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Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology logoLink to Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology
. 2021 Jul 29;46(1):87–102. doi: 10.1007/s12639-021-01417-7

Antischistosomal effects of Ficus carica leaves extract and/or PZQ on Schistosoma mansoni infected mice

Shrouk M A El-Morsy 1, Sayed A M El-Tantawy 1, Eman A El-Shabasy 1,✉
PMCID: PMC8901854  PMID: 35299912

Abstract

Currently, praziquantel (PZQ) is the only drug of choice used for treatment of human schistosomes because of its safety and broad-spectrum activity. It is reported that the repeated chemotherapy is complicated by the occurrence of drug resistance to schistosomiasis. So there is an urgent need to develop new drug combinations therapy. The current study aimed to evaluate antischistosomal activity of F. carica leaves extract alone or in combination with PZQ on Schistosoma mansoni infected mice. Mice were experimentally infected with Schistosoma mansoni and orally administrated 6 weeks’ post-infection with Fig leaves extract and/or PZQ. Schistosoma mansoni (S. mansoni)-infected mice were separated into four groups: untreated (I), treated with PZQ in dose of 200 mg/kg bw (II), treated with Fig leaves extract dose of 400 mg/kg bw (III). Group IV was treated with dose of Fig leaves extract-PZQ as in groups II and III, respectively. The effect was detected parasitologically using ova count technique and oogram pattern in intestine and liver. The greatest antischistosomal effect was achieved using orally administered Fig leaves extract—PZQ as indicated by total worm burden, tissue egg count and oogram pattern. Fig leaves extract + PZQ induced the therapeutic efficacy over the PZQ dose alone in intestine and liver as shown by a complete absence of immature worms, a very high reduction in the total numbers of tissue egg load (59.81% vs. 61.43% & 67.96% vs. 73.46%), mature eggs (37.86 ± 1.4 vs. 34.14 ± 1.9) and increasing in the total number of dead eggs (62.14 ± 1.4vs.67.29 ± 1.76). The results suggested the curcumin in combination with PZQ as a strong schistosomicidal regimen against S. mansoni. In addition, F. carica leaves extract is a promising for PZQ potentiating its antischistosomal action in animal model infected with S. mansoni. Therefore, the present work conclude that combined treatment has a synergetic effect and could be more promising in the management of schistosomiasis.

Keywords: PZQ, Schistosoma, Fig, C57BL/6 mice

Introduction

Schistosomiasis is a parasitic disease arising from infection with blood flukes (trematode worms) of the genus Schistosoma (WHO, 2019). Almost 240 million people worldwide are affected with Schistosomiasis, with over 800 million living in endemic area. (Butrous 2019). At least 5 trematode species are known to infect humans. S. mansoni, S. haematobium, and S. japonicum are the most prevalent species being endemic in worldwide. Disease injury, due to inflammation and fibrosis connected with the parasite's eggs, is usually chronic, and can be painful and debilitating, hampering both personal output and public development (McManus et al. 2018). There is currently no effective vaccine to prevent Schistosoma infection, PZQ is the only available drug used in the treatment and control of schistosomiasis in vitro and in vivo animal and human clinical trials. However, several animal models developed resistance to PZQ after multiple treatments (Shaaban et al. 2019; Mengarda et al. 2020). So, there is an urgent need to develop a new therapeutic strategy for this disease. As a result, there is a growing desire for using plants in therapy instead of synthetic medications, which may have side effects that are even more dangerous than the sickness itself. (Ahmed et al. 2015). In addition, Natural products have historically been regarded as a key role of drug discovery and development (Sharma et al. 2017).

Ficus carica Linn is the most popular member of the genus Ficus, locally known as Anjeer or fig belongs to the family Moraceae. It is generally found in tropical and subtropical regions of the world including southwest Asia and the eastern Mediterranean (Du et al. 2018). Its fruit, have been reported as an important source of sugar, vitamins, organic acid and phenolic compounds, being consumed fresh or dehydrated. Figs showed high amount of fiber, polyphenols and phenolic compounds, such as proanthocyanidins, whereas red wine and tea, which are two good sources of phenolic compounds, contain phenols lower than those in fig (Konyalioglu et al. 2005; Vinson et al. 2005). dried figs are an excellent source of copper, manganese, magnesium, potassium, calcium, and vitamin K (Ahmad et al. 2013). Its fruit, root, and leaves are used in traditional medicine to treat different ailments such as gastrointestinal (colic, indigestion, loss of appetite, and diarrhea), respiratory (sore throats, coughs, and bronchial problems), and cardiovascular disorders and as anti-inflammatory, antispasmodic remedy. In addition, it has hepatoprotective, cytotoxic, hypoglycemic and anthelmintic activity (Khadabadi et al. 2007; Ali et al. 2012; Sharma et al. 2017).

The tegument of schistosomes has been defined as a living, anucleate, and cytoplasmic construction that covers the external surface of the worm (Loukaset al. 2007). The tegument has secretory functions, is involved in the absorption of nutrients, and protects schistosomes from the infected host's immune response (Hellemond et al. 2006; Zhang and Coultas 2013). Scanning electron microscopy (SEM) has become a beneficial device for the survey of the ultrastructural modifications of the surface of the Schistosoma worms in reply to chemotherapy by showing the influence on the tegumental structures (tubercles, spines, intertubercular ridges), oral and ventral suckers (Shaohong et al. 2006).

Scanning electron microscopy (SEM) has become an important method for studying the ultrastructural changes that directly proportional to the potency of antischistosomal drugs (Voge and Bueding 1980) and may explain the process of killing these worms (Hassan et al. 2003). It was suggested that antischistosomal drug induced tegumental damage which might be repaired effectively over the course of 7–14 days after cessation of the drug (Popielet al. 1985). Increased exposure of schistosomal antigens at the parasite surface, as a result of these morphological changes (Harnett and Kusel 1986), followed by losses in the antigenic "disguise" of the worm, the inability to engulf food by oral and ventral suckers and finally in forcing the worms to die (Shaw and Erasmus 1987; Tran et al. 2006; El-Sayad et al. 2017). The main objective of the present work was to study the antischistosomal activity of the extract of Ficus carica leaves and/or PZQ against S. mansoni infected mice.

Materials and methods

Materials

Drug

The Egyptian International Pharmaceutical Industries Company, EIPICO, produced PZQ (PZQ) tablets (Epiquantel). It was administered orally to mice for two consecutive days after six weeks after infection at a dosage of 200 mg / kg body weight.

Plant materials

In the month of August 2018, Ficus carica leaves were collected locally from their natural habitats in Egypt (Delta region). The plant was certified by the Taxonomy professor, Botany section, Science staff, Mansoura University, Mansoura, Egypt.

Ficus carica extract preparation

The leaves were washed numerous times with water and dried for two weeks in the shelter at 25 °C with continuous turning over. In the electric grinder, the dry material was crushed. For ten days with continuous stirring, about 47.23 g of the crushed leaves were soaked in 300 ml of 75% ethanolic alcohol. The sample was filtered with filter paper after ten days. The filtrate was then transferred and condensed, providing 28.2 g of greenish oily crude ethanolic extract (Trease and Evans 1983).

Gas chromatography–mass spectrometry (GC–MS) analysis

The chemical composition of our sample was performed using Trace GC-TSQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG–5MS (30 m × 0.25 mm × 0.25 µ m film thickness). The column oven temperature was initially held at 50 °C and then increased by 5 °C/min to 250 °C hold for 2 min. increased to the final temperature 300 °C by 30 °C/min and hold for 2 min. The injector and MS transfer line temperatures were kept at 270, 260 °C respectively; Helium was used as a carrier gas at a constant flow rate of 1 ml/min. The solvent delay was 4 min and diluted samples of 1 µl were injected automatically using Autosampler AS1300 coupled with GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages over the range of m/z 50–650 in full scan mode. The ion source temperature was set at 200 °C. The components were identified by comparison of their retention times and mass spectra with those of WILEY 09 and NIST 14 mass spectral database (Abd El-Kareem et al. 2016).

Experimental mice and parasites

Infected Biomphalaria alexandrina snails, acquired from Theodor Bilharz Research Institute (TBRI), provided an Egyptian strain of S. mansoni cercariae. The snails were saved in dechlorinated tap water four weeks after infection and then exposed for two hours to artificial light at 28 °C to promote the shedding of cercariae (Tekwu et al. 2017).

For the experiment, black female C57BL/6 mice aged 6–8 weeks, with an average weight of 22–27 g, were used. Mice were acquired in the animal house of the Department of Zoology, Faculty of Science, University of Mansoura, from Misr University of Science & Technology (MUST), Giza, Egypt. Mice were put in wood-chip bedding cages and refreshed every two days. With a 12 h light/dark cycle, they were housed in a temperature-controlled environment.

Animal grouping and mode of treatment

Thirty black female C57BL/6 mice were used in the present study and divided into five groups (six mice per group) as follows:

  1. Control group: Without illness and treatment, this group's mice were healthy.

  2. Infected group: Each mouse of this group was infected subcutaneously with (60 ± 10) newly shed cercariae according to Liang et al. (1987).

  3. Infected PZQ treated group: Mice in this group were treated orally with a full dose of PZQ (200 mg / kg bw) for two consecutive days after six weeks of infection (El-Lakkany et al. 2011).

  4. Infected F treated group: After six weeks post infection, Ficus carica leaves extract (400 mg/kg bw) was orally administered to mice of this group for three days for one week; day after day.

  5. Infected F + PZQ treated group: Mice were orally administered half dose of PZQ drug (200 mg/kg bw) for one day alone then concomitant with F extract (400 mg/kg bw) in the next day.

Methods

Parasitological parameters

Worm recovery

Adult schistosome recovery was evaluated by porto-mesentric perfusion technique accommodated to the method of Duvall and DeWitt (Duvall and Dewitte 1967). Citrate saline (7.5 gm Sodium citrate + 8.5 gm Sodium chloride/L de-ionized water) was used to perfuse the adult worms. Worms have been regained to a clean sieve (previously washed with 70% ethyl alcohol). The perfusion process was ended as the liver, kidney and gut became pale. Worms washed 3 times with Phosphate buffer (pH 7.4): Solution A [NaCl 8.5 gm, KCl0.20 gm, Na2HPO4.12H2O 1.15 &KH2PO40.20 gm in 800 ml Dist. Water], Solution B [CaCl2.2H2O 0.132 gm in 100 ml Dist. Water] and Solution C [MgCl2.6H2O 0.100 gm in 100 ml Dist. Water]. After that, the adult worms were counted as mentioned by (Kamel et al. 1977) and percentage worm reduction was intended by the following formulae termed below and used by Melmanet al. (2009) and Muema et al. (2015).

% Worm Burden Reduction = [(Mean no. of worms from Control group)−(Mean no. of worms from Treated group) × 100%] /[Mean no. of worms from Control group].

Egg count

After scarification of mice, eggs in the liver and intestine were counted, a piece of liver tissue and the small intestine were stored at 20 °C for deposited egg count, where tissues were digested with 4% potassium hydroxide (KOH) as defined by Cheever (1970) and Kamel et al. (1977). Total liver eggs and total intestinal eggs were determined.

Oogram pattern

The pattern assessed degree of ova maturity and viability represented the drug activity that affecting oviposition and maturation. Three parts of the small intestine (each 1 cm in length) were sliced longitudinally after perfusion, washed in saline, partially dried on filter paper and then squeezed between two glass slides. As a rule, in each fragment, one hundred eggs were counted and this was replicated with other fragments until a total of 300 eggs were collected and categorised into three types: immature, mature and dead (Pellegrino et al. 1962).

Scanning electron microscopy examination of the tegument of S. mansoni

S. mansoni, recovered from hepatic and porto-mesentric perfusion, were accumulated in glutaraldehyde buffer solution (25%) that act as a fixative and left overnight at 4 °C.Worms were washed off from fixative residues by being placed in a phosphate buffer overnight at 4° C. After that, increasing concentrations of alcohol (30, 40, & 50%) were used in order to worms passed in for 15 min each. worms were left in 70 percent ethanol, washed twice in 80 & 90 percent ethanol for 30 min, mounted on stainless steel holders, held for about 30 min in a drier, and then exposed to a gold sputtering coat. The portions of Worms were tested using Joel JEM-1200 SEM (Hassan et al. 2003).

Statistical analysis

All statistical studies were behaved using GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, California, USA). Outcomes are offered as mean ± the standard error of the mean (SEM) (n = 6). Statistical contrasts were made by 1-way analysis of variance (ANOVA) succeeded by Neuman-Keuls post-hoc test (Armitage et al. 2008).

Results

Gas chromatography–mass spectrometry (GC–MS) analysis

As reported in Table 1, The volatile components of Ficus carica fruits were extracted by the three extraction methods such as SPME, SD and SE, and then analyzed by GC–MS. Ten volatile compounds were identified by SPME-GC/MS in commercial Egyptian figs. From the results presented herein, the most abundant volatile compounds in fig headspace were amino acids (N-Acetylglycine and L-Norvaline), saturated fatty acids (Tetradecanoic acid and Hexadecanoic acid), unsaturated fatty acids (9,12-Octadecenoic acid, 9-Octadecadienoic acid and Linolenic acid), diterpene alcohol (phytol), phenolic compound (2,3-Dihydroxynaphthoic acid) and alkaloid (2,6-Dimethyl-N-(2-methyl-α -phenylbenzyl) aniline). The current study demonstrated a high content of Hexadecanoic acid and Linolenic acid in fig leaves (Fig. 1).

Table 1.

Listed constituents of F. carica leaves extract sample by (GC–MS) technique

No Compound Name Retention time (RT) Area % Molecular Formula Molecular ion peak Base peak
1 N-Acetylglycine 4.13 3.71 C4H7NO3 117 73
2 L-Norvaline 4.64 0.88 C5H11NO2 117 144
3 Tetradecanoic acid 14.31 0.93 C14H28O2 228 73
4 Hexadecanoic acid 16.77 33.94 C16H32O2 256 73
5 Phytol 17.83 5.64 C20H40O 296 143
6 2,3-Dihydroxynaphthoic acid 18.32 2.5 C11H8O4 204 186
7 9-Octadecadienoic acid 18.95 2.31 C18H34O2 282 73
8 9,12-Octadecenoic acid 19.04 13.15 C18H32O2 280 75
9 Linolenic acid 19.30 25.83 C18H30O2 278 73
10 2,6-Dimethyl-N-(2-methyl-α -phenylbenzyl) aniline 23.77 3.25 C22H23N 301 149
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Fig. 1.

Fig. 1

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Constituents of F. carica leaves extract analyzed by GC/MS technique

Effect of F. carica leaves extract with/without PZQ treatment on tissue egg load in mice infected with S. mansoni.

Reduction in total worm burden in Ficus carica leaves extract treated group was statically significant with 72.6% reduction compared to the control group. On the other hand, S. mansoni infected and treated groups with Ficus carica leaves extract accompanied with PZQ or PZQ only showed extremely significant reduction in total worm burden that reached 100% in worm burden. Generally, S. mansoni was more affected by drugs. They died when treated with PZQ alone, or combined with Ficus carica leaves extract as shown in Table 2 (Fig. 2).

Table 2.

Effect of F. carica leaves extract with/without PZQ treatment on worm burden in mice infected with S. mansoni

Group Worm burden
Male Female Couple Total Reduction %
Infected 1.83 ± 0.79 0.5 ± 0.34 4 ± 0.68 17 ± 1.58 –
Infected + PZQ 0 ± 0 0 ± 0 0 ± 0 0 ± 0 100
Infected + F 1.66 ± 1.11 0 ± 0 3.16 ± 0.87 4.66 ± 1.42 72.6
Infected + F + PZQ 0 ± 0 0 ± 0 0 ± 0 0 ± 0 100
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Results are presented as means ± SE and % of change (n = 6 for each group), (**) % of change related to infected group

b: significant as compared with infected group. PZQ: PZQ F: Ficuss carica

Fig. 2.

Fig. 2

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Mean worm burden

Reduced tissue egg load by different treated groups was reflected histopathologically on the hepatic and intestinal granulomas number. Highest percentage of reduction in intestinal and hepatic tissues was recorded with combined treatment group (73.46% & 61.43%) followed by PZQ treated group (67.96% & 59.81%), and then Ficus carica leaves extract treated group (55.19 & 38.88%) respectively as compared with control infected non treated one with significant difference as shown in Table 3. Collectively, Combined treatment by Ficus carica leaves extract and PZQ induced a significant reduction in the egg count (Fig. 3).

Table 3.

Effect of F. carica leaves extract with/without PZQ treatment on tissue egg load in mice infected with S. mansoni

Mice groups Tissue egg load/gm × 103
Hepatic tissue Intestinal tissue
Egg load Reduction % Egg load Reduction %
Infected 6872 ± 329.6 – 4491 ± 298.4 –
Infected + PZQ 2202 ± 308.3 67.96 1805 ± 332.9 59.81
Infected + F 3079 ± 361 55.19 2745 ± 221.7 38.88
Infected + F + PZQ 1824 ± 309.3 73.46 1732 ± 298.6 61.43
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Results are presented as means ± SE and % of change (n = 6 for each group), (**) % of change related to infected group. b: significant as compared with infected group. PZQ: PZQ F: Ficuss carica

Fig. 3.

Fig. 3

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Number of ova / gm

F. carica leaves extract alone induced a significant decrease in immature and mature eggs in liver and intestine ((40.0 ± 1.52& 36.57 ± 1.45), (40.0 ± 1.52& 36.57 ± 1.45)) respectively and a non-significant difference in numbers of dead ova (5 ± 0.68) as compared to untreated infected one. The complete absence of immature and maximum reduction of mature eggs and dead ova were obtained in the 4th group which was treated with a mixture of the F. carica leaves extract and PZQ ((0.0 ± 0.0&0.0 ± 0.0), (34.14 ± 1.9 & 34.14 ± 1.9), (67.29 ± 1.76&67.29 ± 1.76)) respectively as shown in Table 4 (Fig. 4).

Table 4.

Effect of F. carica leaves extract with/without PZQ treatment on Oogram pattern in mice infected with S. mansoni

Mice groups Oogram pattern (%)
Immature ova Mature ova Dead ova
Infected 49.22 ± 1.03 43.29 ± 1.14 6.22 ± 0.46
Infected + PZQ 0 ± 0 37.86 ± 1.4 62.14 ± 1.4
Infected + F 40 ± 1.52 36.67 ± 1.45 5 ± 0.68
Infected + F + PZQ 0 ± 0 34.14 ± 1.9 67.29 ± 1.76
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Results are presented as means ± SE and % of change (**) % of change related to infected group. PZQ: PZQ F: Ficus carica

Fig. 4.

Fig. 4

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% Egg developmental stages

The surface topography of S. mansoni from the infected untreated group

The male of S. mansoni (adult) was thicker, cylindrical & shorter than the female (adult). The male had a ventral longitudinal cleft; gynaecophoral canal (GC) where the female was holed (Fig. 5a). The oral sucker (OS) was oval and had three distinguished regions; an outer narrow rim (Rm), another inner large zone (IZ), both covered with spines and the mouth opening (MO) (Fig. 5b, c). The ventral sucker (VS) was relaxed with spines. It composed of rim (Rm), inner zone (IZ) and central zone (CZ) (Fig. 5d). The surface between oral and ventral suckers was normal showing hemispherical papillae (HP) and dome papillae (Fig. 5e). The dorsal tegumental surface of the first third region showed normal tubercles (Tu) with (60–65) spines. There were hemispherical papillae (HP) and macule papillae (MaP) (Fig. 5f).

Fig. 5.

Fig. 5

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Micrographs of adult male S. mansoni from infected untreated group: a Paired male (M) and female (F). Note the female emerges from the gynaecophoral canal (GC). b Top view showing normal oral and ventral (OS& VS) suckers. Note the anterior end of female emerges from the gynaecophoral canal. c Top view of anterior region showing oval oral sucker (OS) with mouth opening (MO) and ventral sucker (VS). d The ventral sucker showing rim, inner zone and central zone (Rm, IZ& CZ). e Magnified view of the surface between the oral sucker (OS) and ventral sucker (VS) showing dome papillae (DP) and hemispherical papillae (HP). f Magnified dorsal surface of the first third region showing normal tubercles (Tu). Note macule and hemispherical papillae (Ma& HP) among the tubercles. g Dorsal tegument of middle third region with large numerous and spiny tubercles (Tu). Note presence of dome papillae (DP). h Highly magnified view of the dorsal surface showing two normal spiny tubercles (Tu) and created papillae (CrP)

The tegument in the middle region dorsally composed of normal spiny tubercles (Tu) and showed dome papillae (DP) (Fig. 5g). The right edge of the canal showed normal spiny tubercles (Tu) with hemispherical and macule papillae (HP& MaP respectively) among the tubercles (Figs. 6a, b). Moreover, the left edge of the canal composed of tegumental surface with normal spiny tubercles (Tu) and showed squarish (SP) and macule papillae (MaP) (Fig. 6c). In the posterior region of the body, the two lateral edges of the canal and the inner surface of it showed numerous spines (S). There were numerous hemispherical papillae (HP) beside spines. The tegumental surface surrounded the excretory pore (EP) was smooth and had sensory papillae (Fig. 6d).

Fig. 6.

Fig. 6

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Micrographs of adult male S. mansoni from infected untreated group: a Magnified view of the gynaecophoral canal (right side) showing spiny tubercles (Tu) and macule papillae (MaP) among them. b Highly magnification of the gynaecophoral canal (right side) showing hemispherical and dome shape papillae (HP& DP) among the tubercles (Tu). c Magnified view of the left edge of the gynaecophoral canal with macule and squarish papillae (MaP& SP) between the tubercles (Tu). d Magnification view of ventral surface of the end region showing opened gynaecophoral canal with numerous spines (S) and excretory pore (EP). Note presence of numerous hemispherical papillae (HP)

In female, the lateral surface of the body region after the suckers showed tegumental ridges. There were hemispherical (HP) and macule papillae (MaP) were appeared (Fig. 7a). While the dorsal surface of the first third region showed the normal architecture and there were macule papillae (MaP) (Fig. 7b).Ventrally, There were minute spines, hemispherical and macule papillae (HP& MaP) among them were appeared in the middle region after the suckers (Fig. 7c). The ventral surface of the middle region showed normal architecture of the tegument with small number of spines. Also, Digital (DiP), macule papillae (MaP) and hemispherical papillae (HP) were observed (Fig. 7d).The ventral surface from the begining of the last third region until near the end of the body showed gradually increase in spines (S). There were hemispherical and macule papillae (HP& MaP) were recorded (Figs. 7e, f). In the surface of the end region, there were numerous spines with the appearance of hemispherical, macule papillae (HP& MaP) and dome papillae papillae (DP) (Fig. 7g).

Fig. 7.

Fig. 7

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Micrographs of adult female S. mansoni from infected untreated group: a Lateral surface of the region after the two suckers showing tegumental ridges. Note presence of hemispherical and macule papillae (HP& MaP). b Magnified view of the dorsal surface of the first third region of female showing normal architecture. Note presence of macule papillae (MaP). c Ventral surface of the middle region after the suckers showing hemispherical papillae and macule papillae (HP& MaP). d Magnified ventral view of the middle region showing some minute spines. Note presence of macule, digital and hemispherical papillae (MaP, DiP& HP). e Magnified view of the ventral surface from the beginning of last third region until near the end of the body showing gradually increase of spines (S). Note presence of hemispherical and macule papillae (HP& MaP). f Magnified ventral view of the last third region of female showing numerous spines (S). Note presence of macule papillae (MaP). g End region of the body showing numerous spines. Note presence of hemispherical, macule papillae and dome papillae (HP, MaP& DP)

The surface topography of male S. mansoni worms collected from mice treated with Ficus carica leaves extract (400 mg/kg bw) (F group)

Two weeks after treatment with 400 mg/kg of F. carica leaves extract infected with S. mansoni. All adult males and females examined showed clear changes in shape. Adult male worms were characterized by intensive gross contraction and swelling (Fig. 8a). In the anterior region of the body, the oral and ventral suckers were swelling (Fig. 8b, c, d). The surface between oral and ventral suckers was pitting (Fig. 8c, d). In another specimens, It was flattened in lateral edges appeared like shoulders (Fig. 8e). In the dorsal surface of the sucker’s region sever damage were seen; there was shrinking of the tegument with naked tubercles (Tu), swelling with high loss of tubercles and presence of erosion (E) and peeling (P) (Fig. 8f). Dorsally, there were some nearly naked tubercles (Tu) and presence of blebs (Be) between them (Fig. 8g, h). While others were lost all their spines (Fig. 9a).

Fig. 8.

Fig. 8

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Scanning electron micrographs of adult male S. mansoni from F. carica leaves extract (400 mg/kg) treated group (F group): a Whole mount of male (M). Note contraction and swelling of the body. b The anterior region with swelling of both oral and ventral suckers (OS& VS). c Magnified view of the swollen oral sucker with mouth opening (MO). Note presence of macule papillae (MaP). d Magnified view of the swollen ventral sucker (VS) with extremely damage of the normal architecture. Note presence of macule papillae (MaP). e The anterior region of male showing flattened in lateral edges appeared like shoulders with appearance of macule papillae (MaP). Note deformation of oral and ventral suckers (OS& VS). f Dorsal surface of suckers region showing deformation of the normal architecture. Note shrinking of the tegument with naked tubercles (Tu), erosion (E) and peeling (P). g Dorsal surface of male showing some tubercles (Tu) were nearly naked. Note presence of blebs (Be) and dome papillae (DP) among the tubercles. Highly magnified view of two swollen tubercles (Tu)

Fig. 9.

Fig. 9

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Scanning electron micrographs of adult male S. mansoni from F. carica leaves extract (400 mg/kg) treated group (F group): a Magnified view of another specimen showing naked tubercles (Tu). Note presence of dome shape papillae (DP) among the tubercles. b Lateroventral view of right edge of the gynaecophoral canal showing deformed tegument with peeling (P). Note presence of some partially naked tubercles (Tu). c The region near the end of the right edge of gynaecophoral canal showing swollen tegument with peeling (P). Note presence of macule papillae (MaP). d The left edge of the gynaecophoral canal at the third middle region of male showing deformed tubercles. Note presence of erosion (E), peeling (P) and presence of macule papillae (MaP). e Ventral view of the third middle region of the male showing swollen naked tubercles (Tu). Note presence of erosion (E). f Highly magnified view of the tegument showing peeling and erosion between tubercles. The end region of the body showing alternation of the tegumental architecture. Note presence of peeling (P)

The lateroventral surface of the right edge of the gynaecophoric canal was deformed with peeling (P) and some partially naked tubercles (Tu) (Fig. 9b). Nearly the end of the right edge of the canal, showed there was swelling and peeling (P) with high loss of tubercles (Fig. 9c). The left edge of the gynaecophoric canal of the third middle region was showing deformed tubercles and tegumental erosion (E) and peeling (P) were presented (Fig. 9d). The ventral surface of the 3rd middle region was swelling and presence of swollen tubercles (Tu) without spines (Fig. 9e). Moreover, in high magnification there were peeling (P) and erosion (E) between tubercles (Tu) in some regions (Fig. 9f). Severe damage to the end region of the body was observed; alternation of tegumental architecture with peeling (P) of the tegument and also disappear of tubercles. The tegumental tubercles (Tu) were appeared in irregular rows with loss of spines number (Fig. 9g). In other specimens, the tegument showed surface invagination with peeling (P) and the spines were appeared submerged (Fig. 9h).

In female worms, there were also intensive contractions, shortening of the body (Fig. 10a). The anterior body region (ventrally) was showing shrinked ventral sucker (VS) that pulled to inside and tegumental corrugation between the oral (OS) and ventral (VS) suckers with appearance of hemispherical sensory papillae (HP) (Fig. 10c). The dorsal surface of the sucker’s region was showing highly shrinkage, invagination and peeling (P) (Fig. 10d, e). Tegument in the dorsal surface of the first third region was showing extremely erosion (E) and peeling (P) (Fig. 10b). There was pitted tegumental surface with peeling (P) and invagination (Fig. 10f). In the ventral view of the middle region, there were corrosive tegumental surface, erosion (E) and peeling (P) (Fig. 10g). The tegument in the lateral surface of the last third region was showing high invagination, swelling and peeling (P) (Fig. 10h).

Fig. 10.

Fig. 10

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Scanning electron micrographs of adult female S. mansoni from F. carica leaves extract (400 mg/kg) treated group (F group): a Whole mount of female (F) with contraction and shortening of the body. b Dorsal view of the first third region showing highly deformed tegumental surface due to shrinkage and wrinkling. Note presence of erosion (E) and peeling (P). c Ventral view of the anterior region of the body showing deformed oral sucker (OS) with mouth opening and contracted ventral sucker (VS). Note presence of hemispherical papillae (HP). d Dorsal view of suckers region showing high shrinkage, invagination and peeling (p). Note presence of macule and squarish papillae (MaP& SP). e Magnified view of (d) showing high shrinking. Note presence of large crack and deformed tegumental folds. f Magnified dorsal view of middle region showing peeling (P), corrugation and deformation of the tegumental surface. Note presence of macule and created papillae (MaP& CrP). g Ventral surface of middle region showing pitted tegument with erosion (E) and peeling (P). h Lateral view of last third region showing high invagination, peeling (P) and corrugation that lead to deformation of the tegumental architecture. Note presence of hemispherical and Macule papillae (HP& Map)

Discussion

Even if PZQ is the drug of choice for human schistosomiasis treatment, its therapy can often fail due to potential drug resistance (Melman et al. 2009; Zhang and Coultas 2013; Shaaban et al. 2019). So, there is a necessity for continued research on the way to emerging different chemotherapeutic compounds against schistosomiasis.

Plants are potential causes of novel drugs and many plants have examined for possible antiparasitic properties (Melo et al. 2015; El-Garawani et al. 2016; Shaaban et al. 2019). In the current study, Ficus carica leaves extract was used in order to examine its potential role either alone or in combination with PZQ on treatment of S. mansoni infection. The anti-schistosomicidal effect of F. carica hasn't been studied in many researches, to the best of our knowledge. Only one reported the schistosomicidal activity of F. carica leaves extract on adult S. mansoni worms in vitro (Reda et al. 2016) while its anti-schistosomal activity in an animal host has not been reported. Different extracts (methanolic and aqueous) of leaves of Ficus carica Linn. against Pheritima posthuma in in-vitro anthelmintic were studied. Each extract was studied at 20 mg/ml in the bioassay, which determined the time of paralysis were (3.25 and 2.59 min) respectively, whereas time of death of the worms were (8.01 and 6.36 min) respectively (Patil Amo et al. 2010).

Data obtained in this work showed a significant reduction in total worm burden (72.6%) in group infected and treated with Ficus carica leaves extract as compared to control group. This percentage agreed with Abououf et al. (2018), Awad et al. (2018), Shaaban et al. (2019), Jatsa et al. (2019), Abualmaaty et al. (2020) and Mengarda et al. (2020) who have treated S. mansoni infected mice with (Nigella Sativa oil, Justicia spicigera ethanol extract, Crocus sativus aqueous extract (saffron), Ozoroa pulcherrima Schweinf methanolic extract, Allium sativum extract (allicin) and piplartine) respectively. In this study, there was a significant reduction in egg density in liver (55.19%) and intestine (38.88%) as compared to control. This result disagreed with Aboueldahab and Elhussieny (2016) who have treated with (Curcuma longa) extract and Abualmaaty et al. (2020) but agreed with Shaaban et al. (2019). In the current work, the plant extract had no improved effect on dead ova (5 ± 0.698) as compared to infected untreated group. However, there was a slight significant reduction in mature ova (36.67 ± 1.45) and immature ova (40 ± 1.52). This data may be attributed to the anthelmintic activity of F. carica leaves extract. The GC-mass analysis of the extract exposed the existence of many phytochemical compounds which are directly active against helminthes, altering their viability, mobility and fecundity (Miranda et al. 2012; Kadry 2013; Jatsa et al. 2019).

Our outcomes exposed that; compared with the control-infected population, PZQ (200 mg kg) is still significantly very effective as an anti-schistosomal drug, as confirmed by the complete absence of adult worms from infected mice. This outcome correlates with that recorded after PZQ (300 mg kg) treatment (Shaaban et al. 2019). On the other hand, Aboueldahab and Elhussieny (2016) confirmed that male and female worms were still alive after PZQ (250 mg kg) treatment and PZQ did not display full worm eradication. Compared to infected untreated mice, the count of intestinal and liver ova was significantly contracted in PZQ controlled mice (59.81% and 67.96%) respectively. This remark was also told by Ahmed et al. (2015), Eltaweel et al. (2016) and Shaaban et al. (2019). In addition, no immature ova with a decline in mature ova and a marked rise in dead ova were observed in the PZQ treated group relative to the infected control group. This outcome agreed with Botros et al. (2004); El-kady et al. (2019) and Shaaban et al. (2019).

In the present study, F. carica leaves extract proved to have a non-antagonistic effect on PZQ. This was evident in group treated by combined therapy as complete absence of the adult worms from infected mice when compared with mice treated by monotherapy. This observation went with (Hussein et al. 2017) who have treated infected mice with Curcuma longa and Shaaban et al. (2019). On the other, Aboueldahab and Elhussieny (2016), Abououf et al. (2018) and El-Hawary et al. (2018) reported that after combined treatment male and female worms were still alive. Their plants extract (Curcuma longa extract, Nigella Sativa oil and both Capparis spinosa L. and silymarin) respectively with PZQ were not show complete eradication of worms. Moreover, our observation related to reduction of hepatic and intestinal ova count with combined treatment agreed with Abououf et al. (2018) and Aboueldahab and Elhussieny (2016). F. carica leaves extract combined with PZQ showed the highest reduction effect on liver egg count by 73% that is highly increased to 61% reduction in intestinal egg count. There is a clear transfer of worms to the liver and this may be attributed to the worm's failure to go to its usual portal vein route (Aboueldahab and Elhussieny 2016; Abououf et al. 2018; El-Hawary et al. 2018). In the combination therapy treated group, F. carica leaves extract support the effect of PZQ, as there was full disappearance in immature ova. Moreover, it showed the highest significant rise in the percentage of dead ova and it significantly reduced the percentage of mature ova. This consequence agreed with Hussein et al. (2017), Abououf et al. (2018) and El-Hawary et al. (2018).

Many functions and traits of S. mansoni tegument increased the interest of examining it. Since, it is involved in the intake of nutrients, metabolite secretion and osmotic equilibrium (Rochaet al. 2016). Moreover, development of novel medicines needs a complete knowledge of tegumental components. Therefore, the necessity of researching the schistosome tegument emerges because it serves in its host as a mediator between the parasite and the intravascular environment (El-Shabasy et al. 2015; Kamel and El-Shinnawy 2015). In the study of the ultrastructural alternations on the surface of Schistosoma worms in response to chemotherapy. Scanning electron microscopy has been a helpful tool in illustrating the most alternations on the tegument structures (tubercles, spines and inter-tubercular ridges), oral and ventral suckers. Such ultrastructural damage is directly proportional to the effectiveness of these treatments and may describe the killing mechanism of these worms (El-Taweel et al. 2016). Such ultrastructural damage is directly proportional to the effectiveness of these treatments and may describe the killing mechanism of these worms (El-Taweel et al. 2016).

A number of SEM researches have already established on the tegumental surface of typical S. mansoni (adult) (Miller et al. 1972; Hockley 1973; Hockley and McLaren 1973; Reda et al. 2012, 2019). Spines are extensively scattered in the present study on the oral sucker, ventral sucker, gynaecophoric canal. Moreover, along the tegumental body surface of S. mansoni male and female (some regions) (Kohn et al. 1982; Machado-Silva et al. 1997; Reda et al. 2019). The spines functions are accountable for scraping (attachment to) the tissues of blood vessel for nutrition. In addition to that, the sensory papillae allow oral sucker to choose the suitable site for attachment (Reda and El-Shabasy 2016; Reda et al. 2019). The sensory papillae distribution; on the oral sucker was in agreement with Reda et al. (2019). The structural features of male S. mansoni in control group remained normal and were similar to that in previous studies (Aboueldahab and Elhussieny 2016; Rochaet al. 2016; Reda and El-Shabasy 2016; Silva et al. 2017; Mengarda et al. 2020). The tubercles spines help in adaption to survive in the blood vessels. The porous nature of the surface (regular& irregular pores) leads to increase the surface area. Also, the tegumental absorptive capacity could be increase due to these pores. Female’s tegument (ventrally) shows numerous sensory papillae (larger). For more sensitive directional capabilities, these receptors could provide (Reda et al. 2019).

Examination of adult schistosomes collected from the treated group of F. carica leaves extract revealed noticeable tegumental damage in male and female worms of S. mansoni. Due to the intravascular value of suckers, both male suckers in the current study suffered from tegumental denaturation with internal swelling. Fakahany et al. (2014); Hassan et al. (2016); José et al. (2016); Rocha et al. (2017); Abualmaaty et al. (2020) agreed with this change. Male worms exhibited variations in the tubercles, i.e. reduced in size, collapsed, wrinkled and presented partially or completely spine loss. In addition, encircling tubercles by blebs were detected. Moreover, the tegument lost its normal appearance with shrinking, sharp peeling and erosion. Comparable tegumental damages have been reported for various natural products with antischistosomal activity (Godinho et al. 2014; Kamel and El-Shinnawy 2015; Quelemes et al. 2015; Aboueldahab and Elhussieny 2016; Rocha et al. 2016; Shaaban et al. 2019; Mengarda et al. 2020).

In the present study, female of F. carica leaves extract treated group suffered from shrinked ventral sucker. Moreover, there was corrosive tegumental surface, erosion and peeling. This result agreed with that reported in the study of the same plant extract in vitro (Reda et al. 2016). The alternation of ventral and oral sucker might lead to a lack of the ability to adhere to blood vessels, so it would be more difficult to absorb nutrients from the blood. The damage to the tegument along the body of the worm may have affected the tegument's functions and damaged the worm's defensive mechanism. Thus, the host's immune system could easily strike it (Xiao et al. 2000; Kamel and El-Shinnawy 2015; Shaaban et al. 2019).

Acknowledgements

Many thanks to dr. Waleed Kamel, PhD, School of medicine, Keio University, Tokyo, Japan for revision of our paper (language and grammer).

Author contributions

Prof. SAE participated in the design of the study and reviewed the final editing of the manuscript. Associate Prof. EAE conceived of the study; participated in its design, getting the worms, preparation process and helped to draft & final revision of the manuscript. SME performed the preparation process of worms and participated in the photographing. All authors read and approved the final manuscript.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All deals with animals in this study were carried out according to international valid guidelines of experimental animal studies and research protocol was approved by the local ethical committee of the faculty of Science, Mansoura University with code number Sci-Z-M-2021-33.

Footnotes

Publisher's Note

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Contributor Information

Shrouk M. A. El-Morsy, Email: mahmoudshrouk634@gmail.com

Sayed A. M. El-Tantawy, Email: el-tantawy51@mans.edu.eg

Eman A. El-Shabasy, Email: emanahmah@gmail.com

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