezer at -20°C until of secondary hydatid cysts.
use.Determination of in vitro and in vivo effect
Stock solutions of methanolic and aqueous MATERIAL AND METHODS
crude extracts of D. socotrana were prepared The parasite
by dissolving 0.36 g of the powdered extract Fresh hydatid cysts were obtained from in 10 mL of 50% dimethyl sulfoxide (DMSO).
livers of naturally-infected sheep, which had Concentrations of 1000, 2000, and 5000 been slaughtered at local abattoirs in Sana'a. μg/mL of the stock solutions were used to They were wrapped carefully in clean plastic treat the protoscoleces. The stock solutions bags, placed in an ice box, and transported to of J. unicostata were prepared by dissolving the Department of Biology, Faculty of 0.08 g of the methanolic and aqueous crude Science, Sana'a University, where extracts in 10 mL of 60% DMSO.
protoscoleces were extracted. The outer Concentrations of 500, 750, and 1000 μg/mL surfaces of the cysts were sterilized with of the stock solutions were used for in vitro 70% ethanol before being dissected. treatment of protoscoleces. A mixture of 50% Protoscoleces were extracted according to DMSO and the maintenance medium (1:1000) Smyth (1967). Extracted protoscoleces were was used for D. socotrana and 60% DMSO maintained in a sterile medium made of a and the maintenance medium (1:1000) for J. mixture of Kreb's Ringer Solution (KRS) and unicostata were used as positive controls.
hydatid cyst fluid (4:1), which was prepared Kreb's Ringer solution was used as a negative according to Rotunno et al. (1974) and Al- control.
Eryani (2002). Crystalline penicillin 'G' and The efficacy of methanolic and aqueous streptomycin sulphate were added to the extracts of both plants on the viability of mixture to keep it free from contamination protoscoleces was compared with different (Schwabe et al. 1963, Lorenzini and Ruggieri doses of albendazole sulfoxide (ABZSO), the 1990, Casado et al. 1992, 1996, Hemphill standard medicine used in treating hydatidosis and Gottstein 1995, Urrea-Paris et al. 2000). (Ingold et al. 1999, Urrea-Paris et al. 2000, Stettler et al. 2001).
EurAsian Journal of BioSciences, 2009 EurAsian Journal of BioSciences Barzinji et al.
Both in vitro and in vivo experiments on E. Protoscoleces treated with concentrations of granulosus protoscoleces were performed 1000, 2000, and 5000 μg/mL started during this study. The former were carried out showing decrease in viability at 96, 12, and 3 in sets of 21 Eppendorf tubes at each of the h post treatment, respectively. All treated three concentrations for each of the two plant protoscoleces perished at h 480, 408, and species. Three positive control and three 360 post treatment, respectively (Fig. 1). This negative control tubes were used for each concentration set. 1.0 mL of the maintenance result was highly significant (P<0.05). The medium, supplemented with penicillin and inhibitory effect of an aqueous extract of the streptomycin was added to each tube. same plant was also significant (P<0.05). All Approximately 3000 viable protoscoleces protoscoleces died at h 504 and 408 when were placed in each tube, and incubated at treated with 2000 and 5000 μg/mL, 28°C. The effect of ABZSO was tested in respectively, while 85.2% and 75.0% of the vitro using three tubes for the drug, together untreated controls remained viable until the with three tubes for each of the positive and end of the experiment at h 504 (Fig. 2).
negative controls.
Both methanolic and aqueous extracts of The effects of plant extracts on the in vivo J. unicostata inhibited the viability of development of protoscoleces and secondary protoscoleces. All protoscoleces treated with hydatid cysts was studied in the sixty five 6- the methanolic extract at concentrations of 8 week old experimentally infected mice. The animals were divided into 13 groups with five 500, 750, and 1000 μg/mL, died at hours mice in each group. Mice in each of four 408, 336 and 288, respectively. The majority groups received methanolic and aqueous of protoscoleces in the negative control and extracts of each plant, and one group received positive control groups remained viable until ABZSO by oral administration. Another batch the end of the experiment (Fig. 3). The same of five groups received the same treatment trend was observed in protoscoleces treated but intraperitoneally (IP). Mice in two groups with similar concentrations of the aqueous received oral and IP doses of the positive extracts (Fig. 4).
control. The last group of mice received the ABZSO exhibited a significant effect on the negative control. All animals used in the viability of protoscoleces (P<0.05). 96 h from experiment were sacrificed 60 days post- the onset of treatment, 90.5% of the 3000 treatment and examined for the presence of protoscoleces were dead. All of the remaining secondary hydatid cysts. protoscoleces had died by h 360. Negative The data were expressed as mean values and positive control groups showed a slight ±S.E.M. and tested with one-way ANOVA decline in viability whereas 91.2% and 90.2% followed by the Tukey's test for multiple of the protoscoleces were still viable by h 360 comparisons using Statistical Package for post-treatment. A comparison between the Social Sciences (SPSS) computer package. effects of high concentrations of both D. Results with P<0.05 were considered socotrana and J. unicostata and that of ABZSO is summarized in Fig. 5.
Oral treatment of aqueous and methanolic extracts of the two plants showed varying effects on the growth of secondary hydatid Viability of protoscoleces
cysts, growing on different organs of the Untreated protoscoleces remained viable mice. Generally, the methanolic extract of J. for 24 h prior to treatment, with no significant unicostata exhibited a high noticeable effectdifferences in viability between 0 and 24 h similar to that of ABZSO. However, effects demonstrated by the other extracts were (t= 0.69, P<0.005).
generally weak or not noticeable (Table 1).
In vitro treatment of protoscoleces
Moreover, there was little discernable The viability of protoscoleces was difference between the effects of extracts significantly affected when treated with the administered intraperitoneally and orally methanolic extract of D. socotrana leaves. (Table 2).
EurAsian Journal of BioSciences, 2009 EurAsian Journal of BioSciences Barzinji et al.
Fig. 1. In vitro treated protoscoleces with different
Fig. 4. In vitro treated protoscoleces with different
concentrations of the methanolic extract of concentrations of aqueous extract of J. D. socotrana leaves.
Fig. 2. In vitro treated protoscoleces with different
Fig. 5. Comparison between the highest
concentrations of the aqueous extract of D. concentrations of various in vitro treatments. ABZSO: albendazole sulfoxide (10 μg/mL); Meth. D. socotrana: methanolic extract of D. socotrana (5000 μg/mL); Aq. D. socotrana: aqueous extract of D. socotrana (5000 μg/mL); Meth. J. unicostata: methanolic extract of J. unicostata (1000 μg/mL); Aq. J. unicostata: aqueous extract of J. unicostata (1000 μg/mL).
been claimed. However, the metabolites ofcertain drugs including benzimidazole,mebendazole, albendazole, and albendazolesulfoxide are potentially toxic in some Fig. 3. In vitro treated protoscoleces with different
concentrations of methanolic extract of J. subjects (Davis et al. 1989, Whittaker and Faustman 1991, Pawlowski 1997).
The use of plant extracts against CE has received critical attention in recent years.
Cystic echinococcosis (CE) is a subject of Some studies have shown that extracts of major health concern in many countries. Each certain plant species, belonging to differentconfirmed case could potentially cost the families, may affect the viability ofhealth authorities thousands of dollars protoscoleces and/or the survival of(Nasrieh et al. 2003, Torgerson 2003). secondary hydatid cysts. For instance, seedsAlthough surgery is the most effective choice of Peganum harmala L. (Zygophyllaceae)of treatment, a number of drugs are being affected E. granulosus in mice )Al-Eryaniused and various degrees of success have 2002). Al-Saimary and Zeki (1999) EurAsian Journal of BioSciences, 2009 EurAsian Journal of BioSciences Barzinji et al.
Table 1. Effect of various treatments on the development of secondary hydatid cysts in the different albino
mice organs (oral administration).
Table 2. Effect of various treatments on the development of secondary hydatid cysts in the different albino
mice organs (I.P. administration). demonstrated that extracts of Eucalyptus and organisms (Smyth et. al. 2002). J. unicostataMyrtus communis L., (Myrtaceae), Apium on the other hand was shown to contain agraveolens L. (Umbelliferae) and Trigonella mixture of sterols and ketosteroids and foenum-graecum L. (Leguminosae) can kill the terpenoids (Franke et al. 2004), which might larval stages of Echinococcus sp. Al-Rubaei have an effect on the viability of certain (1999) reported that water extracts of garlic organisms.
Allium sativum L. (Liliaceae) and Artemisia In conclusion, our data suggest that herba-alba Asso (Compositae) affected medicinal plants can be a promising source of viability and killed protoscoleces. Also Al-Aboody (2001) showed that Ruta chalepensis potential anti-protoscoleces. The data suggestL. (Rutaceae) and Plantago lanceolata L. that J. unicostata should be selected for(Plantaginaceae) stopped the viability of further investigation into the potentialprotoscoleces.
discovery of new natural bioactive D. socotrana and J. unicostata were compounds. Studies aimed at the isolation of selected for this study because they are two active anti-protoscoleces constituents are inof the endemic plants on Socotra island that progress and may lead to the discovery ofare used widely for medicinal purposes (Miller compounds with improved therapeutic value. and Morris 2004). The former plant contains agroup of compounds known as curcurbitacins, a special group of highly oxygenated The authors are grateful to Dr. Malcolm tetracyclic triterpenes, with a wide range of Potts for the valuable comments and bioactivity (Dinan et al. 1997, Ito et al. 2002, corrections to the manuscript.
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Dendrosicyos socotrana ve Jatropha unicostata Yaprak Özütlerinin Echinococcus
granulosus Protoskolekslerinin Canliligina Etkisi
Özet
Bu çalisma; Dendrosicyos socotrana ve Jatropha unicostata'nin su ve metanolde elde edilen özütlerinin,
Echinococcus granulosus protoskoleksleri üzerine in vitro ve sekonder hidatit kistlerin gelisimi üzerine in vivo
etkisini incelemektedir. Her yaprak özütünün üç farkli konsantrasyonu kullanildi. Sirasiyla D. socotrana ve J.
unicostata'nin 5000 and 1000 μg/mL yogunluklari en yüksek protoskolisidal aktivite sergiledi ve protoskoleks
canliligini önemli ölçüde azaltti ve/veya durdurdu. Beyaz farelerde, yaprak özütlerinin oral veya intraperitonal
uygulanmasi, sekonder hidatit kistlerinin in vivo gelisimi üzerinde farkedilir oranda inhibitör etki olusturdu. Bu
etkiler, hidatidosis tedavisinde yaygin olarak kullanilan albendazol sülfoksid'inkilerle kiyaslandi.
Keywords: Dendrosicyos socotrana, Echinococcus, hidatidosiz, Jatropha unicostata, Socotra Adasi.
EurAsian Journal of BioSciences, 2009

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