The schistosomicidal activity of ethanolic extracts from branches, leaves, flowers and fruits of Handroanthus impetiginosus (Mart. ex DC.) Mattos (Bignoniaceae) plant and metabolic profile characterization by UPLC-ESI-QTOF analysis

Schistosomiasis, caused by Schistosoma mansoni Sambon, 1907, is a severe and widely distributed parasitic disease, affecting about 200 million people worldwide. The disease is recognized by elevated mortality rates, especially among those living in areas of poor sanitation. Currently, the chemotherapeutic treatment is solely based on using the praziquantel drug. Therefore, there is a need for the discovery of new medicines for the treatment of this parasitosis. Thus, this work aimed to evaluate the schistosomicidal activity of ethanolic crude extracts from the branches, leaves, flowers, and fruits of Handroanthus impetiginosus (Mart ex DC.) Masttos and characterize its metabolic profile by UPLC-ESI-QTOF analysis. Evaluation of plant extract on S. mansoni was carried out in adult worms in vitro , in which the mortality rate was quantified, and the damages in the tegument of the worms were monitored. All extracts induced changes in the viability of adult males of S. mansoni , causing the death of the parasites, which was directly dependent of the concentration.


Introduction
Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma.The infection is directly related to poor sanitation.According to the World

Extracts obtainment
For the crude ethanolic extracts preparation, the branches, leaves, flowers and fruits of H. impetiginosus were left for 15 days to dry separately at room temperature.Afterward, they were desiccated for 48 hours in a circulating air oven at 45ºC, then ground in a vertical rotor mill with fixed knives.-MARCONI -MA680.The powder mass was obtained as follows: 1.945g for branches, 4.570g for leaves, 1.400g for flowers, and 1.925g for fruits.The material was pulverized, macerated, and filtered, and the liquid extract was concentrated in a rotary evaporator.The ethanol extract masses were obtained 500 mg branches (EtOH-Br), 4.570g leaves (EtOH-Le), 1.400g flowers (EtOH-Fl) and 1.925g fruits (EtOH-Fr).

Maintenance of S. mansoni's lifecycle
The biological cycle of S. mansoni, strain LE (Luis Evangelista), is routinely maintained in the Parasitology Research Laboratory of the Research Center in Exact and Technological Sciences, University of Franca, using Balb/c mice as a vertebrate host and snails of the species Biomphalaria blabrata Say, 1818, as an invertebrate host (Hackett, 1993).Adult parasites were obtained by perfusion of the hepatic portal system of mice 54±2 days after infection, with approximately 200 cercariae, according to conditions previously described by Smithes and Terry (1965).After 49 days of the disease, adult worms were recovered through perfusion of the mesenteric and portal veins, according to Smithes and Terry (1965).After collection, the parasites were maintained in RPMI 1640 medium (Inlab Diagnóstica, Diadema, Brazil) buffered with HEPES 20 µM, pH 7.5, supplemented with penicillin (100 UmL) (Cultilab, Campinas, Brazil) and maintained until the use in RPMI-1640 culture medium (Gibco), containing 10% Fetal Bovine Serum (SFB) (Gibco).

In vitro evaluation of schistosomicidal activity
The branches, leaves, flowers, and fruits ethanol extracts were evaluated for changes in the tegument and schistosomicidal activity in vitro against adult male worms of S. mansoni.Adult male worms were recovered from Balb/c mice as described above.Subsequently, an adult worm was transferred per well into a 24-well culture plate containing 2 mL of RPMI 1640 culture medium (Inlab) buffered with 20 µM HEPES, pH 7.5, supplemented with penicillin (100U/mL), streptomycin (100µg/mL) (Cultilab, Campinas, BR) and 10% fetal bovine serum (Cultilab) and incubated in a humidifying atmosphere at 37ºC in the presence of 5% CO 2 .
After 24 hours of incubation, the crude extracts of branches, leaves, flowers and fruits separately previously dissolved in (DMSO) (Sigma-Aldrich) and added to RPMI 1640 (Inlab) medium and then tested at concentrations of 6.25 µg/ mL; 12.5 µg/ml; 50.0 µg/ml; 100.0 µg/ml and 200.0 µg/mL, based on concentrations determined in previous experiments by our research group (Magalhães et al., 2009).The parasites were incubated under the same conditions described above.Each well was observed after 24, 48, and 72 hours of contact with the samples, comparing them with the controls.Mortality and populations.These diseases are considered neglected, as they receive reduced investment in research, development and production of new drugs, vaccines, and the low effectiveness of control programs (Fonseca et al., 2020).
Based on the questions presented, one of the intervention alternatives to deal with the problem is the use of natural products for the treatment of this disease.As a millennial practice, medicinal plants have contributed to medicine been gaining more space as an effective and complementary alternative throughout history (Thomford et al., 2018).In addition to preventing, curing, or minimizing disease symptoms, products of natural origin are more affordable to the population and public health services than chemically synthesized products (Dutta et al., 2019).
The search for anthelmintic compounds, mainly from natural sources, has intensified recently (Jiao et al., 2020;Paula et al., 2020Paula et al., , 2022;;Oliveira et al., 2020a;Lima et al., 2021Lima et al., , 2022)).All this search is summarized in a recently published work, which shows the studies carried out for the treatment of schistosomiasis from the mid-1910s to the year 2016, where more than 100 compounds studied were gathered with information about in vitro and in vivo therapeutic action, toxicity, and pharmacokinetic profiles (Lago et al., 2018).All these efforts may represent hope in controlling schistosomiasis and show the need to search for new compounds to provide effective alternatives to treating the disease.Thus, this research aimed to evaluate the antiparasitic properties of crude ethanolic extracts from different parts of H. impetiginosus (branches, leaves, flowers, and fruits) against S. mansoni.In addition to the investigation of the metabolic profile by UPLC-ESI-QTOF approaches.

Plant material collection
The branches, leaves, flowers and fruits of H. impetiginosus were collected in the urban area at coordinates (20º31'19.19" S, 47º24'36" W).Dr. Milton Groppo performed the botanical identification, and the core material was deposited in the Herbarium of the Department of Biology at FFCLRP-USP with the registration number SPFR 16658.
Brazilian Journal of Biology, 2023, vol. 83, e275824 3/9 The schistosomicidal activity of extracts from Handroanthus impetiginosus and metabolic profile characterization by UPLC-ESI-QTOF integument detachment of male parasites was evaluated using an inverted microscope (Carl Zeiss, Göttingen, Germany).Mortality was determined according to a scale from 0 to 3 where: (3= worms with normal movement.2= decreased motor activity, 1= minimal motor activity with occasional movements, and 0= death of the worms without movement for more than 2 minutes of observation) (Ramirez et al., 2007).
The parasites without movement for more than 2 minutes of observation were washed with RPMI 1640 medium, transferred to culture plates with the same medium without the substances, and monitored as described above.As a negative control, young male worms maintained in RPMI 1640 medium with 0.1% DMSO (Sigma-Aldrich) were used.In the experiment, PZQ at a concentration of 1.56µM was used as a positive control.Three independent experiments were carried out, with five young male worms being evaluated per concentration in each experiment (a total of 25 parasites).Results were expressed as the mean ± standard deviation (SD) of adult male viability.The LC 50 (50% lethal concentration of parasites) was determined by the non-linear regression curve using the dose-response inhibition equation.The test was performed using GraphPad Prism version 6.0 for Windows (GraphPad Software, San Diego, California, USA).

Statistical analysis
Values were expressed as mean ± SD of the samples.A one-way analysis of variance (one-way ANOVA) was used, followed by determining the significance of differences between the control and treated groups (Dunnett post-test).Tests were performed using GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, California, USA).

Mass spectrometer instrument system and analysis
A C18 ACQUITY ®HSS T3 ultra-analytical reversedphase column (1.8 µm, 100 x 2.1 mm, 40 °C) was used for the development of the chromatographic runs in a Xevo-qTOF/MS (Waters Corp., Milford, USA) ultra-performance liquid chromatography coupled to quadrupole time of flight mass spectrometer (MS) instrument (UPLC-QTOF/ MS).The mobile phases contained (A) 1% acidified H 2 O with formic acid and (B) acetonitrile at a flow rate of 0.5 mL/min.Blank samples were a mixture of ACN: water (1:1 v/v).The injection volume consisted of 5 µL of each extracted sample (branches, leaves, flowers and fruits from H. impetiginosus).The resulting chromatographic method gradient was developed as follows: 1% of B and 99% of A (0.1 min), 80% of A and 20% of B (until 7.5 min), 20% of A and 80% of B (until 8.5 min), 1% of A and 99% of B (until 8.6 min) and 99% of A and 1% of B (until 10 min).
Mass data were acquired as MS E independent data acquisition (DIA) using the Masslynxs TM MS Software (Waters Corp., Milford, USA).The ESI (electrospray) ionization source operated in negative and positive modes, and MS data were collected in the centroid mode.The lock spray was calibrated using a leucine-encephalin standard solution at the frequency of 10s with mass ratio (m/z) at m/z 554.2622ESI-in the negative mode and at m/z 556.2768ESI + in the positive ionization mode.The collision energy parameters were performed with alternative high and low energy scans, 10 eV and 40 eV, respectively.The source temperature operating parameters were cone voltage (40V), capillary voltage (3.0 kV), desolvation temperature (300°C), desolvation gas flow (600 L/h) and cone gas flow (30 L/h and 120°C).

MS Data treatment and chemical annotation
The MS Fragment ion spectra (MS E ) raw dataindependent centroided acquisition of the RT-m/z pair and peak area information was processed for each ionization mode using the MS DIAL software 4.80 version.The raw data ions were collected for mass data processing steps, which included the application of peak corrections, data filtering, baseline correction, isotope exclusion ([isotope .For dereplication, the data matrix with m/z-Rt pair and peak area were exported to MS FINDER tool 3.52 after peak blank exclusion and peaks without eligible MS 2 data.The elemental composition was implemented for molecular formula determination.The dereplication was also supported by manually checking the online Dictionary of Natural Products (DNP®) chemical library.An excel format document (.csv) was exported with metabolite annotation.

Metabolic profile and dereplication
Adequate separation of the peaks could be observed in the chromatograms of all H. impetiginosus crude extract samples (Figure 1).The metabolic profile obtained from the UPLC-ESI-QTOF chromatograms of leaves, fruits, flowers, and branches indicated widespread chemical variance in the different vegetal parts of H. impetiginosus extracts.The MS data treatment of both ESI+/ESI-modes allowed the putative identification of the majority known metabolites in the samples.The most shared metabolite classes presented in the extract samples were unveiled using MS finder and DNP libraries matching exact mass and molecular formula, which indicated the presence of most naphthoquinones and flavonoids (Tables 1 and 2).
Studies indicated that naphthoquinones potassium salts of isolapachol and lapachol can exhibit molluscicidal activity in the life cycle of Schistosoma mansoni (Lima et al., 2002).The lapachol, isolapachol or α/β lapachone metabolite could not be differentiated only by mass spectra since they share the same molecular and main fragment ions (m/z 243.1006 and respective m/z 225.0907, m/z 201.0540 and m/z 187.0391) (Figure 2).Although, they were dereplicated in all the samples, especially in the branches and leaves samples.Further studies involving validated MS experiments with standard compounds can implement energy and fragmentation studies aiming univocally differentiation of these quinones by MS technics, which in our case were evidenced at 2 different retention times (Rt 4.76 and 6.68 min) (Figure 1 and 2).
Furthermore, the chemical analyses indicated that naphthoquinones are present in all metabolic content of the analyzed samples.Thus, our study demonstrated that this metabolite class could be encountered in the whole vegetal material of H. impetiginosus, corroborating the literature that the H. impetiginosus specie is rich in the quinone class content (El-Hawary et al., 2021).Besides the quinones, several other metabolite classes reported in the literature were annotated in this dereplication, including the iridoid glycosides, benzoic acid derivatives, furanonaphthoquinones, hydroxyanthraquinones, longchain fatty acids, triterpenoids and phenolic compounds, such as phenylpropanoid glycosides (Table 1 and 2).

Percentage of death in adult male S. mansoni worms
The evaluated extracts reduced motility and caused parasite death depending on concentration and incubation time (Figures 3 and 4).Monitoring parasites kept in culture   The schistosomicidal activity of extracts from Handroanthus impetiginosus and metabolic profile characterization by UPLC-ESI-QTOF for 24h, 48h, and 72h indicated a schistosomicidal effect on adult male S. mansoni worms.For this incubation period, 100% mortality was achieved with extracts at a concentration of 200 µg/mL from flowers for 24h; and with the crude ethanolic extracts of the leaves for 72h.At a 100 µg/mL concentration, the crude extracts that reached 100% mortality in 48h and 72h were from flowers and leaves.

In vitro evaluation of H. impetiginous extracts in the induction of integument changes of adult worms of S. mansoni
The external morphology of the male Schistosoma mansoni presents the gynophore canal (GC), a longitudinal fold in the posterior portion that shelters the female and helps the mating and reproduction.The anterior portion is characterized by a smooth, cylindrical integument, an oral sucker, and a ventral sucker with numerous cylindrical papillae (Figure 5).
Changes promoted by the ethanolic extracts EtOH-Br, EtOH-Le, EtOH-Fl, and EtOH-Fr doses on adult male worms at 24, 48, and 72h are shown in Figures 5a-e.The extract promoted an apparent effect on the musculature of the worms with the evolution of dorsoventral curvature in a time-dependent manner, assuming the form of a corkscrew after 24h of incubation.After 48h of incubation at 12.5 µg/mL of the EtOH-Le, opening, and relaxation in the gynophore canal were observed.After 24h of incubation at 25 µg/mL of the EtOH-Fr, bubbles were observed in the anterior region around the sucker of the worms (Figures 5b and 5c).Moreover, severe changes in the integument were characterized by edema, displacement, wrinkled surface, and erosion, which were observed when incubated at 50 µg/mL of EtOH-Br, and 50 µg/mL of EtOH-Fl for 72h, respectively, throughout the incubation period (Figure 5d and 5e), an evident longitudinal contraction of the muscles occurred, and the worms presented in the form of a corkscrew (Figure 5e).
Furthermore, populations use plants containing naphthoquinones from different locations to treat diseases, from parasitic diseases to different types of cancer.For example, countries with plant species that produce naphthoquinones, such as Brazil, China, and Japan, have been contributing intensively with chemical and pharmacological studies of these species (Rani et al., 2022).Several therapeutic applications are attributed to β-lapachone; in this sense, studies indicate antibacterial, antifungal (Guimaraes et al., 2021), antiviral (Mokarizadeh et al., 2020), antiarthritic (Gong et al., 2021), anti-inflammatory (Oliveira et al., 2020b), trypanocidal (González et al., 2020) and schistosomicidal.Edingl, Tersan, and Waite, in 1947, stated that naphthoquinone derivatives could inhibit aerobic glycolysis in S. mansoni adult worms.
Other studies indicate molluscicidal activity on B. glabrata (intermediate host of S. mansoni) and its eggs and activity on the cercariae of S. mansoni (Jali et al., 2018).
Ortho-naphthoquinone has moderate activity against S.mansoni compared to the reference drug (PZQ).In addition, it exhibits activity in other phases of the parasite's biological cycle, a feat not achieved with praziquantel, which only fights the adult form of the worm (Silva Júnior et al., 2019).The activity of a topical preparation of β-lapachone was tested against S. Mansoni cercariae in rat tails.The preparation showed total blockage of cercariae penetration when applied to the tail 24 hours before infection.Infection depends on the complete penetration of cercariae (Silva Júnior et al., 2019).
There was a decrease in the number of worms by 29.78%, 37.2%, 24.2%, and 40.22% when administered during the skin schistosomulus, pulmonary schistosomulus, juvenile worms, and adult worms phases, respectively.In addition, in all groups, there was a decrease in the number and diameter of hepatic granulomas in the control group.Subsequently, the same research group evaluated the activity of β-lapachone on the integument of S. mansoni worms.They observed that after 24 hours of incubation, exposure to different concentrations of β-lapachone led to the death of 67% to 100% of the worms.Changes in worm motility were also observed, besides scaling, blistering, and tegument rupture (Silva Júnior et al., 2019).However, the pharmacological potential of this naphthoquinone is not indicated for the systemic treatment of these parasites

Conclusion
All extracts caused changes in the viability of adult males of S.mansoni at 49 days.The extracts caused the death of the parasites directly dependent on the concentration and incubation time.The extracts caused contractions in the adult male worms before the schistosomicidal effect.At lethal concentrations, the extracts caused changes in the integument of adult male worms, revealing that the damage caused occurs in a dose-dependent manner.These results open new possibilities for researching herbal medicines to treat neglected tropical diseases, such as schistosomiasis.

Figure 1 .
Figure 1.BPI chromatograms for each type of H. impetiginosus aerial parts extract for the positive ionization mode (ESI+).

Figure 2 .
Figure 2. BPI chromatograms of H. impetiginosus EtOH-leaves extract evidencing high and low energy channels (10 and 40 eV) selected at Rt 4.76 min and m/z 243 in the positive ionization mode (ESI+).EtOH-Le -leaves.

Figure 3 .
Figure 3.Effect of H. impetiginosus extracts on the survival of adult male S. mansoni worms.Adult male worms were incubated in an RPMI medium containing a concentration of 200 µg/mL of 1-branches, 2-leaves, 3-flowers, and 4-fruits.

Figure 4 .
Figure 4. Effect of H. impetiginosus extracts on the survival of adult male S. mansoni worms.Adult male worms were incubated in an RPMI medium containing a concentration of 100 µg/mL of 1-branches, 2-leaves, 3-flowers, and 4-fruits

Table 1 .
List of highest peak areas and respective samples analyzed in the ESI + .EtOH-Le -leaves, EtOH-Fr -fruits., EtOH-Fl -flowers and EtOH-Br -branches.

Table 2 .
List of the highest peak areas and respective samples analyzed in the ESI -.EtOH-Le -leaves, EtOH-Fr -fruits., EtOH-Fl -flowers and EtOH-Br -branches.