Potent Schistosomicidal Constituents from Garcinia brasiliensis

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Aline Pereira Castro, Ana Carolina Alves de Mattos,Neusa Araújo Pereira, Naira Ferreira Anchieta,Matheus Siqueira Silva, Danielle Ferreira Dias, ClaudineiAlves Silva, Giulliano Vilela Barros, Raquel Lopes MartinsSouza, Marcelo Henrique Dos Santos, Marcos José Marques

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Potent Schistosomicidal Constituentsfrom Garcinia brasiliensis

DOI 10.1055/s-0035-1545927Planta Med 2015; 81: 733–741

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Abstract!

Praziquantel is the drug of choice for the treat-ment of schistosomiasis. However, several strainsof Schistosoma mansoni are resistant to prazi-quantel, making it necessary to discover newdrugs that might be used for its treatment. Withthis in mind, the properties of a schistosomicidalethanolic extract of Garcinia brasiliensisMart. ep-icarp, the fractions obtained by partitioning thisextract, including the hexane fractions, ethyl ace-tate fraction, and the aqueous fraction, and theisolated compounds 7-epiclusianone, a majorcomponent from these fractions, and fukugetinwere 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 ofG. brasiliensis Mart. epicarp, its hexane fractions,the ethyl acetate fraction, and 7-epiclusianone,respectively. For 7-epiclusianone, these data wereconfirmed by fluorescent probe Hoechst 33258and resorufin. Additionally, the biocidal effect of7-epiclusianone was even higher than the hexanefractions. Moreover, an inhibitory effect of 7-epi-clusianone on the egg laying of female adult S.mansoni worms was observed in cercariae andschistossomula. Thus, 7-epiclusianone is a prom-ising schistosomicidal compound; however, morestudies are needed to elucidate its mechanism oftoxicity and to evaluate the in vivo activity of thiscompound.

Potent Schistosomicidal Constituents fromGarcinia brasiliensis

Authors Aline Pereira Castro1,3, Ana Carolina Alves de Mattos2, Neusa Araújo Pereira2, Naira Ferreira Anchieta3, MatheusSiqueira Silva1, Danielle Ferreira Dias1, Claudinei Alves Silva1, Giulliano Vilela Barros3, Raquel Lopes Martins Souza3,Marcelo Henrique Dos Santos4, Marcos José Marques3

Affiliations The affiliations are listed at the end of the article

Key wordsl" Clusiaceael" Garcinia brasiliensisl" 7‑epiclusianonel" fukugetinl" Schistosoma mansonil" Schistosomiasis

received February 16, 2015revised February 16, 2015accepted March 14, 2015

BibliographyDOI http://dx.doi.org/10.1055/s-0035-1545927Published online April 23, 2015Planta Med 2015; 81: 733–741© Georg Thieme Verlag KGStuttgart · New York ·ISSN 0032‑0943

CorrespondenceProf. Marcos José MarquesLaboratory of ParasitologyInstitute of Biomedical SciencesFederal University of AlfenasDepartment of Pathology andParasitologyGabriel Monteiro da Silva – 700Alfenas, Minas Gerais 37130000BrazilPhone: + [email protected]

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Introduction!

Schistosomiasis is a neglected tropical diseasethat is caused by trematode flatworms of the ge-nus Schistosoma. It is a disease that threatens mil-lions of people, especially in poor regions [1]. TheWorld Health Organization (WHO) estimated, in2003, that approximately 207 million peoplewere infected with Schistosoma. Moreover, 779million people are at risk of contracting this dis-ease [2], and at least 200000 people die each yeardue to schistosomiasis [3]. Praziquantel (PZQ) iseffective against all clinical forms of schistosomia-sis, However, the emergence of resistant strainshas spurred on the search for new active antihel-minthic compounds, especially those derivedfrom plants [4,5].The search for antiparasitic compounds from nat-ural sources has increased over the last decade[4–6]. In this context, plants remain an importantsource of biologically active compounds, whichcan provide structures for the development ofnew drugs [7].

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Several chemical studies have shown that the ge-nus Garcinia is an important source of naturalproducts with a wide variety of biologically activemetabolites, including polyisoprenylated benzo-phenones, flavonoids, xanthones, and proantho-cyanidins, which have been proven to be effectiveagainst various diseases [8]. Many studies havedemonstrated that Garcinia brasiliensis providesanti-inflammatory [9], antinociceptive [10], anti-oxidant, and antitumor activities [11]. The tetra-prenylated benzophenone 7-epiclusianone (7-epi) (l" Fig. 1) was first isolated from the fruits ofG. brasiliensis [12]. Studies have demonstratedthat the 7-epi extracted from the fruits of G. brasi-liensis is effective against the trypomastigotes ofTrypanosoma cruzi [13] and has vasodilatory[14], antianaphylactic [15], anti-HIV [16], antimi-crobial [17,18], antispasmodic [19], and antipro-liferative effects [20]. Antileishmanial activity[21] and analgesic and anti-inflammatory [10] ef-fects have also been assigned to this benzophe-none. Fukugetin (fuk) (l" Fig. 1) is a bioflavonoidinitially obtained from Garcinia spp, which alsohas demonstrated anti-inflammatory [21] and

istosomicidal Constituents… Planta Med 2015; 81: 733–741

Fig. 1 A Chemical structure of 7-epi. B Chemicalstructure of fuk.

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anti-HIV‑1 activities [22] as well as tumor angiogenesis inhibi-tion [23].Due to the difficulties in treating diseases caused by helminths,especially schistosomiasis, this study evaluated the schistosomi-cidal activity of both an ethanolic extract of G. brasiliensis Mart.epicarp (EEE) and other fractions obtained by additional extrac-tions. The evaluation of the hexane fractions (HF), ethyl acetatefraction (EAF), and aqueous fraction (AF) enables the isolation ofmolecules with a potential therapeutic use in the treatment ofschistosomiasis, as substances with potential antiparasitic activ-ity have been isolated from plants in this family in recent years[24].

Results and Discussion!

Recently, the development of new drugs from medicinal plantsfor the treatment of neglected diseases, including schistosomia-sis, has attracted the attention of many scientific research centersaround the world. For example, plants of the genus Artemisia[25], extracts of Zanthoxylum naranjillo [26], and crude aqueousextracts of Zingiber officinale [27], among others, have been effec-tive against Schsitosoma mansoni. With this in mind, the presentstudy was devised to evaluate the schistosomicidal potential ofEEE and the partition-obtained fractions (HF, EAF, FA) and com-pounds isolated from the fractions, the benzophenone 7-epi andthe bioflavonoid fuk, by means of a bioguided test.The total amount of flavonoid found in the ethanolic epicarp ex-tract of G. brasiliensis was 22.52 µg in quercetin equivalents/g ofthe extract. The flavonoids were known for their beneficial ef-fects on health. An important effect of flavonoids is the scaveng-ing of oxygen-derived free radicals. Moreover, in vitro experi-mental systems also showed that flavonoids possess anti-inflam-matory, antiallergic, antiviral, and anticarcinogenic properties[28]. Many plants rich in flavonoids have demonstrated activityagainst S. mansoni such as Baccharis trimera, popularly knownas carqueja [29], Allium sativum and Allium cepa, popularlyknown as garlic and onion, respectively [30], and Zingiber offici-nale, popularly known as ginger [27].The yields of the EEE and the partitioned fractions HF, EAF, and AFwere 10.0%, 39.0%, 42.0%, and 14.5%, respectively. In l" Fig. 2, theretention times, areas, and quantification of the isolated com-pounds in the extract and the fractions are shown.The analytical curves consisted of six data points, and three rep-licate injections at each concentration level were performed. Thestatistical analysis of the data revealed excellent linearity(r > 0.99) over a concentration range from 10.0 to 300.0mg/mL.

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The limits of detection (LOD) and quantitation (LOQ) were 1.5and 4.5mg/mL for fuk (y = 77296 x-269178/SD = 173217), and0.87 and 2.6mg/mL for 7-epiclusianone (y = 38539 x-84655/SD = 145667), respectively.Chemical analysis of the EEE identified 7-epi as themajor constit-uent (140.02mg/g), followed by fuk (35.86mg/g) (l" Fig. 2A). Inthe HF, 7-epi was identified as the major constituent (278,74mg/g), whereas the fuk peak was not identified (l" Fig. 2B). Achemical analysis of the EAF identified 7-epi as the major constit-uent (209.04mg/g [15]), followed by fuk (24.19mg/g) (l" Fig. 2C).Finally, in the AF, neither 7-epi nor fuk peaks were identified(l" Fig. 2D). These peaks may be compared with the standards ofthese compounds (l" Fig. 2E,F).Those results are in accord with previous results that demon-strated four phenolic components, identified as fukugiside andg-uttiferone A; besides fuk and 7-epi in EEE [10]. These compoundswere characterized by comparison to the standards previouslyisolated [31,32]. The 7-epi, a natural polyisoprenylated benzo-phenone, was first isolated from the fruits of G. gardneriana [12]and has shown activity against trypomastigotes of Trypanosomacruzi in vitro [13], a potent endothelium vasodilator effect [14],and anti-anaphylactic [15], anti-HIV [16], antimicrobial [17],antispasmodic [11], antiproliferative [19], and leishmanicidal ac-tivities [20].Various parameters, including changes in the integrity of the in-tegument andmotility as well as reductions in mating and ovipo-sition, are often evaluated as indicators of biological activity andused in in vitro toxicity studies against adult worms of Schistoso-ma species [4–6,33,34].The schistosomicidal effect of the EEE and its fractions and iso-lated compounds in vitro was analyzed with respect to the timeof incubation and concentration.All adult worms were dead after 24 h exposure to 100.0 µg/mL ofEEE, 50.0 µg/mL of HF, 75.0 µg/mL of EAF, and 14.0 µg/mL of 7-epi.However, the AF and fuk showed no activity at 200.0 µg/mL (thehighest dose tested). In contrast, the worms remained viable inthe negative control group (RPMI-1640 supplementedwith RPMIand methanol). The use of PZQ at 2.0 µg/mL as a positive controlresulted in the death of all of the parasites within 24 h.Mating and egg laying of S. mansoni females werenʼt observed atdoses greater than 50.0 µg/mL and 4.0 µg/mL for the EEE (andtheir fractions) and 7-epi, respectively. Adult male and femaleworms remained separate and no oviposition was observed. Re-garding the reproductive aptitude of the adult worms of S. man-soni, the components of other plants have shown effects on theoviposition of the worm. For example, curcumin, which was ex-tracted from the rhizomes of Curcuma longa [4] and some phlor-

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Fig. 2 Chromatograms obtained by high-per-formance liquid chromatography (HPLC) from EEE(A); HF (B); EAF (C); AF (D); 7-epi (E), and fuk (F)standards. In the attached tables are principle com-ponents found in the extract and the fractionsidentified by HPLC.

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oglucinol derivatives that were obtained from the rhizomes ofthe Dryopteris species [5], demonstrated in vitro effects on egglaying. A decrease in oviposition was also observed using Nigellasativa [35] and a ginger rhizome (Z. officinale) extract [36].Similarly, severe tegumental morphological changes were alsoobserved after the incubating of adult S. mansoni worms withthe extracts and fractions at concentrations above 50.0 µg/mLand with 7-epi at concentrations greater than 10.0 µg/mL. How-ever, no abnormality was observed inworms in the control group(RPMI-1640 supplemented with RPMI and methanol). The dam-age to the tegument of adult S. mansoni worms was confirmedusing a Hoechst 33258 probe, which is a specific marker for cel-lular DNA. To assess the damage of the integument, the wormsexposed to 2.0 µg/mL of PZQ and 14.0 µg/mL (ED100) of 7-epiwere incubated and subsequently placed in contact with theHoechst 33258 probe. Analysis by microscopy showed progres-sive damage on the surface, causing blisters and the detachmentof the tegument (l" Fig. 3). PZQ and 7-epi were able to induce le-sions in the tegument of parasites, and the lesions in S. mansonimales were more pronounced than in females (l" Fig. 3). Changesin the surface of the tegument showed a dose-dependent effect.The Hoechst 33258 probe is very sensitive inmarking the regionswhere damage is present, and any damage or injury to the in-tegument of the worm is precisely marked by this probe [37].This soft tissue damage was similar to the damage reported instudies with PZQ [38].Data obtained in this study demonstrated that the EEE, HF, EAF,and 7-epi exhibited greater activity against adult male S. mansoniworms. Indeed, 7-epi wasmore effective, which demonstrated anED100 of 14.0 µg/mL (l" Fig. 4). The lethality was greater in maleworms after 24 h of incubation with 10.0 µg/mL of 7-epi. There-

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fore, this compound paralyzes the excretory activity in concen-trations more than 12 and 14 µg/mL of 7-epi in males and fe-males, respectively. In several studies, based on the in vitro activ-ity, disparities in drug susceptibility between males and femalesof S. mansoni have been observed. S. mansoni males are oftenmore susceptible than female worms in studies on resistance tooxamniquine [39] and in studies regarding the bioactivity of gin-ger extract [36].In contrast, the results with other compounds, such as fatty ami-noalkanethiosulfuric [34] and 2-[butylamino]-1-phenyl-1-etha-nethiosulfuric acid [6], revealed higher survival rates for malesthan females. Notably, in vitro data obtained in this study demon-strated that the EEE, HF, EAF, and 7-epi exhibited greater activityagainst adult male S. mansoniworms.Serious injury was reported in the integument of female adult S.mansoniworms when treated with artemether [33,40]. The seedtegument is extremely important for successful infection andsurvival in the host and has been amajor target for antischistoso-mal drug development. Therefore, most of the drugs that are cur-rently used against schistosomiasis, including PZQ [38], meflo-quine [40], and artemether [41], act by damaging the integumentof the worm.The results of these experiments revealed that 7-epi was able toparalyze the excretory activity of the adult worms of S. mansoni.This was observed by labeling the parasites with resorufin after aprevious exposure to 7-epi, which prevented the output of theprobe [substrate for P-glycoprotein (Pgp)] to the external envi-ronment. The secretory activity of worms initially exposed to re-sorufin and subsequently incubated with 14.0 µg/mL (ED100) of7-epi was completely inhibited, as characterized by the accumu-lation of the probe in tissues other than the excretory system

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Fig. 4 In vitro effects of 7-epi and fuk against adult S. mansoni;*** p < 0.001.

Fig. 3 In vitro evaluation of the effect of 14.0 µg/mL of 7-epi on the tegument of S. mansoni adultworms. A,B S. mansoni LE not exposed to 7-epi andlabelled with the probe Hoechst 33258. C,D S.mansoni exposed to 7-epi and labelled with theprobe Hoechst 33258. The fluorescent areas indi-cate intense lesions. Each scale bar represents100 µm. E,F S. mansoni exposed to 2 µg/ml PZQand labelled with the probe Hoechst 33258. Thefluorescent areas indicate intense lesions. (Colorfigure available online only.)


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(l" Fig. 5). This compound paralyzed the excretory activity, too, asobserved in other concentrations of 12, 16 and 18 µg/mL. How-


ever, with 12 µg/mL, there was no change observed in the femaleworms.The modulatory effect of some drugs has previously been shownthrough interactions with the cellular transport proteins of theexcretory system in male adult S. mansoni worms [42]. Thus, theresults from present work suggest, for the first time, that 7-epiacted in the excretory system and interfered with the activity ofPgp in an unknown manner. A similar result was observed in thepresence of amiloride (an inhibitor of Na+ and Na+/H+ ATPase)[42]. Some hypotheses can be considered to explain the resultsobtained in this study, such as (i) it is possible that 7-epi depletesenergy by increasing the calcium influx into tissues, leading to anexcessive consumption of ATP, which eventually inhibits Pgp; (ii)the 7-epi substrate can act as a competitive inhibitor of Pgp; (iii)7-epi may bind directly to Pgp, disturbing the excretion of Resor-ufin; (iv) 7-epi can act indirectly on the excretion of resorufin, af-fecting tubular membrane phospholipids and causing the probeto diffuse to places other than the excretory system [43], or (v) asimple contraction induced by 7-epi could cause a delay in theexcretion of the probe due to a lack of a constant intestinal motil-ity from the excretory organ. Thus, the probe would have a great-er opportunity to diffuse to the surrounding tissues.In the present study, we used the loss of motility and tail as mea-sures to evaluate the sensitivity of cercariae when incubatedwith7-epi. At a concentration of 12.5 µg/mL of 7-epi, the cercariaewere immobile after 2 h and 8 h of incubation, and the separation

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Fig. 6 Effect of different concentrations of 7-epi on the viability of cercar-iae, based on the rate (%) of loss of tail. Data are presented as the averageof three experiments; ***p < 0.0001.

Fig. 5 In vitro evaluation of the effect of 14.0 µg/mL of 7-epi on the excretory system of S. mansoniadult worms. A,B S. mansoni labelled with theprobe resorufin and not exposed to 7-epi. C,D S.mansoni labelled with the probe resorufin and ex-posed to 14.0 µg/mL of 7-epi. Arrow: Main tubule(MT), nephridiopore (N), and ramifications of theexcretory system. E,F S. mansoni labelled with theprobe resorufin and exposed to 2 µg/ml of PZQ.(Color figure available online only.)

Fig. 7 Effect of various concentrations of 7-epi on the viability of schisto-somules, based on the rate (%) of loss of movement. Data are presented asaverage of three experiments; ***p < 0.0001.

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of the tails from their bodies occurred in 99.0% of the analyses,whereas in the control groups (pure water, and pure water+ 0.1% DMSO), swimming cercariae (worms that did not experi-ence tail loss) were observed for up to 8 h (l" Fig. 6). The exactmechanism of induction of tail loss is not fully understood. It isbelieved that the process relates to a particular structure be-tween the body and the tail of cercariae. The rearward end ofthe body of cercariae is folded and tapered like a collar on a nar-row structure that connects the body to the tail. This connectivestructure is delicate and can be easily broken [44].

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In the absence of 7-epi, schistosomula viability appeared normal,without any morphological alteration for up to 48 h. Slight mor-phological changes, such as granularity, shortening body with asickle shape, and low activity, were observed after 24 h of incuba-tion with 10.0 µg/mL of 7-epi. At doses of 12.5, 25.0, and 50.0 µg/mL, movement loss in schistosomula occurred after 2, 24, and48 h of incubation, respectively (l" Fig. 7). However, at all of thesedoses, severe morphological changes, such as black granules, andan intense vacuolization around the body associated with a re-



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duction in length, were observed in the first two hours of incuba-tion.Concentrations from 7-epi 25.0 µg/mL resulted in a complete lackof mobility and intense vacuolization after 24 h incubation. Schis-tosomula belonging to the negative control group (RPMI or RPMImethanol 0.1%) demonstrated viability for a standard 48 h. Acomplete lack of mobility and severe vacuolation were also ob-served after PZQ treatment. The effects observed in this studywere similar to those observed in schistosomula incubated with15.0 µg/mL of mefloquine and 100.0 µg/mL of the essential oil ofPiper cubeta [45].The generalized use of folk medicine suggests that natural prod-ucts are harmless; however, the traditional use of these naturalproducts is not satisfactory proof of their efficacy. In this context,the efficacy and safety of natural products require further studybecause the toxicity of the extract and the isolated molecules, 7-epi and fuk, have been demonstrated on murine peritoneal mac-rophages. However, no significant toxicity was observed in mam-malian cell extracts when the isolated molecules were used [20].We demonstrated that 7-epi is an effective compound for useagainst S. mansoni adult worms, cercarie, and schistosomula invitro. With its extensive effects on the mortality rate, reproduc-tive fitness, parasite tegument morphology, and adult S. mansoniworm excretory activity, 7-epi is a promising composite schisto-somicidal. However, further studies are needed to elucidate themechanism(s) of toxicity of 7-epi and evaluate the in vivo activityof this compound.

Materials and Methods!

Plant material and extract preparationThe fruits of G. brasiliensis (Mart.) were collected on the campusof the Federal University of Viçosa-MG, Brazil in February(summer) of 2011. Botanical identification was performed in theHorto Botânico of the Federal University of Viçosa by Dr. João Au-gusto Alves Meira Neto. A voucher specimen (number VIC2604)was deposited at the herbarium of the Federal University of Viço-sa. Epicarps of G. brasiliensis were dried in an oven with a circu-lating air temperature of 40°C for eight days. Dried and pow-dered G. brasiliensis fruit pericarps (1000.0 g) were extracted bymaceration with 3.0 L of ethanol at room temperature, filtered,and then dried using a rotary evaporator under reduced pressureat 45°C. This procedure was repeated five times yielding anamount of 80.0 g of fruit pericarp of the ethanolic extract (EEE).

Isolation and characterizationA portion of the EEE (1.0 g) was then subjected to a liquid/liquidpartition as follows: the sample was dissolved into an ethanol :water solution (1:4, 50.0mL), and after the addition of hexane(50.0mL), the mixture was vigorously stirred. The organic layerwas removed to give the HF (this procedure was performed fivetimes). Some insoluble compounds were retained in the aqueousphase, towhich ethyl acetate (50.0mL) was added and vigorouslystirred. Similarly, the organic layer was removed to produce theEAF (this procedure was performed five times). The HF, EAF, andAF were concentrated at a reduced pressure using a rotary evap-orator andwere then dried in a lyophilizer and stored in a freezer[46]. To isolate the bioactive compounds, the HF and EAF weregenerated from the partition of the EEE and chromatographedon a silica gel (230–400 mesh) column (8 × 100 cm). Sampleswere eluted with increasingly polar mixtures of hexane/ethyl


acetate and ethyl acetate/ethanol to give 50 fractions, and thefractions (200.0mL) were concentrated on a rotavapor at a re-duced pressure. These fractions were pooled in three groups ac-cording to their similarities in thin-layer chromatography (TLC).When necessary, the samples were rechromatographed on a sili-ca gel column as reported above to give 26 fractions (10.0mL)and were then pooled into five groups according to similaritiesin TLC, and recrystallized several times using methanol to obtainpure compounds. 7-Epi was isolated from the HF, and both 7-epiand fuk were isolated from the EAF. Previously, isolated 7-epi andfuk from the Laboratory of Phytochemistry and Medicinal Chem-istry of UNIFAL‑MG were used as standards for identification aswell as for spectral data [10,11]. The purity of 7-epi and fuk(99.0 and 98.0%, respectively) was evaluated by chromatographicanalysis. The procedure adopted for the preparation of the ex-tract, fractions, and isolated compounds is outlined in l" Fig. 8.

Chromatographic analysis and quantificationThe chromatographic analysis of the extracts and fractions, andthe quantification of isolated molecules present in the extractand fractions were performed on a liquid chromatography device(Shimadzu HPLC) using an NST column (Nano Separation Tech-nologies) C18-154 605 (150 × 4.6mm; 5.0mm in particle size)[20]. The mobile phase consisted of a mixture of acetic acid(5mM/L) (eluent A) and methanol/acetic acid (0.1% v/v) (eluentB). The injection volume was 20.0 µL at a flow rate of 1.0mL/min. During the initial 10min, an analysis was performed using50.0% of eluent B followed by an increase in the concentration ofeluent B to 100.0% over 20min. The analysis continued for 30minat a concentration of 100.0% eluent B. After the analysis with elu-ent B was concluded, the columnwas prepared for the next anal-ysis. Chromatograms were obtained at 254 nm and the peakswere compared with the peaks of the compounds that had beenpreviously isolated in the laboratory.For the analytical curves, aliquots of 0.100, 0.300, 0.500, 1.00,2.00, and 3.00mL, from the standard stock solutions of fuk and7-epi (1.00mg/mL) were transferred to separate 10.0mL volu-metric flasks. The volumes were completed with deionized waterprior to injection. Concentration ranges from 10.0 to 300.0 µg/mLof fuk and 7-epi were obtained. Each solution was analyzed intriplicate, and the resulting peak areas were plotted against therespective concentrations.The criteria used to determine the LOD and LOQ were based onthe determination of the slope (S) of the analytical curve andthe standard deviation of responses (SD) in accordance with theformulas LOD = 3.3 SD/S and LOQ = 10 SD/S. The SD was deter-mined from the standard error estimated by the regression line[47]. Extracts samples were assayed against reference standardsand each concentration of fuk and 7-epi was calculated using ananalytical curve.

Determination of total flavonoidsIn a 10.0-mL test tube, 0.5mL of 20.0% (w/v) EEE, 1.5mL of etha-nol, 0.1mL of 10.0% (w/v) AlCl3 ·6H2O, and 0.1mL of 1M potassi-um acetate were combined and mixed. The volume was thenbrought up to a final volume of 5.0mL with H2O. After 30min,the mixture was measured at 425 nm. The standard curve for to-tal flavonoids was generated with a quercetin [Quercetin dehy-drate (≥ 99.0% HPLC) was purchased from Sigma-Aldrich Co.]standard solution (25–120 g/mL) using the same procedure asabove. Total flavonoids were determined as quercetin equivalents

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Fig. 8 Bioassay scheme used for the extractionand purification of the constituents from G. brasi-liensis epicarp.

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(mg quercetin/g extract), and the values are presented as themeans of triplicate analyses [48].

Parasite and experimental animalsThe LE strain of S. mansoni used in this study was routinely main-tained by serial passage in Biomphalaria glabrata and Swissmouse models. The LE strain has been maintained for more than30 years at the Research Center René Rachou/Oswaldo CruzFoundation. All experiments were authorized by the EthicalCommittee for Animal Care of the Federal University of Alfenas(approval number: 534/2013 January 20, 2014) in accordancewith the national and international accepted principles for labo-ratory animal use and care.

In vitro evaluation of the effect of ethanolic extract ofGarcinia brasiliensisMart. epicarp, its partition-obtainedfractions (hexane fractions, ethyl acetate fraction, andaqueous fraction) and the isolated molecules fukugetinand 7-epiclusianone on adult Schistosoma mansoniwormsMice infected with cercariae of S. mansoni (LE strain) were sacri-ficed 45 days post-infection using 3.0% pentobarbital sodium,which was administered intraperitoneally (± 0.3mL/mouse) andperfused, according to the method previously described [49]. Therecovered worms were cultured in 6-well culture dishes (fourpairs per well) with a final volume of 4.0mL of RPMI-1640 me-dium supplemented with 5.0% heat-inactivated fetal bovine se-rum and 1.0% penicillin (10000 IU/mL) and streptomycin(10.0mg/mL) (Sigma). The samples, including EEE, fractions (HF,EAF, and AF), and the isolated compounds 7-epi and fuk wereprepared with methanol in a concentration of 2.0mg/mL. These

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solutions were added to the cultures at different concentrations(EEE and fractions at 50.0, 60.0, 75.0, and 100.0 µg/mL; fuk at50.0, 100.0, and 200.0 µg/mL; 7-epi at 5.0, 10.0, 14.0, 15.0, and25.0 µg/mL) and maintained at 37°C with 5.0% CO2. The cultureswere analyzed at 2 h and 24 h after addition to the samples. In thecontrol groups, the worms were kept under the same conditions,except for the presence of G. brasiliensis-derived samples. After24 h, the worms were washed with culture medium and main-tained under the same conditions, but without the presence ofthe sample, for the remainder of the experiment. Under an in-verted microscope, observations about the worms were docu-mented at 2 and 24 h after the addition of the samples (extract,fraction, and compounds). In addition, the samples were re-moved and the worms were analyzed at 24, 48, 72, and 96 h afterthe first rinsing. A final time point was analyzed eight days afterthe initiation of the experiment.

Evaluation of the excretory system activity of theadult Schistosoma mansoni worm after exposure to7-epiclusianone via resorufin labelingAfter recovery and washing, four couple worms were maintainedin 4.0mL of RPMI-1640 culturemedium supplementedwith 5.0%(v/v) BFS and 1.0% penicillin (10000 IU/mL) and streptomycin(10.0mg/mL). Then, 10.0 µL of resorufin (stock solution 10.0mg/mL in medium) was added to each well and incubated at 37°Cunder 5.0% CO2 for 30min.Afterwards, the worms were washed five times with 2.0mL ofRPMI to remove the excess of probe. Subsequently, (i) 4.0mL ofRPMI-1640 supplemented medium (medium control), (ii)40.0 µL of methanol (solvent control), (iii) 2.5 µL (2.0 µg/mL) of aPZQ (Cestox, MERCK, 100.01% purity) stock solution at 0.8mg/



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mL (compound control), and (iiii) 7-epi at concentrations of12.0 µg/mL, 14.0 µg/mL, and 20.0 µg/mL from a 2.0-mg/mL stocksolution were added into each well.Finally, the worms were incubated at 37°C and 5.0% CO2 for15min. They were once again washed (five times with culturemedium) and transferred to glass slides delimited with vaseline,to prevent leakage, using a small quantity of culture mediumsupplemented with 1.0% sodium pentobarbital at 3.0% (Hypnol,Fontoveter) to inhibit the parasites movements. These tests wereperformed in triplicate.Afterwards, the tests were analyzed with a fluorescence micro-scope (Nikon, Eclipse 80i) using a rhodamine filter for resorufin(maximum excitation/emission of resorufin at 571/585 nm).

Evaluation of damage to the tegument ofthe adult worm of Schistosoma mansoni exposedto a 7-epiclusianone probe after labeling withHoechst 33258The methods utilized to detect tegument damage were describedby Lima et al. [50]. Briefly, the worms were washed five timeswith 2.0mL of RPMI to remove the excess of probe. Subsequently,(i) 4.0mL of RPMI-1640 supplemented medium (medium con-trol), (ii) 40.0 µL of methanol (solvent control), (iii) 2.5 µL (2.0 µg/mL) of a PZQ stock solution at 0.8mg/mL (compound control),and (iiii) 7-epi at concentrations of 12.0 µg/mL, 14.0 µg/mL, and20.0 µg/mL from a 2.0-mg/mL stock solution were added intoeach well and incubated for 24 hours at 37°C with 5.0% CO2. Atthe end of this period, the worms were washed and incubatedfor 15min with 10.0 µL of Hoechst 33258 (stock solution10.0mg/ml). Damage was evaluated by fluorescence microscopyafter washing to remove excess probe from the worms. Parasiteswere put onto the slides with a small amount of culture mediumsupplemented with 1.0% to 3.0% pentobarbital sodium (Hypnol,Fontoveter) to inhibit parasite movement. The slides were thenobserved with a fluorescence microscope (Nikon, Eclipse 80i) us-ing a DAPI filter (excitation/emission maximum of Hoechst 352/455 nm). These tests were performed in triplicate.

In vitro studies of the effect of 7-epiclusianonein cercariaeInfected B. glabrata were induced to eliminate cercariae by ex-posing them to light for 1 h in dechlorinated water. Approxi-mately 100 cercariae (100.0 uL) were placed into a 24-well tissueculture plate with 900.0 µL of dechlorinated water, and exposedto 12.5, 25.0, 50.0, 100.0, and 150.0 µg/mL of 7-epi. After 1, 2, 4,6, and 8 h, motility, contraction, and loss of tail were observed[44] using inverted microscopy. All experiments were performedin triplicate and repeated at least three times usingmethanol andpure water as negative controls. PZQ up to 12.5 µg/mL was usedas a positive control.

In vitro studies of 7-epiclusianone in schistosomulaCercariae were transformed into schistosomula by mechanicaltransformation using a Vortex mixer [51]. Schistosomula werecultured in RPMI-1640 supplemented medium (RPMI-1640+ 5.0% BFS + 100.0 µg/mL of penicillin and streptomycin) and ex-posed to 8.0, 12.0, 24.0, 25.0, 50.0, and 100.0 µg/mL of 7-epi. at37°C under 5.0% CO2. The schistosomula were analyzed after 2,24, and 48 h using inverted microscopy. All experiments wereperformed in triplicate and repeated at least three times using amethanol solution with supplemented RPMI-1640 medium as anegative control and PZQ at 12.5mg/mL as a positive control.


Statistical analysesStatistical evaluation of the results was performed using theSISVAR 5.3 software using analysis of variance (ANOVA) and thetest of SNK was applied to observe significant differences be-tween mean values (p < 0.05).

AcknowledgementsThe authors thank FAPEMIG, CNPq, CAPES, and FINEP for finan-cial support and scholarships, and Dr. Paulo Marcos Zech Coelhofor his help and support.

Conflict of Interest!

The authors declare no conflict of interest.

Affiliations1 Department of Pharmacy, Laboratory of Phytochemistry and MedicinalChemistry, Federal University of Alfenas, Alfenas, Minas Gerais, Brazil

2 Laboratory of Schistosomiasis, Institute René Rachou-Fiocruz, Belo Horizonte,Brazil

3 Department of Pathology and Parasitology, Laboratory of Parasitology,Institute of Biomedical Sciences, Federal University of Alfenas, Alfenas, MinasGerais, Brazil

4 Departament of Chemistry, Federal University of Viçosa, Viçosa-MG, Brazil

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Potent Schistosomicidal Constituents from Garcinia brasiliensis

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