Parasitol Res (2005) 97: 417–419 DOI 10.1007/s00436-005-1470-z
SH O RT CO MM U N IC A T IO N
T. Boonmars Æ W. Khunkitti Æ P. Sithithaworn Y. Fujimaki
In vitro antiparasitic activity of extracts of Cardiospermum halicacabum against third-stage larvae of Strongyloides stercoralis
Received: 30 May 2005 / Accepted: 5 July 2005 / Published online: 7 September 2005 Ó Springer-Verlag 2005
Abstract Extracts of Cardiospermum halicacabum, medicinal plant, were tested in vitro for their effectiveness against third-stage larvae of Strongyloidesstercoralis. Third-stage larvae of S. stercoralis were isolated from cultures of dog’s feces using agar plate culture method. The larvae (1,000 larvae/ml), suspended in phosphate buffer saline solution, pH 7.4, were exposed to aqueous and alcohol extracts (2,000 lg/ml) of C. halicacabum at 37°C with 5% CO2. Ivermectin (250 lg/ ml) and piperazine (2,000 lg/ml) were also used as the reference drugs. The survival of Strongyloides larvae based on its motility was determined daily for 7 days. Strongyloides larvae were viable after contact with ivermectin, piperazine and C. halicacabum (aqueous and alcohol) solutions, but most of them were immobilized, after exposure to aqueous and alcohol extracts of C. halicacabum within 72 and 48 h, respectively, while ivermectin took from 72 to144 h, and piperazine more than 7 days, to achieve the same rate of nonmotility. Clearly, the viability of S. stercoralis larvae was significantly reduced when exposed to extracts of C. halicacabum. Further study is needed on the antiparasitic
T. Boonmars (&) Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand E-mail:
[email protected] Tel.: +66-43-348387 Fax: +66-43-202475 W. Khunkitti Department of Industrial Pharmacy, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand P. Sithithaworn Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand Y. Fujimaki Department of Parasitology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
activity of aqueous and alcohol extracts of C. halicacabum against S. stercoralis.
Strongyloides stercoralis is an important parasitic nematode with worldwide distribution. This parasite may persist for years in a human host and then may disseminate and cause a fatal infection, particularly in immunosuppressed patients who have allergic diseases or who require steroid treatment. Therefore, effective and timely use of anthelmintics is essential. Currently, ivermectin is recommended for treatment of strongyloidiasis, but it is relatively expensive, and so its use is limited. Search for a plant extract from a currently used Thai herbal remedy may provide another effective but less expensive treatment. In this regard, several medicinal plant extracts have been investigated for anthelmintic activity such as an ethanol extract of the white and red barks of Pavetta owareensis, which has a demonstrated curative effect on mice infected with Schistosoma mansoni (Balde et al. 1989). The extract of the ivy, Hedera helex, called hedering, is effective against the trematodes Fasciola hepatica and Dicrococlum spp. (Julien et al. 1995). A chloroform extract of the stem bark of Zanthoxy liebmannianum of Rutaceae, a Mexican folk medicine, has anthelmintic activity but is potentially toxic (Navarrete and Hong 1996). Cardiospermum halicacabum (Family spindaceae), known in Thailand as ‘‘Kokkraorm,’’ is an annual herbaceous creeper 2–3 m high. It is widely distributed in Thailand as well as the Northeast region. The whole plant has been used in traditional medicine for treatment of several diseases; namely, (1) as antiinflammatory (Dhar et al. 1968; Sadigue et al. 1987), (2) an antibiotic against bacteria such as Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Shigella boydii, Xanthomonas oryzae pv. oryzae, Xanthomonas campentris pv. Sesami, Xanthomonas campestris pv. Malvacearum and Xanthomonas campestris pv. (Dhar et al. 1968; Raman
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et al. 1998), (3) an antipyretic (Dhar et al. 1968; GuribFakim and Sewaj 1992) and (4) an analgesic (Dhar et al. 1968). In Ceylon, the aqueous extract of this plant was used as an anthelmintic folk medicine (Gopalakrishnan et al. 1976). In our study, the activity of the extract of C. halicacabum against S. stercoralis thirdstage larvae was assessed in comparison to ivermectin and piperazine. Ivermectin was produced by Shionogi Pharmaceutical Co. Ltd., Osaka, Japan. Sodium chloride, potassium chloride, monosodium hydrogen phosphate and disodium hydrogen phosphate were purchased from CarLo Erba reagenti s.r.l. MonotEdison Group. Streptomycin and penicillin were purchased from M&H Manufacturing Co. Ltd., Thailand. Piperazine came from Jew Brader Co. Ltd., Thailand. C. halicacabum aqueous extract was prepared by boiling 1 kg of dry C. halicacabum in 5,000 ml of water until the volume reduced; it was then lyophilized. C. halicacabum alcohol extract was prepared by maceration of 225 g of the grounded plant in alcohol for 7 days, before being lyophilized. Thirdstage larvae of S. stercoralis were isolated from the fecal culture of laboratory-infected dog’s feces. The feces were placed on agar plates and left at room temperature for 3–5 days (Koga et al. 1991). The third-stage larvae that had migrated into the lid of petri dish were harvested, washed in 0.85% normal saline solution pH 7.4 and centrifuged at 2,000 rpm for 5 min. The pellets were washed three times with 0.1 M phosphate buffered saline solution (PBS; pH 7.4) containing 1,000 i.u. penicillin G sodium/ml and 0.1 mg/ml streptomycin (Sithithaworn et al. 1998). One milliliter of the desired concentration of larval suspension was added to 1 ml of test compound solution to give the required final larval density of 1,000 larvae/ ml in Nunclon-well plates. The PBS with and without 0.05% Tween80 were used as the drug-free media controls. For reference drugs, ivermectin was dissolved in 0.05%w/v Tween80 and piperazine was dissolved with PBS for the final concentration of 250 and 2,000 lg/ml, respectively. The lyophilized aqueous and alcohol extracts of C. halicacabum were similarly adjusted to 2,000 lg/ml. The plates were incubated at 37°C in a humidified incubator with 5% CO2. Viability was discerned by the pH of all the tested compounds and was adjusted to 7.4 prior to addition to the larval suspension. Four replicate wells were allocated for each tested compounds and the survival rates were calculated from the proportions of observed motile worms. Third-stage larvae of S. stercoralis survived in PBS at least 7 days and there was no significant difference in the number of motile larvae cultures in PBS and PBS with Tween80 the vehicle for ivermectin. Forty-one percent and 56% of larvae were active at day 7 of culture. All the larvae were completely nonmotile by day 21 (data not shown). The larvae were actively motile immediately after exposure to ivermectin, piperazine and C. halicacabum extracts, but after which, most of the larvae were rapidly
inactivated as day 1 only 3.08–11.82% in aqueous and alcohol extracts of C. halicacabum and 31.81% of those in ivermectin were motile. No motile larva was observed at days 2 and 3 of culture in alcohol and aqueous extracts of C. halicacabum while for ivermectin, no survival was seen after day 6. Piperazine had little effect on larval motility as it yielded similar profile to those of PBS controls. To examine the temporal effects of C. halicacabum extracts on the larval motility, employing a shorter sampling interval of 3 h performed another set of experiments. The survivals in PBS and piperazine are consistent with the previous experiments. Aqueous extract of C. halicacabum exerted more rapid effect on larval motility than that of the alcohol extract. In order to attain 50% nonmotility or dead it took less than 24 h and more than 36 h for aqueous and alcohol extract of C. halicacabum, respectively. For ivermectin, with reference to PBS plus Tween80 control, no significant effect on larval survival was evident until 72 h of culture. During the first 12 h, the larvae were temporarily inactive before resuming motility again 6 h later. Although the explanation of this observation related to action of ivermectin is unclear, preliminary results of the ongoing studies confirmed the behavior of S. stercoralis L3 in the presence of ivermectin (unpublished observation). The in vitro effect of C. halicacabum on S. stercoralis larvae demonstrated in this study together with its activity on filarial worms (Khunkitti et al. 2000) suggest that this medicinal plant extract deserves further studies on its anthelmintic property. Acknowledgments The study was supported in part by a grant from the Research Foundation of the Khon Kaen University. We gratefully acknowledge the Departments of Parasitology and Animals unit, Faculty of Medicine, Khon Kaen University.
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