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Effect of Lysimachia ramosa (Primulaceae) on helminth parasites: Motility, mortality and scanning electron microscopic observations on surface topography
Veterinary Parasitology 169 (2010) 214–218
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Effect of Lysimachia ramosa (Primulaceae) on helminth parasites: Motility, mortality and scanning electron microscopic observations on surface topography M. Challam, B. Roy *, V. Tandon Department of Zoology, North-Eastern Hill University, Shillong 793 022, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 July 2009 Received in revised form 7 December 2009 Accepted 16 December 2009
The alcoholic extract of Lysimachia ramosa Wall (Primulaceae) was tested in vitro against helminth parasites, Fasciolopsis buski and Ascaris suum, from porcine hosts and Raillietina echinobothrida from domestic fowl. The live adult parasites, collected from a freshly autopsied host, were exposed to different concentrations (5–50 mg) of the test plant extract in physiological phosphate-buffered saline (PBS) having 0.1% dimethyl sulphoxide (DMSO) at 37 1 8C. The treated parasites revealed complete inactivation and flaccid paralysis that was followed by death at varying periods of time. A dose-dependent loss of motility and mortality was observed in all the treated parasites. Scanning electron microscopic observations revealed conspicuous deformity of the surface architecture in all the parasites exposed to the test plant extract. The general tegument in F. buski showed shrinkage and loss of scale-like spines; proglottides all along the strobilar length in R. echinobothrida appeared shrunken and deformed and the cuticular surface of A. suum appeared disorganised, having lost transverse striations. The botanicals of the test plant seem to be effective against all the three types of helminth parasites. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Anthelmintic Lysimachia ramosa Scanning electron microscopy
Helminthic infections are amongst the most common parasitic infections of animals and humans worldwide and are now well recognised as an important veterinary and public health problem, both in developing and in developed countries (Perry and Randolph, 1999; Hafeez, 2003). Survey of the literature revealed that infections of parasites implicating as many as 57 different species of nematode, trematode and cestode worms are commonly prevalent among large livestock animals in northeast India, of which several species are important potential zoonoses as cases of human infections have also been reported (Tandon and Roy, 2005). Although several effective synthetic drugs are available in the market, they are not accessible to the farmers, particularly natives who face problem of affordability and of regular supply (McKeller and Jackson, 2004)
* Corresponding author. Tel.: +91 3642722331; fax: +91 3642550300. E-mail address: [email protected] (B. Roy). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.12.024
and, therefore, rely heavily on ethno-medicines for controlling helminthiasis for their livestock. Meghalaya, one of the hot spots of northeast India, has a wide variety of plants used by natives as curative against worm infections (Rao, 1981). Lysimachia ramosa Wall (Family: Primulaceae) is one such plant, the alcoholic extract of its leaves being widely used by Jaintia and Khasi tribes in the region against intestinal helminth infections. The present investigation, therefore, was taken up to explore and authenticate the possible anthelmintic potential of the plant, if any, on helminth parasites – trematode, cestode and nematode. 1. Materials and methods 1.1. Preparation of ethanol extract Lysimachia ramosa plants were procured from the markets in and around Shillong (Meghalaya) during April–July 2008. The traditionally usable plant parts, that
M. Challam et al. / Veterinary Parasitology 169 (2010) 214–218
215
Table 1 Effect of different concentration of alcoholic extract of Lysimachia ramosa (leaves) on the survival (in h) of nematode, trematode and cestode parasites. Concentration of crude extract/drug (mg ml 1)
Ascaris suum
Fasciolopsis buski
Raillietina echinobothrida
Paralysis
Death
Paralysis
Death
Paralysis
Death
Lysimachia ramosa 5 10 20 30 50
74.12 0.12 69.20 0.18 32.31 0.14 26.02 0.18 22.82 0.23
76.25 0.12 73.42 0.20 37.83 0.17 27.30 0.14 26.45 0.14
3.64 0.14 2.61 0.15 1.55 0.14 1.45 0.14 1.20 0.12
4.21 0.12 3.41 0.15 2.62 0.14 2.32 0.14 2.22 0.12
6.75 0.12 5.80 0.12 4.92 0.07 4.10 0.10 3.40 0.15
7.25 0.12 6.81 0.12 5.24 0.12 4.44 0.15 4.51 0.15
Praziquantel 1 5 10
– – –
– – –
5.12 0.94 4.44 0.14 2.24 0.12
6.23 0.12 5.85 0.10 4.41 0.15
3.12 0.56 2.62 0.39 1.80 0.46
5.05 0.49 3.54 0.56 3.21 0.45
Albendazole 1 5 10
30.21 0.18 26.34 0.14 22.22 0.12
31.21 0.12 28.31 0.14 24.24 0.12
– – –
– – –
– – –
– – –
Control of A. suum, F. buski and R. echinobothrida survived for 102.35 0.22, 21.05 0.22, and 29.50 0.15 h, respectively. Values are expressed as mean SEM (n = 5); P < 0.05; control vs treated.
is, the leaves were washed thoroughly in deionised water and air dried. About 200 g of dried leaves were ground in an electric grinder and then refluxed with ethanol (100 g l 1) for 8 h at 60 8C. The solution obtained was filtered through Whatman filter paper (No. 1) and the volume was reduced through distillation, followed by complete dryness at 50 8C in an oven. The crude extract obtained as a powder material was then refrigerated at 4 8C until further use. One hour prior to experimental assay, varying concentrations of the extract, namely 5, 10, 20, 30 and 50 mg ml 1, were prepared by dissolving in 0.9% phosphate-buffered saline
(PBS, pH 7–7.3) supplemented with 0.1% dimethyl sulphoxide (DMSO). 1.2. Recovery and in vitro treatment of parasites Test parasites, the trematode (Fasciolopsis buski) and the nematode (Ascaris suum) were collected from freshly sacrificed porcine host (Sus scrofa domestica) and the tapeworm (Raillietina echinobothrida) from the intestine of the domestic fowl (Gallus domesticus). The live worms were directly treated with different concentrations of the plant
Fig. 1. Ascaris suum—scanning electron micrographs. (a, b) Untreated control. (a) Anterior end of the worm. (b) Cuticle showing annulations. (c, d) Treated parasites. (c) Body surface showing wrinkles (ethanolic extract of L. ramosa) (scale bar = 50 mm). (d) Anterior end with severe deformity at the lip surface (albendazole) (scale bar = 100 mm).
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extract in PBS having 0.1% DMSO in separate petri dishes. Similar treatment was performed for varying doses of praziquantel and albendazole as broad-spectrum reference drugs; the controls were maintained in a medium containing only PBS with 0.1% DMSO. For each set of treatment, five replicates were used. 1.3. Motility, mortality and scanning electron microscopic analyses Motility of the worms was observed and the time taken for paralysis and death recorded as described earlier (Roy et al., 2008a). The parasites incubated with 50 mg ml 1 extract of PBS were selected for scanning electron microscopy (SEM) observation because of the relatively early paralytic lethal effect of that dose as compared with lower doses. From each medium, individual worms were washed thoroughly in buffer and fixed soon after the onset of paralysis in 4% gluteraldehyde buffered with 0.1 M sodium cacodylate at 4 8C for 4 h, washed in buffer, and followed by secondary fixation in 1% OsO4. The samples were then dehydrated through increasing grades of acetone followed by air drying after treatment with tetramethyl silane as detailed by Roy and Tandon (1991), gold coated using JFC 1100 (Jeol) ion sputtering chamber and viewed in JSM-6360 scanning electron microscope at an electron accelerating voltage of 10–15 kV. 2. Results and discussion Observations on the efficacy of the plant extract and the reference drugs in terms of motility and survivability of the parasites are presented in Table 1. The results indicate that both the plant extract and the two reference drugs showed dose-dependent lethal anthelmintic efficacy. However, at lower concentration, that is, 5 mg ml 1 of plant extract, the nematode did not show any significant mortality with respect to control. The controls A. suum, F. buski and R. echinobothrida maintained in PBS with 0.1% DMSO showed physical activity till the period of 102.35 0.22 h, 21.05 0.22 h, and 29.50 0.15 h, respectively. Stereoscan observations on control A. suum revealed fine surface topography, with three prominent denticulate lips having smooth surface and one labial papilla in the form of a prominent protuberance in each lip (Fig. 1(a)); the cuticle, from the cephalic to the posterior end of the body revealed quite distinct and neatly arranged regular transverse striations in the form of parallel concentric rings running completely round the cylindrical body; clusters of striations are separated by deep groove-like annulations that occur at regular intervals giving the body a segmented appearance (Fig. 1(b)). Worms incubated with 50 mg ml 1 of the plant extract showed prominent wrinkles and disorganisation of the cuticle throughout the body (Fig. 1(c)), including the surface of the lips. Similar deformity of the lip surface and general body cuticle was also observed in the worms treated with the reference drug albendazole (Fig. 1(d)). The untreated control F. buski revealed a normal body contour with scale-like spines on the entire ventral surface and the oral and ventral suckers having radial corrugations
Fig. 2. Fasciolopsis buski—scanning electron micrographs. (a) Untreated control fluke, showing normal surface architecture (scale bar = 10 mm). (b, c) L. ramosa and praziquantel treated parasite respectively, showing pronounced distortion of the tegumental surface (scale bar = 20 mm).
(Fig. 2(a)). By contrast, the flukes treated with 50 mg ml 1 doses of the plant extract and praziquantel as well manifest a pronounced deformity of the body surface, particularly sloughing off of the scales (Fig. 2(b), (c)). The control R. echinobothrida showed the typical cestode fine surface topography, with densely packed microtriches throughout the surface giving it a velvety appearance (Fig. 3(a), (b)). By contrast, the worms treated with the plant extract showed shrunken scolex with severely distorted and folded tegument around the suckers (Fig. 3(c)); the microtriches were eroded and the proglottides appeared deformed with wrinkled surface. The worms exposed to the reference cestocidal drug also revealed similar results (Fig. 3(d)).
M. Challam et al. / Veterinary Parasitology 169 (2010) 214–218
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Fig. 3. Raillietina echinobothrida—scanning electron micrographs. (a, b) Untreated control. (a) A portion of strobila, showing normal surface of proglottides (scale bar = 50 mm). (b) A closer view of the proglottid tegument, revealing a dense cover of microtriches (scale bar = 5 mm). (c, d) The scolex end and strobilar surface, showing distorted and wrinkled tegument in the plant extract treated parasite (scale bar = 50 mm).
The result of the present investigation revealed that the crude extract of L. ramosa caused destructive alterations and deformity in the cuticle of A. suum and also in the tegumental architecture of F. buski and R. echinobothrida as early as 26.45 0.14 h, 2.22 0.12 h and 4.51 0.15 h, respectively, after treatment at a concentration of 50 mg ml 1 of extract of PBS. Pharmacologically active components recorded from different species of the genus Lysimachia are triterpenoid saponins, organic acids and flavones (Liang et al., 2006). Triterpene saponins are known to have cytotoxic activity against human skin fibroblasts and tumour cells (Galanty et al., 2008). Nematocidal and cestocidal activity of saponins have also been established (Ghosh et al., 1993, 1996). Anthelmintic properties of several ethnomedicinal plants such as Calliandra portoricensis, Zingiber officinalis, Zanthoxylum alatum, Flemingia vestita, Psidium guajava, Houttuynia cordata, Lasia spinosa, Alpinia nigra, Spilanthes oleraceae, Millettia pachycarpa and Accacia oxyphylla have been established through in vitro exposure of crude extract of these plant/plant parts against several helminth parasites (Adewuni and Akubue, 1981; Yadav et al., 1992; Roy and Tandon, 1996; Roy, 2001; Temgenmongla and Yadav, 2005; Tangpu and Yadav, 2006; Lalchhandama et al., 2007; Roy et al., 2007, 2008a,b, 2009). The cuticle of nematodes is metabolically active and morphologically specialised for selective absorption of nutrients and osmoregulation. Thus, passive diffusion of anthelmintics through the cuticle (Alvarez et al., 2007) would probably be responsible for destructive changes and deformation of the nematode body surface (Tippawangkosol et al., 2004; Schmahl et al., 2007). In trematodes, the surface papillae act as sensory organs, whereas scales and
suckers are important organs of attachment (Tandon and Roy, 2002). In platyhelminth parasites, the general body surface acts as an absorptive surface; in cestodes, microtriches are known to increase the effective surface area of absorption many fold. In the present investigation, all these structures were observed to be affected and altered by the crude ethanolic extract of L. ramosa. Similar to the present observations, the surface tegument is found to be a principal target site for different synthetic drugs and natural anthelmintic products (Geary et al., 1992; Martin et al., 1997; Roy et al., 2009). Drugs such as albendazole and its related compound enter the parasite body through simple diffusion and cause disruption of the tegumental and muscle layers (Mottier et al., 2003; Markoski et al., 2006). Histomorphological and ultrastructural changes caused by anthelmintic agents with respect to tegumental deformity have been well documented in different helminths (McKinstry et al., 2003; Xiao et al., 2003; Meany et al., 2004). Crude extract of F. vestita and its active component genistein produced prominent structural alterations in R. echinobothrida extending from destruction of tegument, microtriches and suckers to vacuolisation and muscle distortion (Tandon et al., 1997; Pal and Tandon, 1998). Naguleswaran et al. (2006) observed that synthetic derivatives of genistein, Rm 6423 and Rm 6426 induced considerable damage in Echinococcus granulosus and E. multiloculoris, making them non-viable by inducing truncation of microtriches, nuclear pyknosis and vesiculations; in addition, Rm 6423 specifically induced dramatic breakdown of the structural integrity of the metacestode germinal layer. The crude alcoholic extract of L. ramosa thus seems to have anthelmintic effect. However, the precise mechanism by which the lethal action is exerted is not clear. Therefore,
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identification of the active principle of the plant-derived component is a prerequisite to ascertain the anthelmintic efficacy and actual mode of action of the botanicals on the parasite. Acknowledgements This study was supported by University Grants Commission (New Delhi) – supported project to BR. Infrastructural facilities provided by UGC-DSA programme to the Department of Zoology, North-Eastern Hill University, Shillong are also acknowledged. References Adewuni, C.O., Akubue, P.I., 1981. Preliminary studies on the anthelmintic properties of the aqueous extract of Calliandra portoricensis (Jacq.) Benth. Bull. Anim. Health Proc. Afri. 29, 171–175. Alvarez, L.L., Mottier, M.L., Lannsse, C.E., 2007. Drugs transfer into target helminth parasites. Trends Parasitol. 23, 97–104. Galanty, A., Michalik, M., Sedek, L., Podolak, I., 2008. The influence of LTS4, a saponoside from Lysimachia thyrsiflora L., on human skin fibroblasts and human melanoma cells. Cell. Mol. Biol. Lett. 13, 585–598. Geary, T.G., Klien, R.D., Vanover, L., Bowman, J.L., Thompson, D.P., 1992. The nervous system of helminths as target of drugs. J. Parasitol. 78, 215–230. Ghosh, M., Sinhababu, S.P., Sukul, N.C., Mahato, S.B., 1993. Antifilarial effect of two triterpenoid saponins isolated from Acacia auriculiformis. Indian J. Exp. Biol. 31, 604–606. Ghosh, M., Sinhababu, S.P., Sukul, N.C., Ito, A., 1996. Cestocidal activity of Acacia auriculiformis. J. Helminthol. 70, 171–172. Hafeez, M.D., 2003. Helminth parasites of public health importance – Trematode. J.P.D. 27, 69–75. Lalchhandama, K., Roy, B., Dutta, B.K., 2007. In vitro anthelmintic activity of Acacia oxyphylla: changes in the tegumental enzymes of the cestode, Raillietina echinobothrida. Pharmacologyonline 2, 307–317. Liang, B., Tiang, J., Xu, L., Yang, S., 2006. Triterpenoid saponins from Lysimachia davurica. Chem. Pharm. Bull. 54, 1380–1383. Martin, R.J., Robertson, A.P., Bjorn, H., 1997. Target sites of anthelmintics. Parasitology 114, 111–124. Markoski, M.M., Trinddade, E.S., Cabrera, G., 2006. Praziquantel and albendazole damaging action on in vitro developing Mesocestiodes corti (Platyhelminthes: cestoda). Parasitol. Int. 55, 51–61. Mottier, M.L., Alvarez, L.I., Pis, M.A., Lanusse, C.E., 2003. Transtegumental diffusion of benzimizole anthelmintics into Moniezia benedeni: correlation with their octanol–water partition coefficients. Exp. Parasitol. 103, 1–7. McKeller, Q.A., Jackson, F., 2004. Veterinary anthelmintics: old and new. Trends Parasitol. 20, 454–461. McKinstry, B., Fairweather, I., Brennan, G.P., Forbes, A.B., 2003. F. hepatica: tegumental surface alterations following treatment in vitro and in vivo with nitroxynil (Trodax). Parasitol. Res. 91, 251–263. Meany, M., Fairweather, I., Brennan, G.P., Forbes, A.B., 2004. Transmission electron microscope study of the ultrastructural changes induced in the tegument and gut of Fasciola hepatica following in vivo drug treatment with closulon. Parasitol. Res. 92, 232–241. Naguleswaran, A., Spicher, M., Vonlaufen, N., Ortega-Mora, L.M., Torgerson, P., Gottstein, B., Hemphill, A., 2006. In vitro metacestocidal activities of genistein and other isoflavones against Echinococcus
multilocularis and Echinococcus granulosus. Antimicrob. Agents Chemother. 50, 3770–3778. Pal, P., Tandon, V., 1998. Anthelmintic efficacy of Flemingia vestita (Fabaceae): genistein induced alteration in the ultrastructure of tegument in the cestode, Raillietina echinobothrida. J.P.D. 22, 104–109. Perry, B.D., Randolph, T.F., 1999. Improving the assessment of the economic impact of parasitic diseases and their control in production animals. Vet. Parasitol. 84, 145–168. Rao, R.R., 1981. Ethnobotany of Meghalaya: medicinal plants used by Khasi and Garo tribes. Ecol. Bot. 35, 4–9. Roy, B., 2001. Stereoscan observations on the surface alteration of Orthocoelium dinniki induced by extract of Spilenthes oleraceae L. Rev. Parasitol. 18, 9–14. Roy, B., Tandon, V., 1991. Usefulness of tetramethyl silane in the preparation of helminth parasites for scanning electron microscopy. Rev. Parasitol. 8, 405–413. Roy, B., Tandon, V., 1996. Effect of root tuber extract of Flemingia vestita, a leguminous plant, on Artyfechinostomum sufratyfex and Fasciolopsis buski: a scanning electron microscopic study. Parasitol. Res. 82, 248– 252. Roy, B., Dasgupta, S., Tandon, V., 2008. Ultrastructural observations on tegumental surface of Raillietina echinobothrida and its alteration caused by root peel extract of Millettia pachycarpa. Microsc. Res. Tech. 71, 810–815. Roy, B., Dasgupta, S., Tandon, V., 2009. Ultrastructural observations on Fasciolopsis buski and its alteration caused by shoot extract of Alpinia nigra. Microsc. Res. Tech. 72, 61–66. Roy, B., Lalchhandama, K., Dutta, B.K., 2007. Anticestodal efficacy of Acacia oxyphylla on Raillietina echinobothrida: light and electron microscopic study. Pharmacologyonline 1, 279–287. Roy, B., Lalchhandama, K., Dutta, B.K., 2008. Scanning electron microscopic observations on the in vitro anthelmintic effects of Millettia pachycarpa on Raillietina echinobothrida. Phcog. Mag. 4 (13), 20–26. Schmahl, G., Mehlhorn, H., Klimpel, S., Krieger, K.J., 2007. Efficacy of a combination of imidacloprid plus moxidectin against larva and adult stages of nematodes (Trichuris muris, Angiostrongylus cantonensis) in rodents. Parasitol. Res. 101, S85–S92. Tandon, V., Pal, P., Roy, B., Rao, H.S.P., Reddy, K.S., 1997. In vitro anthelmintic activity of root tuber extract of Flemingia vestita, an indigenous plant in Shillong, India. Parasitol. Res. 83, 492–498. Tandon, V., Roy, B., 2002. SEM Pictorial Guide to Trematodes of Live Stock in India. Regency Publication, New Delhi, pp. 1–72. Tandon, V., Roy, B., 2005. Status of helminthiasis in livestock of Northeast India. Proc. Nat. Acad. Sci., India 75B, 229–244. Tangpu, V.T., Yadav, A.K., 2006. Anticestocidal efficacy of Psidium guajava against experimental Hymenolepis diminuta infection in rats. Ind. J. Parmacol. 38, 29–32. Temgenmongla, Yadav, A.K., 2005. Anticestodal efficacy of folklore medicinal plants of Naga tribes in North-east India. Afr. J. Trad. CAM. 2, 129–133. Tippawangkosol, P., Choochote, W., Na-Bangchang, K., Jitpakdi, A., Pitasawat, B., Riyong, D., 2004. The in vitro effect of albendazole, ivermectin, diethycarbamazine, and their combination against infective third-stage larvae of nocturnally subperiodic Brugia malayi (Narathiwat strain): scanning electron microscopy. J. Vector Ecol. 29, 101– 108. Xiao, S.H., Guo, J., Chollet, J., Tanner, Wu.M., Utzinger, J., 2003. Effect of artemether on Schistosoma japonicum: dose-efficacy relationship, and changes in worm morphology and histopathology. Chin. J. Parasitol. Parasit. Dis. 22, 148–153. Yadav, A.K., Tandon, V., Rao, H.S.P., 1992. In vitro anthelmintic activity of fresh tuber extract of Flemingia vestita against Ascaris suum. Fitoterapia 63, 395–398.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Effect of Lysimachia ramosa (Primulaceae) on helminth parasites: Motility, mortality and scanning electron microscopic observations on surface topography M. Challam, B. Roy *, V. Tandon Department of Zoology, North-Eastern Hill University, Shillong 793 022, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 July 2009 Received in revised form 7 December 2009 Accepted 16 December 2009
The alcoholic extract of Lysimachia ramosa Wall (Primulaceae) was tested in vitro against helminth parasites, Fasciolopsis buski and Ascaris suum, from porcine hosts and Raillietina echinobothrida from domestic fowl. The live adult parasites, collected from a freshly autopsied host, were exposed to different concentrations (5–50 mg) of the test plant extract in physiological phosphate-buffered saline (PBS) having 0.1% dimethyl sulphoxide (DMSO) at 37 1 8C. The treated parasites revealed complete inactivation and flaccid paralysis that was followed by death at varying periods of time. A dose-dependent loss of motility and mortality was observed in all the treated parasites. Scanning electron microscopic observations revealed conspicuous deformity of the surface architecture in all the parasites exposed to the test plant extract. The general tegument in F. buski showed shrinkage and loss of scale-like spines; proglottides all along the strobilar length in R. echinobothrida appeared shrunken and deformed and the cuticular surface of A. suum appeared disorganised, having lost transverse striations. The botanicals of the test plant seem to be effective against all the three types of helminth parasites. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Anthelmintic Lysimachia ramosa Scanning electron microscopy
Helminthic infections are amongst the most common parasitic infections of animals and humans worldwide and are now well recognised as an important veterinary and public health problem, both in developing and in developed countries (Perry and Randolph, 1999; Hafeez, 2003). Survey of the literature revealed that infections of parasites implicating as many as 57 different species of nematode, trematode and cestode worms are commonly prevalent among large livestock animals in northeast India, of which several species are important potential zoonoses as cases of human infections have also been reported (Tandon and Roy, 2005). Although several effective synthetic drugs are available in the market, they are not accessible to the farmers, particularly natives who face problem of affordability and of regular supply (McKeller and Jackson, 2004)
* Corresponding author. Tel.: +91 3642722331; fax: +91 3642550300. E-mail address: [email protected] (B. Roy). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.12.024
and, therefore, rely heavily on ethno-medicines for controlling helminthiasis for their livestock. Meghalaya, one of the hot spots of northeast India, has a wide variety of plants used by natives as curative against worm infections (Rao, 1981). Lysimachia ramosa Wall (Family: Primulaceae) is one such plant, the alcoholic extract of its leaves being widely used by Jaintia and Khasi tribes in the region against intestinal helminth infections. The present investigation, therefore, was taken up to explore and authenticate the possible anthelmintic potential of the plant, if any, on helminth parasites – trematode, cestode and nematode. 1. Materials and methods 1.1. Preparation of ethanol extract Lysimachia ramosa plants were procured from the markets in and around Shillong (Meghalaya) during April–July 2008. The traditionally usable plant parts, that
M. Challam et al. / Veterinary Parasitology 169 (2010) 214–218
215
Table 1 Effect of different concentration of alcoholic extract of Lysimachia ramosa (leaves) on the survival (in h) of nematode, trematode and cestode parasites. Concentration of crude extract/drug (mg ml 1)
Ascaris suum
Fasciolopsis buski
Raillietina echinobothrida
Paralysis
Death
Paralysis
Death
Paralysis
Death
Lysimachia ramosa 5 10 20 30 50
74.12 0.12 69.20 0.18 32.31 0.14 26.02 0.18 22.82 0.23
76.25 0.12 73.42 0.20 37.83 0.17 27.30 0.14 26.45 0.14
3.64 0.14 2.61 0.15 1.55 0.14 1.45 0.14 1.20 0.12
4.21 0.12 3.41 0.15 2.62 0.14 2.32 0.14 2.22 0.12
6.75 0.12 5.80 0.12 4.92 0.07 4.10 0.10 3.40 0.15
7.25 0.12 6.81 0.12 5.24 0.12 4.44 0.15 4.51 0.15
Praziquantel 1 5 10
– – –
– – –
5.12 0.94 4.44 0.14 2.24 0.12
6.23 0.12 5.85 0.10 4.41 0.15
3.12 0.56 2.62 0.39 1.80 0.46
5.05 0.49 3.54 0.56 3.21 0.45
Albendazole 1 5 10
30.21 0.18 26.34 0.14 22.22 0.12
31.21 0.12 28.31 0.14 24.24 0.12
– – –
– – –
– – –
– – –
Control of A. suum, F. buski and R. echinobothrida survived for 102.35 0.22, 21.05 0.22, and 29.50 0.15 h, respectively. Values are expressed as mean SEM (n = 5); P < 0.05; control vs treated.
is, the leaves were washed thoroughly in deionised water and air dried. About 200 g of dried leaves were ground in an electric grinder and then refluxed with ethanol (100 g l 1) for 8 h at 60 8C. The solution obtained was filtered through Whatman filter paper (No. 1) and the volume was reduced through distillation, followed by complete dryness at 50 8C in an oven. The crude extract obtained as a powder material was then refrigerated at 4 8C until further use. One hour prior to experimental assay, varying concentrations of the extract, namely 5, 10, 20, 30 and 50 mg ml 1, were prepared by dissolving in 0.9% phosphate-buffered saline
(PBS, pH 7–7.3) supplemented with 0.1% dimethyl sulphoxide (DMSO). 1.2. Recovery and in vitro treatment of parasites Test parasites, the trematode (Fasciolopsis buski) and the nematode (Ascaris suum) were collected from freshly sacrificed porcine host (Sus scrofa domestica) and the tapeworm (Raillietina echinobothrida) from the intestine of the domestic fowl (Gallus domesticus). The live worms were directly treated with different concentrations of the plant
Fig. 1. Ascaris suum—scanning electron micrographs. (a, b) Untreated control. (a) Anterior end of the worm. (b) Cuticle showing annulations. (c, d) Treated parasites. (c) Body surface showing wrinkles (ethanolic extract of L. ramosa) (scale bar = 50 mm). (d) Anterior end with severe deformity at the lip surface (albendazole) (scale bar = 100 mm).
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extract in PBS having 0.1% DMSO in separate petri dishes. Similar treatment was performed for varying doses of praziquantel and albendazole as broad-spectrum reference drugs; the controls were maintained in a medium containing only PBS with 0.1% DMSO. For each set of treatment, five replicates were used. 1.3. Motility, mortality and scanning electron microscopic analyses Motility of the worms was observed and the time taken for paralysis and death recorded as described earlier (Roy et al., 2008a). The parasites incubated with 50 mg ml 1 extract of PBS were selected for scanning electron microscopy (SEM) observation because of the relatively early paralytic lethal effect of that dose as compared with lower doses. From each medium, individual worms were washed thoroughly in buffer and fixed soon after the onset of paralysis in 4% gluteraldehyde buffered with 0.1 M sodium cacodylate at 4 8C for 4 h, washed in buffer, and followed by secondary fixation in 1% OsO4. The samples were then dehydrated through increasing grades of acetone followed by air drying after treatment with tetramethyl silane as detailed by Roy and Tandon (1991), gold coated using JFC 1100 (Jeol) ion sputtering chamber and viewed in JSM-6360 scanning electron microscope at an electron accelerating voltage of 10–15 kV. 2. Results and discussion Observations on the efficacy of the plant extract and the reference drugs in terms of motility and survivability of the parasites are presented in Table 1. The results indicate that both the plant extract and the two reference drugs showed dose-dependent lethal anthelmintic efficacy. However, at lower concentration, that is, 5 mg ml 1 of plant extract, the nematode did not show any significant mortality with respect to control. The controls A. suum, F. buski and R. echinobothrida maintained in PBS with 0.1% DMSO showed physical activity till the period of 102.35 0.22 h, 21.05 0.22 h, and 29.50 0.15 h, respectively. Stereoscan observations on control A. suum revealed fine surface topography, with three prominent denticulate lips having smooth surface and one labial papilla in the form of a prominent protuberance in each lip (Fig. 1(a)); the cuticle, from the cephalic to the posterior end of the body revealed quite distinct and neatly arranged regular transverse striations in the form of parallel concentric rings running completely round the cylindrical body; clusters of striations are separated by deep groove-like annulations that occur at regular intervals giving the body a segmented appearance (Fig. 1(b)). Worms incubated with 50 mg ml 1 of the plant extract showed prominent wrinkles and disorganisation of the cuticle throughout the body (Fig. 1(c)), including the surface of the lips. Similar deformity of the lip surface and general body cuticle was also observed in the worms treated with the reference drug albendazole (Fig. 1(d)). The untreated control F. buski revealed a normal body contour with scale-like spines on the entire ventral surface and the oral and ventral suckers having radial corrugations
Fig. 2. Fasciolopsis buski—scanning electron micrographs. (a) Untreated control fluke, showing normal surface architecture (scale bar = 10 mm). (b, c) L. ramosa and praziquantel treated parasite respectively, showing pronounced distortion of the tegumental surface (scale bar = 20 mm).
(Fig. 2(a)). By contrast, the flukes treated with 50 mg ml 1 doses of the plant extract and praziquantel as well manifest a pronounced deformity of the body surface, particularly sloughing off of the scales (Fig. 2(b), (c)). The control R. echinobothrida showed the typical cestode fine surface topography, with densely packed microtriches throughout the surface giving it a velvety appearance (Fig. 3(a), (b)). By contrast, the worms treated with the plant extract showed shrunken scolex with severely distorted and folded tegument around the suckers (Fig. 3(c)); the microtriches were eroded and the proglottides appeared deformed with wrinkled surface. The worms exposed to the reference cestocidal drug also revealed similar results (Fig. 3(d)).
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Fig. 3. Raillietina echinobothrida—scanning electron micrographs. (a, b) Untreated control. (a) A portion of strobila, showing normal surface of proglottides (scale bar = 50 mm). (b) A closer view of the proglottid tegument, revealing a dense cover of microtriches (scale bar = 5 mm). (c, d) The scolex end and strobilar surface, showing distorted and wrinkled tegument in the plant extract treated parasite (scale bar = 50 mm).
The result of the present investigation revealed that the crude extract of L. ramosa caused destructive alterations and deformity in the cuticle of A. suum and also in the tegumental architecture of F. buski and R. echinobothrida as early as 26.45 0.14 h, 2.22 0.12 h and 4.51 0.15 h, respectively, after treatment at a concentration of 50 mg ml 1 of extract of PBS. Pharmacologically active components recorded from different species of the genus Lysimachia are triterpenoid saponins, organic acids and flavones (Liang et al., 2006). Triterpene saponins are known to have cytotoxic activity against human skin fibroblasts and tumour cells (Galanty et al., 2008). Nematocidal and cestocidal activity of saponins have also been established (Ghosh et al., 1993, 1996). Anthelmintic properties of several ethnomedicinal plants such as Calliandra portoricensis, Zingiber officinalis, Zanthoxylum alatum, Flemingia vestita, Psidium guajava, Houttuynia cordata, Lasia spinosa, Alpinia nigra, Spilanthes oleraceae, Millettia pachycarpa and Accacia oxyphylla have been established through in vitro exposure of crude extract of these plant/plant parts against several helminth parasites (Adewuni and Akubue, 1981; Yadav et al., 1992; Roy and Tandon, 1996; Roy, 2001; Temgenmongla and Yadav, 2005; Tangpu and Yadav, 2006; Lalchhandama et al., 2007; Roy et al., 2007, 2008a,b, 2009). The cuticle of nematodes is metabolically active and morphologically specialised for selective absorption of nutrients and osmoregulation. Thus, passive diffusion of anthelmintics through the cuticle (Alvarez et al., 2007) would probably be responsible for destructive changes and deformation of the nematode body surface (Tippawangkosol et al., 2004; Schmahl et al., 2007). In trematodes, the surface papillae act as sensory organs, whereas scales and
suckers are important organs of attachment (Tandon and Roy, 2002). In platyhelminth parasites, the general body surface acts as an absorptive surface; in cestodes, microtriches are known to increase the effective surface area of absorption many fold. In the present investigation, all these structures were observed to be affected and altered by the crude ethanolic extract of L. ramosa. Similar to the present observations, the surface tegument is found to be a principal target site for different synthetic drugs and natural anthelmintic products (Geary et al., 1992; Martin et al., 1997; Roy et al., 2009). Drugs such as albendazole and its related compound enter the parasite body through simple diffusion and cause disruption of the tegumental and muscle layers (Mottier et al., 2003; Markoski et al., 2006). Histomorphological and ultrastructural changes caused by anthelmintic agents with respect to tegumental deformity have been well documented in different helminths (McKinstry et al., 2003; Xiao et al., 2003; Meany et al., 2004). Crude extract of F. vestita and its active component genistein produced prominent structural alterations in R. echinobothrida extending from destruction of tegument, microtriches and suckers to vacuolisation and muscle distortion (Tandon et al., 1997; Pal and Tandon, 1998). Naguleswaran et al. (2006) observed that synthetic derivatives of genistein, Rm 6423 and Rm 6426 induced considerable damage in Echinococcus granulosus and E. multiloculoris, making them non-viable by inducing truncation of microtriches, nuclear pyknosis and vesiculations; in addition, Rm 6423 specifically induced dramatic breakdown of the structural integrity of the metacestode germinal layer. The crude alcoholic extract of L. ramosa thus seems to have anthelmintic effect. However, the precise mechanism by which the lethal action is exerted is not clear. Therefore,
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identification of the active principle of the plant-derived component is a prerequisite to ascertain the anthelmintic efficacy and actual mode of action of the botanicals on the parasite. Acknowledgements This study was supported by University Grants Commission (New Delhi) – supported project to BR. Infrastructural facilities provided by UGC-DSA programme to the Department of Zoology, North-Eastern Hill University, Shillong are also acknowledged. References Adewuni, C.O., Akubue, P.I., 1981. Preliminary studies on the anthelmintic properties of the aqueous extract of Calliandra portoricensis (Jacq.) Benth. Bull. Anim. Health Proc. Afri. 29, 171–175. Alvarez, L.L., Mottier, M.L., Lannsse, C.E., 2007. Drugs transfer into target helminth parasites. Trends Parasitol. 23, 97–104. Galanty, A., Michalik, M., Sedek, L., Podolak, I., 2008. The influence of LTS4, a saponoside from Lysimachia thyrsiflora L., on human skin fibroblasts and human melanoma cells. Cell. Mol. Biol. Lett. 13, 585–598. Geary, T.G., Klien, R.D., Vanover, L., Bowman, J.L., Thompson, D.P., 1992. The nervous system of helminths as target of drugs. J. Parasitol. 78, 215–230. Ghosh, M., Sinhababu, S.P., Sukul, N.C., Mahato, S.B., 1993. Antifilarial effect of two triterpenoid saponins isolated from Acacia auriculiformis. Indian J. Exp. Biol. 31, 604–606. Ghosh, M., Sinhababu, S.P., Sukul, N.C., Ito, A., 1996. Cestocidal activity of Acacia auriculiformis. J. Helminthol. 70, 171–172. Hafeez, M.D., 2003. Helminth parasites of public health importance – Trematode. J.P.D. 27, 69–75. Lalchhandama, K., Roy, B., Dutta, B.K., 2007. In vitro anthelmintic activity of Acacia oxyphylla: changes in the tegumental enzymes of the cestode, Raillietina echinobothrida. Pharmacologyonline 2, 307–317. Liang, B., Tiang, J., Xu, L., Yang, S., 2006. Triterpenoid saponins from Lysimachia davurica. Chem. Pharm. Bull. 54, 1380–1383. Martin, R.J., Robertson, A.P., Bjorn, H., 1997. Target sites of anthelmintics. Parasitology 114, 111–124. Markoski, M.M., Trinddade, E.S., Cabrera, G., 2006. Praziquantel and albendazole damaging action on in vitro developing Mesocestiodes corti (Platyhelminthes: cestoda). Parasitol. Int. 55, 51–61. Mottier, M.L., Alvarez, L.I., Pis, M.A., Lanusse, C.E., 2003. Transtegumental diffusion of benzimizole anthelmintics into Moniezia benedeni: correlation with their octanol–water partition coefficients. Exp. Parasitol. 103, 1–7. McKeller, Q.A., Jackson, F., 2004. Veterinary anthelmintics: old and new. Trends Parasitol. 20, 454–461. McKinstry, B., Fairweather, I., Brennan, G.P., Forbes, A.B., 2003. F. hepatica: tegumental surface alterations following treatment in vitro and in vivo with nitroxynil (Trodax). Parasitol. Res. 91, 251–263. Meany, M., Fairweather, I., Brennan, G.P., Forbes, A.B., 2004. Transmission electron microscope study of the ultrastructural changes induced in the tegument and gut of Fasciola hepatica following in vivo drug treatment with closulon. Parasitol. Res. 92, 232–241. Naguleswaran, A., Spicher, M., Vonlaufen, N., Ortega-Mora, L.M., Torgerson, P., Gottstein, B., Hemphill, A., 2006. In vitro metacestocidal activities of genistein and other isoflavones against Echinococcus
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