In Vitro Assessment of the Cercaricidal Activity of Sida acuta Burm. F. and Sida rhombifolia Linn. (Malvaceae) Hydroethanolic Extracts, Cytotoxicity, and Phytochemical Studies

Despite the global efforts, schistosomiasis remains a public health problem in several tropical and subtropical countries. One of the major challenges in the fight against schistosomiasis is the interruption of the parasite life cycle. Here, we evaluated the anticercarial, cytotoxicity, and phytochemical profiles of Sida acuta (HESa) and Sida rhombifolia (HESr) hydroethanolic extracts (Malvaceae). Schistosoma mansoni cercaria was collected from fifteen Biomphalaria pfeifferi-infected snails. Twenty-five cercariae were incubated in duplicate with different concentrations (31.25–1,000 μg/mL) of HESa or HESr. The cercaria viability was monitored at 30 min time intervals for 150 min, and the concentration-response curve of each plant extract was used to determine their respective lethal concentration 50 (LC50). Additionally, the cytotoxicity profile of each plant extract was evaluated on the Hepa 1–6 cell line at a concentration range of 15.625–1,000 µg/mL using the WST-8 assay method and its inhibitory concentration 50 (IC50) was calculated. Moreover, phytochemical characterization of each plant extract was carried out by HPLC-MS. Both extracts exhibited cercaricidal activity in a time- and concentration-dependent manner. At 30 min time point, HESa (LC50 = 28.41 ± 3.5 µg/mL) was more effective than HESr (LC50 = 172.42 ± 26.16 µg/mL) in killing S. mansoni cercariae. Regarding the cytotoxicity effect of both extracts, the IC50 of HESa (IC50 = 109.67 µg/mL) was lower than that of HESr (IC50 = 888.79 µg/mL). The selectivity index was 3.86 and 5.15 for HESa and HESr, respectively. Fifteen compounds were identified from HESa and HESr after HPLC-MS analysis. N-Feruloyltyramine, a polyphenol, and thamnosmonin, a coumarin, were identified in both extracts. HESa and HESr displayed cercaricidal activity and were not toxic on Hepa 1–6 cell line. Based on the selectivity index of these extracts, S. rhombifolia extract could be more effective on S. mansoni cercariae than S. acuta extract. This study could provide baseline information for further investigations aiming to develop plant-based alternative drugs against S. mansoni.


Introduction
Schistosomiasis, also known as bilharzia, is an infectious disease caused by trematodes flatworms of the genus Schistosoma. It is known as one of the most prevalent tropical diseases worldwide. It is estimated that 229 million people required preventive treatment in 2018 and close to 800 million are at risk of infection [1]. After an infected person releases Schistosoma eggs into the water by defecation or urination, the ripe miracidia hatch out and invade the intermediate host freshwater snail where they form sporocysts. ey then matured to give cercariae that emerges from snails and swims to penetrate the skin of humans and/or animals that are definitive hosts [2]. erefore, to control schistosomiasis, the life cycle of the parasite can be interrupted by killing cercariae and miracidia [3][4][5]. e use of chemical compounds to control the aquatic snails, cercariae, or miracidia is not recommended because of their adverse effects on the environment [6]. A large number of plant families with potential schistosomicidal activity have been identified through plant screening, which represents a continuous effort to find new bioactive molecules. e anticercarial activities of several plants such as Rauwolfia vomitoria [7], Nigella sativa [8] Balanites aegyptica, Azadirachta indica, Nauclea latifolia, Morinda lucida, Phyllanthus amarus, Vernonia amygdalina [9], and Ozoroa pulcherrima [10] have been reported. In several countries, Sida L. (Malvaceae) has been used for centuries in traditional medicines for the prevention and treatment of different diseases such as diarrhea, dysentery, gastrointestinal and urinary infections, cancer, malaria, and helminth infections [11][12][13][14][15][16][17][18]. Moreover, during the last decade, Jatsa et al. [19][20][21] have demonstrated the in vitro and in vivo anti-schistosomal potency of Sida pilosa. In addition, previous investigations on two of the plants of the genus Sida, namely Sida acuta and Sida rhombifolia, reported their anthelmintic and larvicidal activities [22][23][24]. Considering the above, this study was therefore carried out to evaluate the in vitro cercaricidal activity of S. acuta and S. rhombifolia hydroethanolic extracts against Schistosoma mansoni cercaria, their cytotoxicity profile, and determine their phytoconstituents.

Collection, Preparation, and Extraction Process of Plant
Specimens.
e whole plant of S. acuta (Figure 1(a)) was collected in February 2017 at Nkoemvone near Ebolowa in the South Region while the aerial parts of S. rhombifolia (Figure 1(b)) were harvested in March 2017 at Bazou, a village of Ndé division in the West region of Cameroon. e plant specimens were identified and authenticated at Cameroon's National Herbarium under a voucher specimen HNC n°46188 (S. acuta) and HNC n°20113 (S. rhombifolia). Each plant species was air-dried for 2 weeks, and the dried plant materials were ground into powder. Exactly 400 g of each pulverized plant material was macerated with 800 mL of ethanol/water mixture (70/30) at room temperature for 48 h. e macerates were filtered, and the filtrate was concentrated using a rotary evaporator (Buchi Rotavapor, R 200) under reduced pressure at 40°C. e crude extract was finally air-dried, and the yield of obtained hydroethanolic extracts of S. acuta (HESa) and S. rhombifolia (HESr) was calculated.

Snails Infection and Preparation of the Cercarial
Suspension. Schistosome cercariae were obtained from experimentally infected juvenile Biomphalaria pfeifferi snails that were maintained in the snail laboratory at the Centre Schistosomiasis and Parasitology of Yaoundé, Cameroon, under standard laboratory conditions of 18-26°C. Briefly, each snail was exposed for 12 hours to 4-5 miracidia obtained after hatching of eggs isolated from S.-mansoni-infected mice liver. Four weeks later, 14 infected snails, known to be shedding cercariae, were pooled into a glass Beaker with 20 mL of distilled water and allowed to shed cercariae by exposing them to artificial light for 60 min. After the exposition time, snails were removed from the beaker and 100 μL of the cercariae suspension was transferred on three microscope slides. A volume of 100 µL of Lugol was added on each slide to stain and fix cercariae, and the slides were transferred onto a light microscope to estimate the number of cercariae per 100 µL, and the volume of the cercariae suspension containing approximately 25 viable cercariae was determined [7,9].

In Vitro Cercaricidal Assay.
e concentrations ranging from 1,000 to 31.25 µg/mL are widely used to evaluate the cercaricidal activity of medicinal plant crude extracts [7,9]. According to this protocol, serial dilutions (1,000, 500, 250, 125, 62.5, and 31.25 μg/mL) of each of the test plant extract were prepared using distilled water as diluent. A volume of 1 mL of each dilution was added into a well of a 24-well culture plate. Niclosamide-olamide 5% (1 µg/mL; Jiangsu Aijin Agrochemical Co. Ltd., China) was used as a reference control. Approximately 25 cercariae were pipetted into each of the wells. Cercaria mortality was observed for 150 min at 30 min intervals since the infectivity of cercariae is known to be rapidly lost after 12 h [25]. e bottom of each well was observed at 4X magnification using an inverted microscope and cercariae survival and mortality were recorded at specific 30 min time intervals (30,60,90,120, and 150 min). Cercariae were presumed dead when they stopped movement and sank or their tail was detached [26]. All experiments were carried out in duplicate and repeated twice. e viability percentage and the lethal concentration 50 (LC 50 ) values of plant extracts on schistosome cercariae were calculated at each time point.

Cytotoxicity Assay of the Extracts against Hepatocyte Cell
Lines. Cytotoxicity of S. acuta and S. rhombifolia extracts was investigated on C57/L mouse melanoma liver cells line (Hepa 1-6, ATCC CRL-1830) using WST-8-based assay as previously described by Murata et al. [27]. e cells were cultured in high-glucose Dulbecco's Modified Eagle

Qualitative Determination of Compounds Using LC-DAD-(HR) ESI-MS
Sample Preparation. HESa and HESr were dissolved in HPLC grade methanol at a concentration of 5 mg/mL and then filtrated through a syringe-filter-membrane. Aliquots of 5 µL were injected into the UPLC-DAD/MS Dionex Ultimate 3000 HPLC (Germany).

HPLC-MS Conditions.
High-resolution mass spectra were obtained with a quadrupole-time-of-flight (QTOF) spectrometer (Bruker, Germany) equipped with a HESI source. e spectrometer was operated in positive mode (mass range: 100-1,500, with a scan rate of 1.00 Hz) with automatic gain control to provide high-accuracy mass measurements within 0.40 ppm deviation using Na formate as calibrant. e following parameters were used for experiments: spray voltage of 4.5 kV and the capillary temperature of 200°C.

Statistical Analysis.
Graph drawing and statistical analysis were performed using GraphPad Software version 8.01 (GraphPad Software, San Diego, USA). e results were expressed as means ± SEM, and Student's t-test and two-way ANOVA followed by Tukey multiple comparison post-test were used to determine the significance of differences between mean values. A p-value less than 0.05 was considered statistically significant.

Cercaricidal Activity of Plant
Extracts. e mortality of S. mansoni cercariae following in vitro exposure to different concentrations of S. acuta and S. rhombifolia extracts was both time-and concentration-dependent increased. In the absence of the plant extract, cercariae showed normal viability without any morphological changes (tail loss) for up to 2 h (Figures 2(a) and 3(a)).

Cercaricidal Activity of Sida acuta Hydroethanolic
Extract. As shown in Figure 2(b), following 30 min incubation of cercariae with S. acuta hydroethanolic extract with 1,000, 500, or 250 µg/mL, 100% of the cercariae died. At the same time, we recorded mortality rates of 83%, 88%, and 91% of cercariae incubated with 125, 62.5, and 31.25 µg/mL of the hydroethanolic extract of S. acuta, respectively. However, from 60 min to 150 min, we obtained a mortality rate of 100% with all concentrations of the extract. At 30 minutes time point, the cercaricidal activity of HESa at all concentrations was significantly higher (p < 0.01) than the niclosamide-olamide one (Figure 2(b)).

Cercaricidal Activity of Sida rhombifolia Hydroethanolic Extract.
e incubation of cercariae with S. rhombifolia hydroethanolic extract at 1,000, 500, and 250 µg/mL induced after 30 min, mortality rates of 99%, 97%, and 66%, respectively. At the 60 min time point, we observed a mortality rate of 100% from the cercariae incubated with niclosamide-olamide or HESr at 1,000 and 500 µg/mL, while the same rate (100%) was obtained after 90 min and 120 min of incubation respectively with 250 and 125 µg/mL of HESr. e concentrations of 62.5 and 31.25 µg/ mL showed a 100% cercariae mortality rate at the 150 min time point. In addition, at 30 minutes time-point, HESr at 500 and 1,000 µg/mL showed a significantly high cercaricidal activity (p < 0.001) in comparison to niclosamide one (Figure 3(b)). Table 1, the cecaricidal median lethal concentration of S. acuta extract was almost constant from 60 to 150 mins of incubation. Conversely, the LC 50 profile was gradually decreased after cercariae incubation with S. rhombifolia extract. e LC 50 of HESa was then 28.41 ± 3.45 µg/mL and 18.63 ± 0.28 µg/mL after 30 min and 60 min of incubation respectively, while that of HESr were 172.42 ± 26.16 µg/mL and 56.60 ± 4.07 µg/ mL at the same time points. After 150 min of incubation, both extracts disclosed the same LC 50 (18.15 ± 0.00 µg/mL).

Effect of Sida acuta and Sida rhombifolia Hydroethanolic
Extracts on Mouse Hepatic (Hepa 1-6) Cell Growth. As indicated in Figure 4, the results of the effect of HESa and HESr extracts on the growth of the Hepa 1-6 cells at different concentrations (15.625-1,000 µg/mL) showed a decrease in the viability of cells in a concentration-dependent manner after 24 h of incubation. e inhibitory rates of S. acuta and S. rhombifolia extracts are shown in Table 2. At 31.25 µg/mL, the inhibitory rate was 28.69% and 14.78% for HESa and HESr, respectively. e same tendency was observed at 125 µg/mL where, after 24 h of incubation, inhibitory rates of 51.66% and 29.91% were recorded for HESa and HESr, respectively. Furthermore, the incubation of cells with the highest concentration (1,000 µg/mL) shows inhibition rates of 85.69% for HESa and 51.49% for HESr (Table 2). Based on their inhibitory activity on the Hepa 1-6 cells, the IC 50 of HESa was 109.67 ± 6.99 µg/mL, and that of HESr was 888.79 ± 29.94 µg/mL.
Based on their cercaricidal activity and their cytotoxicity, the selectivity index of S. acuta and S. rhombifolia hydroethanolic extracts was calculated. At 30 min post-incubation, this index was 3.86 for HESa and 5.15 for HESr.

Phytochemical Constituents of Sida acuta and Sida rhombifolia Hydroethanolic Extracts.
Qualitative phytochemical analysis of hydroethanolic extracts of S. acuta (HESa) and S. rhombifolia (HESr) revealed the presence of some secondary metabolites such as flavonoids, polyphenols, saponins, alkaloids, tannins, anthocyanins, triterpenes, sterols, anthraquinones, coumarins, and cardiac glycosides (Table 3). e HPLC-MS analysis of S. rhombifolia and S. acuta hydroethanolic extracts confirmed the presence of alkaloids, polyphenols, and flavonoids. e HPLC-MS chromatogram recorded for HESr was composed of peaks of chemical compounds, with retention times below 10 min ( Figure 5) while those of HESa were below 15 min ( Figure 6). e combination of the MS spectral data and the information from the literature allows tentative identification of 23 compounds (numbered 1-23 on the chromatograms).
e peak eluted at 0.34 min (compound 1), corresponding to the molecular ion [M + Na]+ detected at m/z 203 was credited to glucose with a molecular mass of 203.05 ( Figure 5(a)). Compound 2 appears at RT 3.06 min and produced the molecular ion [M + H]+ detected at m/z 219. According to the obtained data, it was possible to infer the molecular mass of 219.09 and identify it as quindoline, an alkaloid ( Figure 5(b)). Compound 3 was observed at RT 3.13 min with a molecular ion [M + Na]+ detected at m/z 503 and was identified as 20-hydroxyecdysone, an ecdysteroid with the molecular mass of 503.29 ( Figure 5(c)). Concerning compound 4 identification, it appeared at RT 3.50 min with a molecular ion [M + H]+ detected at m/z 314 and was identified as the polyphenol, N-feruloyltyramine with 314.13 as molecular mass ( Figure 5(d)). e peak eluted at 4.39 min was allotted to compound 8 with a molecular ion [M + Na]+ at m/z 617 and was identified as tiliroside, a flavonoid with 617.12 as molecular mass ( Figure 5(h)). e analysis of the HeSr spectrum also allowed to identify two coumarins, thamnosmonin (compound 9,  Figure 3: Effect of Sida rhombifolia hydroethanolic extract on Schistosoma mansoni cercariae viability (a) and mortality rate (b). All bars are expressed as mean ± SEM. * p < 0.05 and * * * p < 0.001, significantly different from controls (distilled water). ### p < 0.001, significantly different from reference control (niclosamide).   Table 5).
e chemical structures of all identified compounds from S. acuta and S. rhombifolia hydroethanolic extracts are shown in Figure 7

Discussion
e present investigation showed that hydroethanolic (30: 70) extracts of S. acuta whole plant and S. rhombifolia aerial parts disclosed cercaricidal activity against S. mansoni cercariae in both time-and concentration-dependent manner.
is confirms the larvicidal activity of isolated compounds from S. rhombifolia previously highlighted by Islam et al. [23]. Previous studies conducted by some authors have also reported the cercaricidal activity of Glinus lotoides fruits aqueous extract [29], R. vomitoria stem bark, roots ethanolic extract [7], and O. pulcherrima roots methanolic extract and fractions [10]. Other studies have also reported a time-and concentration-dependent molluscicidal, cercaricidal, and/or schistosomicidal activity of various plant      Evidence-Based Complementary and Alternative Medicine extracts such as A. indica and V. amygdalina [9], Millettia thonningii [30,31], and Jatropha elliptica [32]. Based on the median lethal concentration LC 50 and at any defined period, the cercaricidal activity of S. acuta hydroethanolic extract was more important than that of S. rhombifolia. As compared with results from previous studies, S. acuta and S. rhombifolia hydroethanolic 30:70 extracts were more potent, regarding the LC 50 and the effective time, in killing S. mansoni cercariae than G. lotoides fruits aqueous extract [29], R. vomitoria stem bark and roots ethanolic extracts [7], A. indica and V. amygdalina methanolic extracts [9] but less effective than O. pulcherrima roots methanolic extract [10]. e cercaricidal activity of S. acuta and S. rhombifolia extracts might be due to their secondary metabolites such as flavonoids, alkaloids, and saponins. Abo-Zeid and Shohayeb [8] have shown the anti-miracidial and anti-cercarial activities of total alkaloids, saponins, and volatile oil extracted from N. sativa seeds hydroethanolic extract. It has also been demonstrated that alpinumisoflavone and robustic acid, two isoflavonoids isolated from M. thonningii seeds exhibit in      Evidence-Based Complementary and Alternative Medicine vitro cercaricidal activity [30,31]. Moreover, Dos Santos et al. [32] reported a strong cercaricidal activity of diethyl 4phenyl-2,6-dimethyl-3,5 pyridine dicarboxylate, a pentasubstituted pyridine alkaloid from the rhizome of J. elliptica with an LC 100 of 2 µg/mL. e cercaricidal potential of these secondary metabolites could strongly support the efficacy of S. acuta and S. rhombifolia extracts in killing S. mansoni cercariae. Indeed, phytochemicals belonging to flavonoids, polyphenols, coumarins, alkaloids, and steroids were isolated from S. rhombifolia hydroethanolic 30:70 extract in this study. e cercaricidal activity of S. acuta and S. rhombifolia may be due to their ability to damage the cercariae tegument and disturb its motor activity. Xiao et al. [33] have previously demonstrated that exposure of S. japonicum cercariae to praziquantel results in intensive disturbance in motor activity and lysis of cercarial tissues, followed by an extensive release of gland contents and separation of the tail from the body. ese observations were  Evidence-Based Complementary and Alternative Medicine 13 followed by the cercariae surface damages that are characterized by the decrease of the membranous glycocalyx, the swelling, and the degeneration of mitochondria distributed in the muscle and parenchymal cells as well as the lysis of the tegumental muscular layer. In fact, in our study, the incubation of S. mansoni cercaria with S. acuta and S. rhombifolia hydroethanolic extracts resulted in the separation of their tail from their body. e genus Sida L. is one of the most diverse in the Malvaceae family, with about 200 species scattered in every part of tropical and subtropical regions in the world [18,34,35]. In addition, 142 chemical constituents belonging to various classes have been reported for Sida sp. Alkaloids, flavonoids, and ecdysteroids were predominant and reported mostly from S. acuta, S. cordifolia, S. rhombifolia, S. glutinosa, and S. spinosa. [36]. In our study, alkaloids were the major bioactive principles of S. acuta and S. rhombifolia hydroethanolic 30:70 extracts. Moreover, among the 15 compounds identified in those extracts, only thamnosmonin and xanthyletin (coumarins), and tiliroside (flavonoid) were recently identified in S. rhombifolia hydroethanolic extract [37]. Some of these compounds have been previously identified and/or isolated from the Sida L. genus. In fact, quindoline has been isolated from S. cordifolia and S. rhombifolia [38,39], while 20-hydroxyecdysone has been isolated from S. spinosa [40] S. cordifolia [41], S. tuberculata [42][43][44], and S. acuta [37]. Furthermore, N-feruloyltyramine and di-(2-ethylhexyl) phthalate have been isolated from S. acuta [45] and scoparone from S. rhombifolia [46]. Moreover, Das et al. [47] identified chrysin in S. glutinosa and Silva et al. [48] isolated luteolin from S. galheirensis. Acacetin and kaempferol were isolated from S rhombifolia [38,39], while cryptolepine and cryptolepinone were characterized from S. acuta [38,39]. ese  phytoconstituents exhibited various pharmacological activities such as anti-microbial, anti-plasmodial, vasorelaxant, anti-oxidant, and anti-inflammatory [38,42,44,49,50].
Regarding the cytotoxic activity of both extracts on the growth of the Hepa 1-6 cells, S. acuta extract disclosed the highest anti-proliferative activity than S. rhombifolia. Antiproliferative activity of a plant extract may be linked to its phytochemical constituents. It has been shown that quindoline and cryptolepinone, as well as N-trans-feruloyltyramine identified in S. acuta and S. rhombifolia hydro ethanolic extracts have a significant anti-proliferative activity on mouse hepatoma cells (Hepa 1c1c7) by inhibiting the quinone reductase activity [46].

Conclusions
S. acuta and S. rhombifolia hydroethanolic extracts disclosed cercaricidal activity and were not toxic on Hepa 1-6 cell line. However, Based on the LC 50 , the cercaricidal activity was more pronounced with S. acuta extract. e cercaricidal activity of these plant extracts may be linked to the presence of several secondary metabolite such as alkaloids, flavonoids, and coumarins. Based on their activity and their selectivity index, S. rhombifolia extract could be more effective on S. mansoni cercariae than S. acuta extract. is study could provide baseline information for further investigations aiming to develop plant-based alternative drugs against S. mansoni.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this study.