Experimental Parasitology 122 (2009) 289–298
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Fasciola gigantica: Anthelmintic effect of the aqueous extract of Artocarpus lakoocha Naruwan Saowakon a, Tawewan Tansatit b, Chaitip Wanichanon a, Waraporn Chanakul c, Vichai Reutrakul c, Prasert Sobhon a,* a
Department of Anatomy, Faculty of Science, Mahidol University, RamaVI Rd., Bangkok 10400, Thailand Faculty of Veterinary Science, Mahidol University, Salaya Campus, Nakon Pathom 73170, Thailand c Department of Chemistry, Faculty of Science, Mahidol University, RamaVI Rd., Bangkok 10400, Thailand b
a r t i c l e
i n f o
Article history: Received 12 December 2008 Received in revised form 6 March 2009 Accepted 14 April 2009 Available online 22 April 2009 Keywords: Fasciola gigantica Anthelmintic drug Artocarpus lakoocha Tegument
a b s t r a c t The effect of the crude extract of Artocarpus lakoocha (70% composition is 2,4,30 ,50 - tetrahydroxystilbene -THS) on adult Fasciola gigantica was evaluated after incubating the parasites in M-199 medium containing 250, 500, 750 and 1000 lg/ml of the crude extract, or triclabendazole (TCZ) at the concentrations of 80 and 175 lg/ml as the positive control, for 3, 6, 12 and 24 h, using relative motility (RM) assay and observation by scanning electron microscope (SEM). Decreased contraction and motility were first observed after 3 h incubation with TCZ at the concentration 80 and 175 lg/ml. TCZ markedly reduced the parasite’s motility at the concentration of 175 lg/ml at 6 h, and killed the worms after 12 h exposure. The crude extract of A. lakoocha at all concentrations reduced the parasite’s motility similar to TCZ at 3 h incubation. In 250 and 500 lg/ml of the crude extract, the values were decreased from 3 to 12 h, then they were stable between 12 and 24 h and reduced to the level approximately 30–40% of the control. At 750 and 1000 lg/ml concentrations the crude extract rapidly reduced the RM values from the start to 12 h and killed the parasites between 12 and 24 h incubation. The crude extract also inhibited the larval migration by 75% and 100% at the concentrations of 250–500 and 750–1000 lg/ml, respectively. TCZ and the crude extract caused sequentially changes in the tegument including swelling, followed by blebbings that later ruptured, leading to the erosion and desquamation of the tegument syncytium. As the result, lesion was formed which exposed the basal lamina. The damage appeared more severe on the dorsal than the ventral surface, and earlier on the anterior part and lateral margins when compared to the posterior part. The severity and rapidity of the damages were enhanced with increasing concentration of the crude extract. Hence, the crude extract of A. lakoocha, may exert its fasciolicidal effect against adult F. gigantica by initially causing the tegumental damage. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Fasciolosis, caused by the liver fluke of Fasciola spp., is a serious disease that causes substantial production and economic losses in sheep, cattle and other ruminants in many countries (Fairweather and Boray, 1999; Rivera et al., 2004). As it is a zoonotic disease, human may also be infected; thus it is considered to be a serious public health problem as well (Wiwanitkit et al., 2002). It has been estimated that 2.4–17 million people are infected and more than 90 million people are at risk (Keiser et al., 2005). The infection has caused loss worldwide estimated at US$ 2000 million per annum (McManus and Dalton, 2006; Spithill and Dalton, 1998). In Thailand, the annual loss, has been estimated around 350–400
Abbreviations: DMSO, dimethyl sulphoxide; THS, 2,4,30 ,50 -tetrahydroxystilbene. * Corresponding author. Fax: +662 354 7168. E-mail addresses:
[email protected] (N. Saowakon),
[email protected] (P. Sobhon). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.04.011
million baht, mainly due to weight and meat loss, reduction in dairy product and fertility of the animals (Sukhapesan et al., 1990). At present, effective vaccines are not yet available (LópezAbán et al., 2007; Sexton et al., 1990; Sobhon et al., 1998; Wedrychowicz et al., 2007), therefore, anthelmintic drugs are the main method employed for controlling the fluke infection. Triclabendazole (TCZ) is the drug of choice and the most effective flukicide against both juvenile and adult flukes, and it has been used to control fasciolosis since 1983 (Keiser et al., 2007a). However, the resistance to this drug has emerged and may pose a serious problem as no other effective drug is available (Fairweather, 2005; Keiser et al., 2007b). Thus new anthelmintics are urgently needed. In this report we have investigated a novel anthelmintic effect of the extract from Artocarpus lakoocha Roxb. a medicinal herb commonly used by indigenous people in Thailand and Laos as anthelmintics (Charoenlarp et al., 1981, 1989). The brown powder (called Puag-Haad in Thai) is a product of the aqueous extraction of A. lakoocha Roxb. prepared by boiling the wood chips and then evaporating water
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away. This preparation has been used as a traditional anthelmintic drug for treatment of tapeworm infection in Thailand. Seventy percent of the crude aqueous extract of A. lakoocha is composed of a phenolic compound, trans-2,4,30 ,50 -tetrahydroxystilbene (THS) (Likhitwitayawuid et al., 2006; Mongkolsuk et al., 1957; Poopyruchpong et al., 1978). Recent studies reported that THS exhibited antiherpetic, anti-HIV (Likhitwitayawuid et al., 2005), anti-inflammatory (Chung et al., 2003), anti-oxidation (Lorenz et al., 2003), and anti-apoptotic activities, and it also showed neuroprotective action (Andrabi et al., 2004). Furthermore, the chemical structure of THS is similar to that of halogenated-phenolic fasciolocides. Hence, it is possible that THS could act as a drug for the treatment of liver fluke infection in cattle and human, as it was shown in a previous in vitro study that the crude extract of A. lakoocha could inhibit motility and caused morphological changes in the tegument of the fish trematode, Haplorchis taichui (Wongsawad et al., 2005). Hence, the aims are to investigate the effects of the crude extract of A. lakoocha on motility, tegumental surface changes, and the killing of adult F. gigantica following the in vitro incubation, and on migration of the newly excysted juvenile (NEJ). 2. Materials and methods 2.1. Parasites Adult liver flukes were collected from the bile duct and gall bladder of cattle which were killed at a local slaughter house in Phatumthani province, Thailand. They were washed several times with 0.85% NaCl solution. Only intact and actively mobile worms were used immediately for this study. 2.2. Drugs The crude extract of A. lakoocha was prepared in the Department of Chemistry, Faculty of Science, Mahidol University, Bangkok, Thailand (lot no. VR-11817). The concentration used in this study was based on the report by Wongsawad et al. (2005), which demonstrated that the lowest concentration of the crude extract that could killed the adult H. taichui in vitro was 250 lg/ml. The stock solution was prepared by dissolving 6 g powder of the crude extract in ten milliliters of dimethyl sulphoxide (DMSO). The M-199 medium (Sigma) containing antibiotics (penicillin 50 IU/ ml, streptomycin 50 lg/ml, gentamycin 30 IU/ml), was mixed with the stock solution so that the final concentrations of the crude extract in the medium were at 250, 500, 750, and 1000 lg/ml for use in this experiment. A commercial anthelmintic, triclabendazole (TCZ) (FasinexÒ 10%, Ciba Geigy) was dissolved in the medium at concentrations of 80 and 175 lg/ml and used as the positive controls. The latter concentration of TCZ was equivalent to 175 lg/ ml of THS in the crude extract at the concentration 250 lg/ml, as THS makes up 70% of the crude extract (Mongkolsuk et al., 1957; Poopyruchpong et al., 1978). The negative control was carried out by incubating the worms in the culture medium containing 0.1% (v/v) DMSO and antibiotics similar to those listed above. 2.3. Assay methods for drug’s activities 2.3.1. Motility assay of adult fluke Two hundred and eighty adult flukes were randomly assigned to seven groups (40 flukes per group): group 1 was the negative control and groups 2–3 were treated with TCZ as the positive controls. Flukes in groups 4–7 were incubated in M-199 culture medium containing various doses of the crude extract as mentioned above. The worms were incubated for 24 h in an incubator with 5% CO2 at 37 °C. After 3, 6, 12, and 24 h incubation time (10
parasites per each observation time), motility was assessed by examination under a stereomicroscope, and the tegumental alterations were observed under light (LM) and scanning electron (SEM) microscopes. Motility scores were assigned by using the following criteria: 3 = movement of the whole body, 2 = movement of only part of the body, 1 = immobile but not dead and unstained with the vital dye – 1% methylene blue in 0.85% NaCl solution, and 0 = immobile and stained with the vital dye. The efficacies of the tested drugs against adult F. gigantica were calculated as the relative motility (RM) value using the formula listed below (Kiuchi et al., 1987). A small RM value indicated stronger drug activity, and when all flukes died this value was 0.
P
nN N MI test 100 RM value ¼ MI control
Motility index ðMIÞ ¼
n = motility score, N = number of flukes with the score of n. 2.3.2. Larval migration inhibition assay The metacercariae of F. gigantica were collected from infected snails, Lymnaea rubiginosa. Metacercariae were excysted, and the newly excysted juveniles (NEJs) were separated from the empty cyst walls, unexcysted metacercariae and debris by transferring to the excystment tower placed within the 24-well plate and incubated at 37 °C as previously described (Wilson et al., 1998). The active NEJs migrated through the membrane of the excystment tower fitted in the well of the microplate. After NEJs migrated through the membrane of the excystment tower, they were collected in a fresh M-199 medium containing the antibiotics as mentioned earlier. The 400 active NEJs were added to each 15 ml test-tube containing 1 ml medium and the crude extract of A. lakoocha at the concentration 250, 500, 750 and 1000 lg/ml, or TCZ at the concentration 175 lg/ml. The NEJs incubated in the culture medium containing 0.1% (v/v) DMSO and antibiotics were used as the negative control. The NEJs were incubated at 37 °C for 2 h. Then, each sample was transferred to the excystment tower fitted within 24-well plate. Any air trapped between the tower and plate was removed by tapping the plate. The plates were covered with lids and were incubated at room temperature for overnight. Then, the excystment towers were removed, and NEJs that passed through the membrane of the excystment tower into each well were counted and the percents of migration inhibition were calculated according to the formula shown below, and each experiment was repeated three times.
% larval migration inhibition ¼
ðnumber of controlled NEJ number of tested NEJÞ 100 number of controlled NEJ
Number of controlled NEJ = number of NEJs in control solution without the drugs that migrated through the excystment tower into the collecting well. Number of tested NEJ = number of NEJs in drug-treated group that migrated through the excystment tower into the collecting well. 2.3.3. Observations of tegumental changes by LM using semithin sections, and SEM For LM and SEM studies another two hundred and eighty adult flukes were divided into seven groups and treated similarly as mentioned in Section 2.3.1. At 3, 6, 12, 24 h, 5 flukes were taken
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for LM or SEM observations at each incubation time. For LM observation, the flukes were cut and fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M PBS buffer pH 7.4 at 4 °C for 24 h. After washing several times, the tissue blocks were dehydrated through a graded series of ethanol, infiltrated with propylene oxide, and embedded in Araldite 502 resin. The plastic blocks were cut at 700 nm thickness in the MT-2 Sorvall ultramicrotome, stained with 1% methylene blue, observed and photographed under the light microscope, Nikon DXM-1200F. For scanning electron microscopy (SEM), the fluke samples, taken from each incubation time, were fixed in the same fixative as for LM, cut into three pieces, each covering about one-third of the body length, i.e., the anterior part (covering an area from the oral sucker to the level of Mehlis’s gland), the remaining part was divided equally into the middle one-third and the posterior one-third. These body parts were washed with phosphate buffer, and posted-fixed in 1% OsO4 for 2 h. They were washed repeatedly in cold double distilled water, dehydrated through a graded series of ethanol, dried in a Hitachi HCP-2 critical point drying machine using liquid CO2, mounted on aluminum stubs, coated with platinum and palladium in a Hitachi E-102 ion–sputtering apparatus set at 10–15 mA for 7 min. Specimens were observed and photographed in a Hitachi S-2500 scanning electron microscope, operating at 15 kV. 3. Results 3.1. Motility scores All liver flukes in the control group showed active movement throughout the duration of the experiment while flukes treated with TCZ and crude extract exhibited reduced motility in a concentration and time-dependent manner (Fig. 1). The initial reduction of motility (decreased RM value) occurred at 3 h in TCZ at the concentration of 80 lg/ml. Then RM value declined gradually from 3 h until 24 h incubation. Flukes incubated in TCZ at the concentration of 175 lg/ml, exhibited reduced motility at a more rapid rate than the previous dose, as the RM value dropped rapidly from the start and the parasites became completely immobile and killed at 12 h. When incubated in the crude extract of A. lakoocha at various concentrations (250–1000 lg/ml), the worm motility and RM value decreased throughout the experimental period. In 250 and 500 lg/ml of the crude extract the worms exhibited gradual reduction of RM value at 3 h, and the decrease was sharp from 3 to 12 h, then RM value was stable between 12 and 24 h and reduced to the level approximately 30–40% of the control. Although, flukes were immobile during this period, most were still alive and unstained by vital dye. When incubated in 750 and 1000 lg/ml of the crude extract, the worms showed rapid reduction of RM value from the
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start to 12 h. The rates of reduction of RM values in the two doses were approximately equal to that of 175 lg/ml TCZ. All of the worms ceased motility between 12 and 24 h, and most of them took up the vital dye which indicated that they were killed. 3.2. Larval migration inhibition assay The NEJs were incubated with various concentrations of the crude extract of A. lakoocha, as mentioned in Section 2.3.2 and the percents of larval migration inhibition (LMI) through the membrane of the excyst towers as compared to the treatment with TCZ were shown in Fig. 2. The LMI of NEJs treated with 250 and 500 lg/ml of the crude extract was found to be 74%, and 76%, respectively. When NEJs were incubated with 750 and 1000 lg/ml of the crude extract LMI was 100%. TCZ at the concentration 175 lg/ml also showed 100% LMI. 3.3. Light microscopic (LM) and scanning electron microscopic (SEM) observations of the tegument 3.3.1. LM observations of the tegument cross section The tegument of negative control adult parasites showed even cytoplasmic syncytium that was joined by the processes of tegumental cells. There were numerous spines embedded in the syncytium (Fig. 3A), which was supported by muscle layers and the basal lamina. In contrast, the samples treated with the crude extract of A. lakoocha, at concentration 250 lg/ml at 3 h incubation started to show damage which appeared as numerous vacuoles in the tegument syncytium (Fig. 3B and C), while the underlying structures still appeared normal. At the concentration 750 lg/ml at 3 h, the tegument showed numerous blebbings (Fig. 3D) and disruption (Fig. 3E) on the surface, but muscle underlying the tegument still exhibited normal appearance. At 24 h incubation in 750 lg/ml of the crude extract, there was a general disruption of the tegument (Fig. 3F), as the components in the tegumental syncytium were degenerated and the tegument itself was partly sloughed off. The muscle cells which were located underneath the basement membrane showed less severe morphological changes, including the lighter staining and vacuolization of the cytoplasm. This implies that the most affected target organ from the treatment with the crude extract is the tegument. Therefore, we focused our observation on the changes of the tegumental surface by SEM. 3.3.2. Observation of changes in the tegument by SEM The typical intact tegument surface of adult parasite exhibited microridges and grooves with numerous serrated spines as reported earlier by our group (Dangprasert et al., 2001; Sobhon et al., 1998). Spines were most numerous on the anterior part espe-
Fig. 1. (A) Relative motility (RM) values of the control and the experimental flukes treated with triclabendazole (TCZ) and the crude extract of A. lakoocha at various concentrations and durations. Each point in the graph represents the response from 10 flukes.
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Fig. 2. The percents of larval migration inhibition of NEJs of F. gigantica incubated in various doses of TCZ and the crude extract of A. lakoocha. Each column of the histogram represents 400 newly excysted juveniles (NEJ). The experiment is triplicated.
cially around the oral and ventral suckers (Fig. 4A) and reduced in number on posterior part of the body. There were clusters of sensory papillae scattering throughout the body surface between spines (Fig. 4B). Abnormal changes were not observed on the tegumental surface of the negative control flukes incubated up to 24 h in the medium containing 0.1% DMSO (Figs. 4A and 6A). 3.3.2.1. Sequence of changes of the tegumental surface. The changes of the tegument induced by TCZ and the crude extract followed a similar sequence. The first sign of change was the swelling of the surface marked by deep furrows (Figs. 4C, inset, 5A). This was followed by the blebbing of the surface as the result of the formation of small bulbous structures on the affected surface (Fig. 4C). Then the blebs were disrupted at their apex (Fig. 5C) followed by focal erosion of the surface (Figs. 4E and 5D) that resulted in the sloughing of larger area of the surface, which later appeared as patches of lesions devoiding of the tegumental syncytium (Figs. 4F and 5E). In the final stage the basal lamina was disrupted, and the spines which used to be anchored to the basal lamina, were dislodged (Fig. 5F). The degree and severity of changes in the tegument as affected by TCZ and the crude extract were illustrated in Figs. 6B–D. 3.3.2.2. Effect of triclabendazole. TCZ at concentrations 80 and 175 lg/ml, caused similar damages to the flukes but the damages occurred earlier and were more severe at the latter dose (Fig. 6B). At 3 h incubation, on the ventral surface, disrupted blebs were observed on the anterior region while erosion and blebbing appeared on the middle and posterior parts. On the dorsal surface, disrupted blebs were prominent at the anterior two-third, whereas the posterior region showed only swollen surface. After 6 h incubation, the degree of damage was more severe, as blebs and disrupted blebs were observed on the anterior part while lesion and extensive erosion were observed on the middle and posterior parts of the ventral surface. On the dorsal surface, the tegument of anterior two-third exhibited erosion while disruption of blebs could be observed on the posterior part. After 12 h incubation, almost all of both dorsal and ventral surfaces exhibited completely sloughed tegument with spines dislodged from their sockets, while the posterior part exhibited erosion on the ventral surface and disrupted
blebs on the dorsal surface. Following 24 h incubation, the basal lamina of the whole surface was disrupted and the spines were dislodges. 3.3.2.3. Effect of the crude extract of A. lakoocha. The sequence of changes and severity of tegumental damage of adult F. gigantica treated with the crude extract of A. lakoocha were also consisted of 6 levels as in TCZ treatment: (i) swelling, (ii) blebbing, (iii) disruption, (iv) erosion, (v) lesion, and (vi) basal lamina disruption with spine dislodging as shown in Fig. 5. Fig. 6C and D illustrated the sequence of changes and the extent of damage of the tegument on the ventral and dorsal surfaces. The crude extract at concentrations 250 and 500 lg/ml showed similar pattern of changes. Following 3 h incubation (Fig. 6C), the ventral surface of the tegument showed swelling and many deep furrows (Figs. 5A and 6C) at the middle region of the ventral surface, and blebs on the posterior region. On the dorsal surface, the tegument appeared swollen and blebs were observed on the anterior region and lateral margins of the posterior region. After 6 h incubation, there were swelling and blebbing on the middle part of the ventral surface. Disrupted blebs were observed on the anterior region. On the dorsal surface, disrupted blebs and swollen surfaces were observed at the anterior and lateral margins of the middle regions. More severe changes occurred after 12 h incubation, as the disrupted blebs were expanded on the anterior and lateral margins of the middle and posterior regions of both surfaces (Figs. 5C and 6C). The swollen tegument was observed on the midline of middle and posterior regions of both surfaces. At 24 h incubation, most tegumental surface on the anterior and posterior parts of both ventral and dorsal surfaces was eroded and became spineless. The only remaining tegument with disrupted blebs was observed in the middle region of ventral surface. Almost all of the dorsal surface except the edge of the middle region showed complete lesion, and all spines were dislodged, exposed basal lamina still bared spines in their sockets (Fig. 6C). The flukes treated with the crude extract at the concentrations 750 and 1000 lg/ml, showed similar sequence of changes and the extent of damages on the tegumental surface, with more severe destruction occurred at the earlier incubation time (Figs. 5F and 6D). At 3 h incubation with 750 and 1000 lg/ml of the crude
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Fig. 3. Light micrographs showing the histology of the tegument of adult F. gigantica. (A) The negative control parasite incubated in M-199 medium containing 0.1% DMSO for 24 h, showing spines (Sp) embedded in the intact tegument (T), and muscle layers (M) lying underneath the basal lamina (BL). (B) In the parasite treated with the crude extract of A. lakoocha, at the concentration 250 lg/ml, at 3 h incubation, showing vacuoles (arrow) in the tegument but the muscle (M) and other structures underneath the basement membrane appear intact. (C) Higher magnification from the boxed area in B, showings numerous vacuoles (arrow) in the tegument (T). (D) A fluke treated with the crude extract at the concentration 750 lg/ml at 3 h incubation, showing the bleb formation (Bl) on the surface. (E) Blebs (Bl) and disrupted blebs (Db) appear on the tegumental syncytium close to the surface. The neuron (N) which is located underneath the muscle (M) layers shows small vacuoles in its cytoplasm. (F) After 24 h incubation with 750 lg/ml of the crude extract, the tegument shows extensive degeneration and sloughing (arrow) from the basal lamina (BL).
extract, disrupted blebs and erosion were prominently on anterior two-third of the ventral and dorsal surfaces. Severe lesion was observed on the posterior region of the ventral surface while the dor-
sal surface exhibited severe disruption of basal lamina and dislodging of spines. After 6 h incubation, the parasites showed exposed basal lamina on both surfaces, but the ventral surface
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Fig. 4. Scanning electron micrographs (SEMs) of the surface of the control flukes (A and B) and flukes treated with triclabendazole (TCZ) at the concentration of 80 lg/ml (C– F). (A) The ventral surface of the anterior region of the control fluke, showing oral (Os) and ventral suckers (Vs) and the genital pore (Gp). (B) At higher magnification, the tegument at middle region of the ventral surface of the same parasite shows rows of serrated spines (Sp) and sensory papillae (arrow). The surface between spines exhibits parallel ridges (ri), intervened by grooves. (C) Medium magnification of the tegumental surface at the middle region of the dorsal surface of a parasite after incubation in TCZ at 80 lg/ml for 3 h, showing severe swelling of tegument, marked by deep furrows (arrow), and the surface is covered with numerous blebs (Bl). Inset demonstrates the posterior region of the ventral surface after 3 h incubation, showing deep furrow (arrow) and swollen surface. (D) At 6 h incubation with 80 lg/ml of TCZ, the tegument surface reveals severe tegumental erosion at the middle region of the ventral surface. The swollen tegument with deep furrow (arrow) is shown at the lateral margin. The empty spine sockets (SS) are evident on the exposed basal lamina, and this is surrounded by disrupted blebs (Db). (E) At 24 h incubation with 80 lg/ml of TCZ, the tegument is completely sloughed off from the middle part of the dorsal surface, exposing a large area of basal lamina (BL) bearing the empty spine sockets (SS). Pieces of tegument on the same part are sloughed off (arrow). (F) At 24 h incubation with 80 lg/ml of TCZ, the anterior region of the dorsal surface shows severe erosion of the tegument (arrow), which eventually becomes lesion (Le). Spines are still anchored to the basal lamina.
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Fig. 5. SEMs showing the sequence and severity of the tegumental changes following the treatment of adult F. gigantica with the crude extract of A. lakoocha at the concentration 250–1000 ll/ml. These changes occur in tandem and are classified into 6 level, (i) swelling, (ii) blebbing, (iii) disruption, (iv) erosion, (v) lesion and (vi) basal lamina disruption and spine dislodging. (A) At 3 h incubation with 250 lg/ml of A. lakoocha, the ventral middle region showing swollen surface divided by deep furrows (arrow), and spines (Sp) are still intact. (B) The middle region of ventral surface showing numerous blebs (Bl), erosion (Er) and lesion (Le). (C) At 12 h incubation with 250 lg/ ml of A. lakoocha, blebs (Bl) and disrupted blebs (Db) appear at the middle region of the ventral surface. (D) Following 24 h incubation in the crude extract at 500 lg/ml, there are disrupted blebs (Db) and extensive erosion (Er) of the ventral anterior region which lead to the desquamation of the tegument. (E) The ventral lateral margin after treatment with 500 lg/ml of the crude extract for 24 h, showing extensive erosion (arrows), exposed basal lamina (BL) that bears spines (Sp) and some of which are dislodged from their sockets (SS). (F) The dorsal anterior region following the treatment with 1000 lg/ml, for 3 h, showing the exposed and disrupted basal lamina, all spines become dislodged, leaving only spine sockets (SS).
appeared less affected. After 12 h incubation parasites treated with both concentrations exhibited severe lesion and disruption of the
basal lamina throughout both the ventral and dorsal surfaces (Fig. 6D).
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Fig. 6. The illustrations of the sequence of tegumental changes and the extent of damages as observed by SEM on the ventral (V) and the dorsal (D) surfaces of the control flukes (A) and experimental flukes (B–D). (A) 0.1% DMSO (control), (B) flukes treated with triclabendazole (TCZ) at the concentration 175 lg/ml, (C) flukes treated with the crude extract of A. lakoocha (AL) at concentration 250 lg/ml, and (D) with AL at concentration 1000 lg/ml.
4. Discussion Our work is the first to demonstrate the comparative effects of triclabenedazole (TCZ) and the crude extract of A. lakoocha on the adult F. gigantica. The results showed that TCZ was more active in causing damage than the crude extract which contains 70% of 2,4,30 ,50 -tetrahydroxystilbene (THS). It was observed that TCZ at the concentration of 175 lg/ml which is equivalent to THS concentration in 250 lg/ml of the crude extract, (Mongkolsuk et al., 1957; Poopyruchpong et al., 1978), caused greater and faster reduction of the parasite motility. However, at higher concentration at 750 and 1000 lg/ml the crude extract exhibited similar rate of motility reduction and parasite killing effect as TCZ at 175 lg/ml. Similarly, the results of larval migration inhibition assay showed that the
effect of TCZ on the inhibition of the NEJs migration was 25% greater than the crude extract at equivalent dose (175 lg/ml of TCZ versus 250 lg/ml of the crude extract). But at the higher doses (>750 lg/ml) the extract of A. lakoocha could also inhibit the migration of NEJs at 100% as well. These results indicate that the crude extract, and probably THS as it is the major constituent could kill the immature as well as mature F. gigantica infection at proper dosages. LM and SEM observations could be used to determine the target of the drugs as morphological changes could be observed. In this study, LM was used to observe the earliest change in a limited area of the tegumental syncytium while tegumental surface changes which reflected the changes in the tegument cytoplasm could be observed over a much wider area by SEM. These changes occurred
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in definite sequences in response to both TCZ and the crude extract, and were consisted of swelling, blebbing, that was later disrupted, leading to erosion and desquamation of the tegument, resulting in the lesion, and finally the exposure and disruption of basal lamina and the dislodging of spines. Following these tegumental changes, the parasite became immobile. Regional differences in response to the crude extract were also observed, with the dorsal surface being more severely affected than the ventral, and the anterior and middle regions, as well as the lateral margins of the flukes generally more affected than the posterior region. The surface changes observed in the present study resembles that demonstrated on F. hepatica treated with clorsulon (Meaney et al., 2003), and with nitroxynil (Trodax) (McKinstry et al., 2003), and also similar to changes observing on the surface of H. taichui treated with aqueous extract of A. lakoocha (Wongsawad et al., 2005). In contrast, the severe damage were observed on the posterior region after treatment of F. hepatica with halogenated phenols i.e., bithionol and hexachlorophene (Gusel’nikova, 1974) and with albendazole sulphoxide (Buchanan et al., 2003), with triclabenedazole sulphoxide (Stitt and Fairweather, 1993), and with praziquatel on Opisthochis viverrini (Apinhasmit and Sobhon, 1996). The regional differences in responses to various anthelmintics could not be definitely explained, as it may depend on the thickness, variation in architecture, routes of drug uptake and drug metabolism in different parts of the tegument. As for the mechanism of action of the crude extract, it is likely that the swelling and blebbing which are the earliest signs of changes could be elicited by the osmotic imbalance, due to Na+ influx into the syncytium, followed later by swelling, blebbing, disruption, erosion and lesion. Once the surface layer is totally destroyed the drug could penetrate deeper into the muscular layer and cause motility reduction and finally death. As THS, the major constituent of the crude extract (up to 70%) (Mongkolsuk et al., 1957; Poopyruchpong et al., 1978), has similar chemical structure to the halogenated phenol group of drugs, such as nitroxynil, it could act via similar mechanism. It was reported that nitroxynil acts as an uncoupler of oxidative phosphorylation (Fairweather et al., 1984; McKinstry et al., 2003). As the result, the decreased production of ATP would affect Na+–K+ pump, leading to the influx of Na+ and water, and consequent the swelling of the syncytium as observed in the study of F. hepatica treated with nitroxynil (McKinstry et al., 2003). The tegument of adult F. gigantica has numerous mitochondria in the middle and lower parts of the tegument especially in close association with the basal membrane infoldings (Sobhon et al., 2000). These mitochondria may be closely involved in providing the energy for ion (Na+–K+) transports (Fairweather et al., 1984). Thus, the crude extract of A. lakoocha which contains very high content of THS could cause the tegument changes by affecting the oxidative phosphorylation in mitochondria. In contrast, TCZ, a benzimidazole derivative, binds to the b-tubulin molecules, and disrupts microtubule-mediated process. As a result, the transport of secretory granules from the tegument cell bodies towards the tegument is blocked by TCZ and benzimidazole derivatives. This could lead to progressive damage of the tegumental surface, especially the replacement of the rapidly turn-over surface membrane. Consequently, the surface membrane could be weakened and disrupted (Stitt and Fairweather, 1993). This could cause water influx into the tegument and results in the osmotic imbalance as well, which elicits similar sequence of tegumental changes as occur in the case of nitroxynil and THS in the crude extract. Further study of the effect of the extract, especially that of the pure compound THS, on other cellular and subcellular targets in the adult F. gigantica will be studied by using similar assay, as well as transmission electron microscopy and other means. Moreover, the effects of the crude extract of A. lakoocha as well as pure THS compound on other trematode parasites
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