**2. Antischistosomal drugs**

For the control of schistosomiasis, which at present is dependent on chemotherapy, it is not satisfactory to have only one single effective treatment (Caffrey, 2007; Doenhoff et al., 2008; Fenwick et al., 2003). Ideally, other antischistosomal drugs would be available so that the classical strategy of alternating treatments to avoid the development of resistance could be used. Unfortunately, the other drugs used before the advent of PZQ, oxamniquine and metrifonate, are restricted in their use. Metrifonate, a drug that exhibits activity against *S. haematobium*, has recently been withdrawn from the market because of medical, operational, and economic criteria (Reich & Fenwick, 2001; Utzinger et al., 2003). Oxamniquine is the only alternative antischistosomal drug, but it is effective only against *S. mansoni*. In the 1970s, oxamniquine was used for individual and mass treatment of schistosomiasis, with satisfactory results regarding efficacy and tolerance. However, its use is currently declining and is being replaced by praziquantel (Cioli, 2000; Reich & Fenwick, 2001; Utzinger et al.,

Natural products, mainly plants, have been the source of medicines for thousands of years. The discovery of pure compounds as active principles in plants was first described at the beginning of the 19th century, and the art of exploiting natural products has become part of the molecular sciences (Kayser et al., 2003). Several extracts or bioactive constituents from living organisms have been used in many communities worldwide against parasitic diseases, including schistosomiasis, and in the past decades, natural products have attracted renewed interest (Kayser et al., 2003; Mølgaard et al., 2001; Ndamba et al., 1994; Sanderson

*In vitro* screening systems are useful and affordable ways to discover potential anthelmintic candidates for *in vivo* tests (Keiser, 2010; Ramirez et al., 2007; Yousif et al., 2007). Because a molecular-target approach is still rarely employed in schistosomicidal drug discovery, a more common strategy has been the complementary approach of whole-organism phenotypic screening *in vitro* to measure compound efficacy (Keiser, 2010; Ramirez et al., 2007; Yousif et al., 2007). In this context, screening for natural products that are active against schistosome is important in the establishment of future strategies for new

Considerable efforts are ongoing to develop novel schistosomicidal agents. As a result, many natural compounds with promising antischistosomal properties have been identified (Braguine et al., 2009; de Moraes et al., 2011; Magalhães et al., 2009, 2010; Moraes et al., 2011; Mølgaard et al., 2001; Parreira et al., 2010; Sanderson et al., 2002). The efficacy of these new compounds against schistosome is defined using three strategies: a) curative, by killing the adult worm; b) prophylactic, by killing schistosomula; and c) suppressive, by inhibiting worm egg-laying. Thus, several parameters, such as motor activity, tegumental changes, and oviposition, are often evaluated as indicators of biological activity and toxicity in

This chapter reviews the present state of *in vitro* drug screening strategies used to discover new compounds active against *S. mansoni*, the most important species infecting humans, with an emphasis on natural products. Also highlighted are the best practices and challenges for drug screenings. Furthermore, information is provided about toxicity, susceptible *Schistosoma* stages, and other interesting laboratory studies on potential antischistosomal compounds, both

For the control of schistosomiasis, which at present is dependent on chemotherapy, it is not satisfactory to have only one single effective treatment (Caffrey, 2007; Doenhoff et al., 2008; Fenwick et al., 2003). Ideally, other antischistosomal drugs would be available so that the classical strategy of alternating treatments to avoid the development of resistance could be used. Unfortunately, the other drugs used before the advent of PZQ, oxamniquine and metrifonate, are restricted in their use. Metrifonate, a drug that exhibits activity against *S. haematobium*, has recently been withdrawn from the market because of medical, operational, and economic criteria (Reich & Fenwick, 2001; Utzinger et al., 2003). Oxamniquine is the only alternative antischistosomal drug, but it is effective only against *S. mansoni*. In the 1970s, oxamniquine was used for individual and mass treatment of schistosomiasis, with satisfactory results regarding efficacy and tolerance. However, its use is currently declining and is being replaced by praziquantel (Cioli, 2000; Reich & Fenwick, 2001; Utzinger et al.,

antischistosomal drug discovery to control schistosomiasis (Yousif et al., 2007).

et al., 2002; Tagboto & Townson, 2001).

studies with schistosome species.

**2. Antischistosomal drugs** 

natural products and natural product-derived compounds.

2001, 2003). As there is currently no available vaccine for this disease in people (Bergquist et al., 2008), chemotherapy may now be at a crucial point.

Chemotherapy against schistosomiasis was reviewed extensively by Cioli et al. (1995), with an emphasis on compounds that were used in the past. Additionally, Cioli (1998) summarised some interesting laboratory studies on potential antischistosomal compounds and the possible emergence of praziquantel-resistant schistosomes. More recently, Ribeirodos-Santos et al. (2006) reviewed results from a comprehensive search of the scientific literature for substances and compounds tested for schistosomiasis therapy over the past century. The authors gathered information on the therapeutic action in humans or animal models and the mechanisms of action of over 40 drugs.

Briefly, antimonial compounds were introduced in 1918, and this group of drugs has been the major point of schistosome chemotherapy for approximately 50 years. However, they cause numerous side effects, such as nausea, vomiting, diarrhoea, anorexia, and cardiovascular, hepatic, and dermatological disturbances. Lethality from cardiac syncope and anaphylactic shock was also reported. Emetine, a drug used to treat amoebiasis, was employed in the second decade of the past century, but the doses required against schistosomiasis were at the very limit of toxicity. The introduction of 2,3-dehydroemetine reduced the toxicity of the parent compound, but patients had to be hospitalised over a month for treatment. Thus, the use of 2,3-dehydroemetine as an antischistosomal agent was abandoned (Cioli et al., 1995). Only in the 1960s was there a breakthrough in the treatment of schistosomiasis, with the rise of metrifonate, nitrofurans, lucanthone, niridazole, hycanthone, and, finally, oxamniquine. In the 1970s, several schistosomicidal drugs emerged, such as tubercidin, amoscanate, PZQ and its benzodiazepine derivative Ro11- 3128, and oltipraz. Nevertheless, the therapeutic doses of most of these drugs were found to cause major side effects. PZQ, an isoquinoline-pyrazine derivative, immediately proved to be superior to any other schistosomicidal drug and quickly became the drug of choice in most endemic areas (Cioli et al., 1995; Fenwick & Webster, 2006). Because of the reliance on a single drug for the treatment and control of schistosomiasis and the considerable concern regarding the development of PZQ resistance, it is timely to review potential alternatives, with an emphasis on natural products.

#### **2.1 Antischistosomals: Natural product and natural product-derived compounds**

The use of natural products for curative and therapeutic purposes has a long history, and compounds derived from natural products have made a big impact on the pharmaceutical industry (Newman, 2003; Newman & Cragg, 2007). In addition to microbes and plants, there has been growing interest in other living organisms, such as arthropods and amphibians, as important sources of biologically active compounds (Kayser et al., 2003). However, the potential for using living beings as sources of new antischistosomal drugs is still poorly explored. In recent decades, there has been a growing interest in the scientific community to search for extracts and pure compounds, especially those derived from plants, that exhibit potential schistosomicidal properties, as one alternative method to the conventional chemical control.

Plants have been traditionally used in the treatment of different diseases, including schistosomiasis, especially in Africa and Asia (Ndamba et al., 1994). In general, medicinal plants are prepared by traditional healers, who have empirical knowledge and

Antischistosomal Natural Compounds: Present Challenges for New Drug Screens 337

Here, data on natural products and related natural product-derived compounds are reviewed, especially those from recent years or that have received considerable attention

Relevant notes

Artemisinin derivatives: artemether (1982) and artenusate (1983); effective against immature schistosome in experimentally infected animals; morphological alteration on the tegument (Utzinger et al., 2001)

Effective in mice infected with *S. mansoni*  (Baldé et al., 1989)

*In vitro* activity on *Schistosoma* adult worms; inhibitory effect on egg-laying; female more susceptible than male; not tested on schistosomula (Barth et al., 1997)

Active against *S. mansoni* in mice (Ogboli, 2000)

There is a great debate about the efficacy and effectiveness of myrrh in the treatment of schistosome infections, both in laboratory and clinical settings (Abdul-Ghani et al., 2009; Badria et al., 2001; Botros et al., 2004, 2005; Sheir et al., 2001)

Active on *S. mansoni*-infected mice; crushed seed also has *in vitro* effects against *S. mansoni* miracidia, cercariae, and adult worms, and an inhibitory effect on egg-laying; not tested on schistosomula (Mahmoud et al., 2002; Mohamed et al., 2005)

*In vitro* against *S. mansoni* adult worms; no egg production was observed for experimental worms; not tested on schistosomula. Schistosomicidal activity against *S. mansoni* cercariae and miracidia has been previously described (Lyddiard et al., 2002)

due to their antischistosomal properties (Table 1).

Extract/ Compound and Biological Source

Artemisinin, active principle from the plant *Artemisia annua* L. (Asteraceae)

Extracts from the plant *Pavetta owariensis* P. Beauv (Rubiaceae) contain proanthocyanins

Goyazensolide isolated from the plant *Eremanthus goyazensis* (Gardner) Sch. Bip. (Compositae)

Extract of leaf from the plant *Vernonia amygdalina* Del (Compositae)

Mirazid myrrh, an oleo-gumresin from the stem of the plant *Commiphora molmol*  (Burseraceae)

<sup>2002</sup>Oil from the plant *Nigella sativa* L. (Ranunculaceae)

> Extract of the seeds and isoflavonoids from the plant *Millettia thonningii* (Schum. et Thonn.) Baker (Leguminosae)

\* Dates of introduction or publication are only approximate.

Table 1. *In vitro* and *in vivo* antischistosomal characteristics of natural products.

Date \*

1980

1989

1997

2000

2001

2002

cultural communities throughout the world. For example, in Zimbabwe, Ndamba et al. (1994) investigated the herbal remedies used in the treatment of schistosomiasis. Based on interviews with 286 traditional healers, they composed a list of 47 plant species most widely used to treat urinary schistosomiasis. Based on this survey, the seven most commonly used plants, *Abrus precatorius* (Leguminosae) *Ozoroa insignis* (Anacardiaceae), *Dicoma anomala* (Cornpositae), *Ximenia caffra* (Oleaceae), *Lannea edulis* (Anacardiaceae), *Elephantorrhiza goetzei* (Leguminosae) and *Pterocarpus angolensis* (Leguminosae), were collected, prepared as described by the traditional healers, their efficacy was evaluated using laboratory animals previously exposed to *S. haematobium* cercariae, and the activity from the extract of *P. angolensis* bark was almost comparable to that of praziquantel. Later, Mølgaard et al. (2001) screened extracts of 23 plant species, popularly used against schistosomiasis in Zimbabwe, for their anthelmintic effect against schistosomula of *S. mansoni*, and the best results against larval forms were obtained with stem and root extracts from *Abrus precatorius* (Fabaceae) and stem bark from *Elephantorrhiza goetzei* (Mimosaceae). All families and names of the plants that are used by traditional healers to treat urinary schistosomiasis in Zimbabwe are described by Ndamba et al. (1994).

Some of the most interesting antischistosomal compounds are derivatives of artemisinin, such as artemether and artesunate (Utzinger et al., 2001; Xiao et al., 2002). They are highly effective in the treatment of malaria and have also been shown to exhibit antischistosomal properties. Artemisinin is a sesquiterpene lactone with an endoperoxide group, which was isolated from the leaves of *Artemisia annua* L. This plant has been used for centuries in Chinese traditional medicine as antidote to many different ailments (Lee, 2007; Utzinger et al., 2001). Artemisinin has been used as an antimalarial since the early 1970s, and its antischistosomal activity was discovered in 1980 by a group of Chinese scientists. In 1982, antischistosomal properties were conrmed for artemether, the methyl ether derivative of artemisinin. Interestingly, artemether has been shown to be active against immature schistosome in experimentally infected animals, but it is less effective against adult worms (Utzinger et al., 2001). Signicant progress has been made with artemether and its potential for the control of schistosomiasis, which has been reviewed by Utzinger et al. (2001). The mechanism of action of artemisinin and its derivatives appears to involve an interaction with heme, which cleaves the endoperoxide bridge of the drug to produce carbon-centred free radicals that then alkylate parasite proteins (Golenser et al., 2006). In addition, scanning electron microscopy showed that artemether caused extensive and severe damage to the tegument in 21-day-old *S. mansoni* harboured in mice (Xiao et al., 2000). Considering that artemether and praziquantel exhibit the highest activity against schistosomula and adult worms, respectively, combined treatment has been proposed to enhance the reduction in worm burden (Utzinger et al., 2003). Currently, new trials to use artemisinin and its synthetic derivatives as lead molecules for drug discovery against schistosomiasis and various other diseases are rapidly growing, and the studies are ongoing (Lee, 2007; Utzinger et al., 2003, 2007; Xiao et al., 2002). Likewise, research on other natural products and natural product-derived compounds against schistosome has been performed by many groups. Accordingly, several plants with antischistosomal properties have been described in the literature (Braguine et al., 2009; de Moraes et al., 2011; Magalhães et al., 2009, 2010; Moraes et al., 2011; Mohamed et al., 2005; Mølgaard et al., 2001; Parreira et al., 2010; Sanderson et al., 2002).

cultural communities throughout the world. For example, in Zimbabwe, Ndamba et al. (1994) investigated the herbal remedies used in the treatment of schistosomiasis. Based on interviews with 286 traditional healers, they composed a list of 47 plant species most widely used to treat urinary schistosomiasis. Based on this survey, the seven most commonly used plants, *Abrus precatorius* (Leguminosae) *Ozoroa insignis* (Anacardiaceae), *Dicoma anomala* (Cornpositae), *Ximenia caffra* (Oleaceae), *Lannea edulis* (Anacardiaceae), *Elephantorrhiza goetzei* (Leguminosae) and *Pterocarpus angolensis* (Leguminosae), were collected, prepared as described by the traditional healers, their efficacy was evaluated using laboratory animals previously exposed to *S. haematobium* cercariae, and the activity from the extract of *P. angolensis* bark was almost comparable to that of praziquantel. Later, Mølgaard et al. (2001) screened extracts of 23 plant species, popularly used against schistosomiasis in Zimbabwe, for their anthelmintic effect against schistosomula of *S. mansoni*, and the best results against larval forms were obtained with stem and root extracts from *Abrus precatorius* (Fabaceae) and stem bark from *Elephantorrhiza goetzei* (Mimosaceae). All families and names of the plants that are used by traditional healers to treat urinary schistosomiasis in Zimbabwe are described by

Some of the most interesting antischistosomal compounds are derivatives of artemisinin, such as artemether and artesunate (Utzinger et al., 2001; Xiao et al., 2002). They are highly effective in the treatment of malaria and have also been shown to exhibit antischistosomal properties. Artemisinin is a sesquiterpene lactone with an endoperoxide group, which was isolated from the leaves of *Artemisia annua* L. This plant has been used for centuries in Chinese traditional medicine as antidote to many different ailments (Lee, 2007; Utzinger et al., 2001). Artemisinin has been used as an antimalarial since the early 1970s, and its antischistosomal activity was discovered in 1980 by a group of Chinese scientists. In 1982, antischistosomal properties were conrmed for artemether, the methyl ether derivative of artemisinin. Interestingly, artemether has been shown to be active against immature schistosome in experimentally infected animals, but it is less effective against adult worms (Utzinger et al., 2001). Signicant progress has been made with artemether and its potential for the control of schistosomiasis, which has been reviewed by Utzinger et al. (2001). The mechanism of action of artemisinin and its derivatives appears to involve an interaction with heme, which cleaves the endoperoxide bridge of the drug to produce carbon-centred free radicals that then alkylate parasite proteins (Golenser et al., 2006). In addition, scanning electron microscopy showed that artemether caused extensive and severe damage to the tegument in 21-day-old *S. mansoni* harboured in mice (Xiao et al., 2000). Considering that artemether and praziquantel exhibit the highest activity against schistosomula and adult worms, respectively, combined treatment has been proposed to enhance the reduction in worm burden (Utzinger et al., 2003). Currently, new trials to use artemisinin and its synthetic derivatives as lead molecules for drug discovery against schistosomiasis and various other diseases are rapidly growing, and the studies are ongoing (Lee, 2007; Utzinger et al., 2003, 2007; Xiao et al., 2002). Likewise, research on other natural products and natural product-derived compounds against schistosome has been performed by many groups. Accordingly, several plants with antischistosomal properties have been described in the literature (Braguine et al., 2009; de Moraes et al., 2011; Magalhães et al., 2009, 2010; Moraes et al., 2011; Mohamed et al., 2005; Mølgaard et

Ndamba et al. (1994).

al., 2001; Parreira et al., 2010; Sanderson et al., 2002).

Here, data on natural products and related natural product-derived compounds are reviewed, especially those from recent years or that have received considerable attention due to their antischistosomal properties (Table 1).


\* Dates of introduction or publication are only approximate.

Table 1. *In vitro* and *in vivo* antischistosomal characteristics of natural products.

Antischistosomal Natural Compounds: Present Challenges for New Drug Screens 339

As shown in Table 1, several *in vitro* studies have been conducted to search for new natural substances with schistosomicidal activity. These natural products and natural productderived compounds mostly come from plants. Extensive phytochemical investigations of many species have revealed the presence of a large number of novel compounds belonging to different classes (Kayser et al., 2003; Kato & Furlan, 2007; Parmar et al., 1997; Prassad et al., 2005). For example, various secondary metabolites have been isolated from the family Piperaceae, and these plants have generated great interest as a result of their biologically active metabolites, such as pyrones, terpenes, lactones, chromenes, chalcones, lignoids, amides, and alkaloids (Kato & Furlan, 2007; Parmar et al., 1997). Regarding the variety of biological properties in particular, Moraes et al. (2011) demonstrated the *in vitro* schistosomicidal activity of piplartine, an amide found in several *Piper* species. The authors showed that at low concentrations (9.5 µM) this amide can kill *S. mansoni* adult worms (male and female coupled) and that the sub-lethal concentration of piplartine (6.3 µM) caused a 75% reduction in egg production. Additionally, piplartine was not cytotoxic against mammalian cells when given at concentrations up to three times higher than what is needed for a schistosomicidal effect (31.5 µM). Furthermore, *Piper* species are widely distributed in tropical and subtropical regions of the world, and they are among the most important medicinal plants used in various systems of medicine (Jaramillo & Manos, 2001; Parmar et

Relevant notes

Effective in *S. mansoni*-infected mice (Araújo et al., 2011)

*In vitro* against *S. mansoni* adult worms; reduction in egg-laying; causes alterations on the tegument of worms; not tested on schistosomula; not toxic in mammalian cells (Moraes et al., 2011)

*In vitro* against *S. mansoni* adult worms; reduction in egg-laying; causes alterations on the tegument of worms; not tested on schistosomula; not toxic in mammalian cells (de Moraes et al., 2011)

*In vitro* against *S. mansoni* adult worms; causes alterations on the tegument of worm; not tested on schistosomula; not toxic in mammalian cells (Leite et al., 2011)

*In vitro* against *S. mansoni* adult worms; not tested on schistosomula (Ferreira et al., 2011)

Date \*

2011

2011

2011

2011

2011

Table 1. Continued

Extract/ Compound and Biological Source

Sulfated polysaccharide α-D-glucan extracted from lichen *Ramalina celastri* (Spreng.) Krog. & Swinsc

Piplartine, an amide isolated from plant *Piper tuberculatum* Jacq. (Piperaceae)

Dermaseptin 01, an antimicrobial peptide found in the skin of frog of the genus *Phyllomedusa* (Hylidae)

Epiisopiloturin, an alkaloid isolated from plant *Pilocarpus microphyllus* Stapf ex Holm (Rutaceae)

Extract from plants of the *Artemisia* genus (Asteraceae)


Table 1. Continued


Table 1. Continued

338 Current Topics in Tropical Medicine

Relevant notes

*In vitro* male worms seemed more susceptible than female; reduction in egg output; activity against *S. mansoni* in mice was conflicting between Mostafa et al. (2011) and Sanderson et al. (2005); morphological alteration on the tegument; not tested on schistosomula

Effective on *S. mansoni*-infected mice (El-Ansary et al., 2007; El-Banhawey et al., 2007); the *in vitro* schistosomicidal activity of curcumin, the major constituent in the rhizome, and reduction in egg production has been reported (Magalhães et al., 2009)

Active against *S. mansoni* in mice (50 mg/kg) and not effective in high dose (100 mg/kg); affects the development and maturity of *S. mansoni* eggs in mice and seems to be an agent in protecting hepatic tissue against oxidative damage due to *S. mansoni* infection. *In vitro* allicin (2011), the main constituent of garlic, causes alterations on the tegument of male worm in high doses (10 to 20 µg/ml), but toxicity not assessed; not tested on schistosomula (El Shenawy et al., 2008; Lima et al., 2011; Riad et al., 2007)

Effective in *S. mansoni* mice model (Jatsa et al., 2009)

*In vitro* against *S. mansoni* adult worms; reduction in egg-laying; not tested on schistosomula; not toxic in mammalian cells (Braguine et al., 2009)

*In vitro* against *S. mansoni* adult worms; reduction in egg-laying; not tested on schistosomula; not toxic in mammalian cells (Magalhães et al., 2010)

*In vitro* against *S. mansoni* adult worms; reduction in egg-laying; not tested on schistosomula; not toxic in mammalian cell (Parreira et al., 2010)

*In vitro* against *S. mansoni* adult worms; reduction in egg-laying; not tested on schistosomula; not toxic in mammalian cells (de Melo et al., 2011)

Date \*

2005

2007

2009

2009

2010

2010

2011

Table 1. Continued

Extract/ Compound and Biological Source

Extract of rhizomes from the plant *Zingiber ofcinale* Roscoe (Zingiberaceae)

> Extract from plant *Curcuma longa* L. (Zingiberaceae)

<sup>2007</sup>Extract from garlic *Allium sativum* L. (Liliaceae)

> Extract from the plant *Clerodendrum umbellatum* Poir (Verbenaceae)

Extract from the plant *Zanthoxylum naranjillo*  Griseb (Rutaceae) and its isolated compounds

Phloroglucinol compounds from plants of the *Dryopteris* genus (Dryopteridaceae)

Essential oil from the plant of *Baccharis dracunculifolia* DC. (Asteraceae)

Essential oil from plant *Ageratum conyzoides* L. (Asteraceae)

As shown in Table 1, several *in vitro* studies have been conducted to search for new natural substances with schistosomicidal activity. These natural products and natural productderived compounds mostly come from plants. Extensive phytochemical investigations of many species have revealed the presence of a large number of novel compounds belonging to different classes (Kayser et al., 2003; Kato & Furlan, 2007; Parmar et al., 1997; Prassad et al., 2005). For example, various secondary metabolites have been isolated from the family Piperaceae, and these plants have generated great interest as a result of their biologically active metabolites, such as pyrones, terpenes, lactones, chromenes, chalcones, lignoids, amides, and alkaloids (Kato & Furlan, 2007; Parmar et al., 1997). Regarding the variety of biological properties in particular, Moraes et al. (2011) demonstrated the *in vitro* schistosomicidal activity of piplartine, an amide found in several *Piper* species. The authors showed that at low concentrations (9.5 µM) this amide can kill *S. mansoni* adult worms (male and female coupled) and that the sub-lethal concentration of piplartine (6.3 µM) caused a 75% reduction in egg production. Additionally, piplartine was not cytotoxic against mammalian cells when given at concentrations up to three times higher than what is needed for a schistosomicidal effect (31.5 µM). Furthermore, *Piper* species are widely distributed in tropical and subtropical regions of the world, and they are among the most important medicinal plants used in various systems of medicine (Jaramillo & Manos, 2001; Parmar et

Antischistosomal Natural Compounds: Present Challenges for New Drug Screens 341

*In vitro* studies with schistosomula, juvenile and adult worms of *S. mansoni* are frequently used in screening strategies for the discovery of new antischistosomal drugs (Abdulla et al., 2009; Keiser, 2010; Mølgaard et al., 2001; Peak et al., 2010; Ramirez et al., 2007; Smout et al., 2010; Yousif et al., 2007). Parasites at different stages might show differences with regard to drug sensitivity. The *in vitro* methods currently utilised have recently been reviewed, and following the establishment of the *S. mansoni* life cycle in the laboratory, *in vitro* parasite culture techniques were developed (Keiser, 2010; Ramirez et al., 2007). For *in vitro* trials, parasites of different ages are used, such as 3-h-old and 1-, 3-, 5- and 7-day-old schistosomula, 21 day-old juveniles, and 42- to 56-day-old adults. Figure 1 shows the life cycle of *S. mansoni* in the laboratory, illustrating the collection points for *in vitro*

Fig. 1. Life cycle of *S. mansoni*, illustrating the collection points for *in vitro* chemotherapeutic studies. Black arrow: maturation of parasite within final host. Blue arrow: aquatic phase

**4. Parasite culture system** 

chemotherapeutic studies.

al., 1997). In addition to the wide geographical distribution and their use in folk medicine, the interest in these compounds and plant extracts is based on the fact that it is easy to isolate secondary metabolites and to propagate the plant, which has a short reproductive cycle. Thus, considering the *in vitro* schistosomicidal activity of the amide piplartine, the importance of more research on the biological activity of the natural compounds isolated from the family Piperaceae and other plants is apparent.
