In vitro and in silico evaluation of the schistosomicidal activity of eugenol derivatives using biochemical, molecular, and morphological tools.

Background: Eugenol shows both antibacterial and antiparasitic activities, suggesting that it might be evaluated as an option for the treatment of praziquantel-resistant schistosome. Methods: The in vitro activities of three eugenol derivatives (FB1, FB4 and FB9) on adult worms from Schistosoma mansoni were examined by fluorescence and scanning electron microscopy to analyze effects on the excretory system and integument damage, respectively. Biochemical tests with verapamil (a calcium channel antagonist) and ouabain (a Na+/K+-ATPase pump inhibitor) were used to characterize eugenol derivative interactions with calcium channels and the Na+/K+-ATPase, while in silico analysis identified potential Na+/K+-ATPase binding sites. Results: The compounds showed effective doses (ED50) of 0.324 mM (FB1), 0.167 mM (FB4), and 0.340 mM (FB9). In addition, FB4 (0.322 mM), which showed the lowest ED50, ED90 and ED100 (p < 0.05), caused the most damage to the excretory system and integument, according to both fluorescence and scanning electron microscopy analysis. The death of adult worms was delayed by ouabain treatment plus FB1 (192 versus 72 hours) and FB9 (192 versus 168 hours), but the response to FB4 was the same in the presence or absence of ouabain. Besides, no changes were noted when all of the eugenol derivatives were combined with verapamil. Moreover, FB1 and FB9 inhibited Na+/K+-ATPase activity according to in silico analysis but FB4 did not show a time-dependent relationship and may act on targets other than the parasite Na+/K+-ATPase. Conclusion: Eugenol derivatives, mainly FB4 when compared to FB1 and FB9, seem to act more effectively on the integument of adult S. mansoni worms.

In vitro and in silico evaluation of the schistosomicidal activity of eugenol derivatives using biochemical, molecular, and morphological tools

Background Eugenol shows both antibacterial and antiparasitic activities, suggesting that it might be evaluated as an option for the treatment of praziquantel-resistant schistosome. Methods The in vitro activities of three eugenol derivatives (FB1, FB4 and FB9) on adult worms from Schistosoma mansoni were examined by fluorescence and scanning electron microscopy to analyze effects on the excretory system and integument damage, respectively. Biochemical tests with verapamil (a calcium channel antagonist) and ouabain (a Na+/K+-ATPase pump inhibitor) were used to characterize eugenol derivative interactions with calcium channels and the Na+/K+-ATPase, while in silico analysis identified potential Na+/K+-ATPase binding sites. Results The compounds showed effective doses (ED50) of 0.324 mM (FB1), 0.167 mM (FB4), and 0.340 mM (FB9). In addition, FB4 (0.322 mM), which showed the lowest ED50, ED90 and ED100 (p < 0.05), caused the most damage to the excretory system and integument, according to both fluorescence and scanning electron microscopy analysis. The death of adult worms was delayed by ouabain treatment plus FB1 (192 versus 72 hours) and FB9 (192 versus 168 hours), but the response to FB4 was the same in the presence or absence of ouabain. Besides, no changes were noted when all of the eugenol derivatives were combined with verapamil. Moreover, FB1 and FB9 inhibited Na+/K+-ATPase activity according to in silico analysis but FB4 did not show a time-dependent relationship and may act on targets other than the parasite Na+/K+-ATPase. Conclusion Eugenol derivatives, mainly FB4 when compared to FB1 and FB9, seem to act more effectively on the integument of adult S. mansoni worms.(AU)

Camu-camu (Myrciaria dubia) seeds as a novel source of bioactive compounds with promising antimalarial and antischistosomicidal properties.

Parasitic diseases have attracted worldwide attention of their consequent impact on mortality and morbidity. Accordingly, several plants have been screened for antiparasitic activity aiming to create new alternatives for treatment. These diseases have been neglected and have not attracted worldwide attention (nowadays), the health concerns are focused in chronic diseases, but it is necessary to focus on parasitic diseases and look for prophylactic alternatives, such as plant extracts. Although camu-camu (Myrciaria dubia) seeds are a rich source of antioxidant antimutagenic, cytotoxic, anti-inflammatory, antimicrobial, antihypertensive and neuroprotective compounds, nothing is known about their antiparasitic effects. Thus, in the present study we aimed to evaluate five extracts of camu-camu seeds (100% water, 100% ethyl alcohol, 50% water + 50% ethyl alcohol, 25% water + 75% ethyl alcohol, and 75% water + 25% ethyl alcohol) in relation to their in vitro antimalarial, antischistosomicidal, leishmanicidal and anti-hemolytic effects. The extracts exhibited antischistosomicidal (ED50 values from 418.4 to >1000.0 µg/mL) and antimalarial activities (IC50 values from 24.2 to 240.8 µg/mL) for both W2 and 3D7 strains in all intra-erythrocytic stages. Correlation analysis showed that the toxic effects may mainly be attributed to methylvescalagin (r = -0.548 to -0.951, p < 0.05) and 2,4-dihydroxybenzoic acid (r = -0.612 to -0.917, p < 0.05) contents. Moreover, the anti-hemolytic effect was associated to methylvescalagin (r = -0.597, p < 0.05). No toxic effects were observed for leishmaniasis and IMR90 normal cells. Herein, methylvescalagin was the bioactive compound of greatest interest once it presented simultaneous relation with antiparasitic and anti-hemolytic activities.

Doxycycline hyclate: A schistosomicidal agent in vitro with immunomodulatory potential on granulomatous inflammation in vivo.

We investigated the effect in vitro and in vivo of doxycycline hyclate (Dx), a broad-spectrum antibiotic inhibitor of matrix metaloproteinases (MMPs), on adult Schistosoma mansoni worms and granulomatous liver inflammation in infected mice. Adult S. mansoni worms in culture treated with different concentrations of Dx (50-180 µg/mL) were studied for eight days to assess its morphology, eggs production, and mortality. Uninfected mice and those infected with S. mansoni, untreated and treated with praziquantel (Pz; 200 mg/kg) or Dx (50 mg/kg), were evaluated for 60 days. Our results indicated that Dx induced dose-dependent integumentary lesions (bubbles, tubercle collapse, spicule disappearance, peeling, and erosion), reduced mating rate and eggs-laying in adult S. mansoni worms. The effective lethal dose required to kill 50% of worms was 112.0 µg/mL Dx (DL50). In mice, S. mansoni infection induced hepatomegaly, intense IL-4, IL-10, TNF-α and TGF-ß production, granulomatous inflammation and hepatic glycogen depletion. The number and size of the granulomas was similar in untreated and Dx-treated animals. Untreated animals showed a predominance of productive granulomas, and intense MMP-2 and MMP-9 activities. Dx-treated mice exhibited a significant increase in IL-4 levels, tissue inflammation, proportion of involutive granulomas, and hepatic collagenogenesis, as well as attenuated MMP-2 and MMP-9 activities. Our findings indicated that Dx is toxic to adult S. mansoni worms in vitro. However, in vitro beneficial effects were not reproduced in vivo, since Dx treatment increased liver granulomatous inflammation and collagenogenesis in S. mansoni-infected mice by a process potentially associated with Dx-mediated hepatic MMP-2 and MMP-9 inhibition.

In vivo schistosomicidal activity of 7-epiclusianone and its quantification in the plasma of healthy and Schistosoma mansoni infected mice using UPLC-MS/MS.

BACKGROUND: The tetraprenylated benzophenone 7-epiclusianone (7-epi) is a substance isolated from the fruits of Garcinia brasiliensis and in vitro studies have demonstrated that 7-epi is effective against Schistosoma mansoni adult worms. HYPOTHESIS/PURPOSE: Here we report the in vivo evaluation of 7-epi and its pharmacokinetic in healthy and Schistosoma mansoni infected mice. STUDY DESIGN AND METHODS: In this work, we assayed the schistosomicidal activity of 7-epi at the dose of 100 mg/kg and 300 mg/kg body weight/day in S. mansoni experimentally infected mice. Besides, two groups of animals were treated and a detailed analysis of plasma samples was performed by liquid chromatography coupled to mass spectrometry (LC-MS/MS). RESULTS: The worm burden showed a reduction in the infected mice after treatment with 300 mg/kg for five days (p < .05). And we found an increase of AUC0-∞ (20846 vs. 32438 ng.h/ml) and a decrease of total apparent clearance (0.006 vs. 0.004 l/h/kg) of 7- epi in the infected group compared to the healthy group. Consequently, the half-life increased (1.73 vs. 6.11 h) and Cmax was reduced (5427.5 vs. 3321.0 ng/ml) in the infected group compared to the healthy group. In addition, histopathological investigations were performed analysing liver samples from healthy and infected mice. CONCLUSION: The results showed significant schistosomicidal in vivo activity at 300 mg/kg. In addition, livers from S. mansoni infected mice showed a greater number of egg granulomas and the changes in the pharmacokinetic parameters in this group could be associated with the pathology of the murine experimental schistosomiasis.

Photodynamic therapy for Schistosoma mansoni: Promising outcomes.

The purpose of this study was to assess, for the very first time, the effects of photodynamic therapy (PDT) on Schistosoma mansoni in vitro by measuring reactive oxygen species (ROS) generation throughout the treatment, as well as the behavior of the parasites (mating, motility and contraction/shortening), and damage to their tegument and excretory systems. The parasites were divided into 4 groups: control, photosensitizer, laser and PDT. Light irradiation was delivered with an InGaAlP low-level laser device operating at 660nm, with 40mW and 100J/cm2. For PDT, different toluidine blue dye (TBO) concentrations and times of exposure were utilized. Interestingly, TBO-mediated PDT was able to kill S. mansoni (P<0.001) due to the significant amount of ROS released that inflicted damages in the tegument and excretory system, as well as contraction and cessation of motility. In addition, males of S. mansoni were shown to be more sensitive to PDT if compared to their corresponding females when the optimal TBO concentration of 31.2µL was considered (P=0.0126). PDT presents two major advantages: not inducing microbial resistance and also lacking adverse effects. Therefore, PDT may become a promising therapeutic alternative for schistosomiasis in the near future, especially for cases of allergy and resistance to praziquantel.

In vitro evaluation of the schistosomicidal effect of the extracts, fractions and major 3-hydroxy-2,6-dialkyl-substituted piperidine alkaloids from the flowers of Senna spectabilis (Fabaceae).

In this work, we present the in vitro schistosomicidal activity evaluation of the most active dichloromethane fraction (FDm) (ED50=83.5µg/mL) and of a mixture of the major alkaloids ((-)-cassine/(-)-spectaline, C/E) (ED50=37.4µg/mL) from the flowers of Senna spectabilis against adult worms and cercariae. We also demonstrate other toxic effects including paralysis of the adult worms, inhibition of the secretory activity, tegument lesions and cercaricidal activity. In the association test of Praziquantel (PZQ)-C/E, we observed up to 80% mortality of Schistosoma mansoni in comparison to PZQ monotherapy. Due to the diversity of the toxic effects, the schistosomicidal activity of C/E is likely a result of a multitarget mechanism involving the tegument, secretory system and neuromotor action.

Potent Schistosomicidal Constituents from Garcinia brasiliensis.

Praziquantel is the drug of choice for the treatment of schistosomiasis. However, several strains of Schistosoma mansoni are resistant to praziquantel, making it necessary to discover new drugs that might be used for its treatment. With this in mind, the properties of a schistosomicidal ethanolic extract of Garcinia brasiliensis Mart. epicarp, the fractions obtained by partitioning this extract, including the hexane fractions, ethyl acetate fraction, and the aqueous fraction, and the isolated compounds 7-epiclusianone, a major component from these fractions, and fukugetin were tested in vitro on adult worms of S. mansoni. Mortality, damage to membranes, and excretory system activity were observed at 100.0, 50.0, 75.0, and 14.0 µg/mL for the ethanolic extract of G. brasiliensis Mart. epicarp, its hexane fractions, the ethyl acetate fraction, and 7-epiclusianone, respectively. For 7-epiclusianone, these data were confirmed by fluorescent probe Hoechst 33 258 and resorufin. Additionally, the biocidal effect of 7-epiclusianone was even higher than the hexane fractions. Moreover, an inhibitory effect of 7-epiclusianone on the egg laying of female adult S. mansoni worms was observed in cercariae and schistossomula. Thus, 7-epiclusianone is a promising schistosomicidal compound; however, more studies are needed to elucidate its mechanism of toxicity and to evaluate the in vivo activity of this compound.