Original Papers
733
Authors
Aline Pereira Castro 1, 3, Ana Carolina Alves de Mattos 2, Neusa Araújo Pereira 2, Naira Ferreira Anchieta 3, Matheus Siqueira Silva 1, Danielle Ferreira Dias 1, Claudinei Alves Silva 1, Giulliano Vilela Barros 3, Raquel Lopes Martins Souza 3, Marcelo Henrique Dos Santos 4, Marcos José Marques 3
Affiliations
The affiliations are listed at the end of the article
Key words " Clusiaceae l " Garcinia brasiliensis l " 7‑epiclusianone l " fukugetin l " Schistosoma mansoni l " Schistosomiasis l
Abstract !
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
Introduction !
received revised accepted
February 16, 2015 February 16, 2015 March 14, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1545927 Published online April 23, 2015 Planta Med 2015; 81: 733–741 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Marcos José Marques Laboratory of Parasitology Institute of Biomedical Sciences Federal University of Alfenas Department of Pathology and Parasitology Gabriel Monteiro da Silva – 700 Alfenas, Minas Gerais 37130000 Brazil Phone: + 55 35 32 99 10 67 [email protected]
Schistosomiasis is a neglected tropical disease that is caused by trematode flatworms of the genus Schistosoma. It is a disease that threatens millions of people, especially in poor regions [1]. The World Health Organization (WHO) estimated, in 2003, that approximately 207 million people were infected with Schistosoma. Moreover, 779 million people are at risk of contracting this disease [2], and at least 200 000 people die each year due to schistosomiasis [3]. Praziquantel (PZQ) is effective against all clinical forms of schistosomiasis, However, the emergence of resistant strains has spurred on the search for new active antihelminthic compounds, especially those derived from plants [4, 5]. The search for antiparasitic compounds from natural sources has increased over the last decade [4–6]. In this context, plants remain an important source of biologically active compounds, which can provide structures for the development of new drugs [7].
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.
Several chemical studies have shown that the genus Garcinia is an important source of natural products with a wide variety of biologically active metabolites, including polyisoprenylated benzophenones, flavonoids, xanthones, and proanthocyanidins, which have been proven to be effective against various diseases [8]. Many studies have demonstrated that Garcinia brasiliensis provides anti-inflammatory [9], antinociceptive [10], antioxidant, and antitumor activities [11]. The tetraprenylated benzophenone 7-epiclusianone (7" Fig. 1) was first isolated from the fruits of epi) (l G. brasiliensis [12]. Studies have demonstrated that the 7-epi extracted from the fruits of G. brasiliensis is effective against the trypomastigotes of Trypanosoma cruzi [13] and has vasodilatory [14], antianaphylactic [15], anti-HIV [16], antimicrobial [17, 18], antispasmodic [19], and antiproliferative effects [20]. Antileishmanial activity [21] and analgesic and anti-inflammatory [10] effects have also been assigned to this benzophe" Fig. 1) is a bioflavonoid none. Fukugetin (fuk) (l initially obtained from Garcinia spp, which also has demonstrated anti-inflammatory [21] and
Castro AP et al. Potent Schistosomicidal Constituents …
Planta Med 2015; 81: 733–741
Downloaded by: University of Illinois. Copyrighted material.
Potent Schistosomicidal Constituents from Garcinia brasiliensis
Original Papers
Fig. 1 A Chemical structure of 7-epi. B Chemical structure of fuk.
anti-HIV‑1 activities [22] as well as tumor angiogenesis inhibition [23]. Due to the difficulties in treating diseases caused by helminths, especially schistosomiasis, this study evaluated the schistosomicidal activity of both an ethanolic extract of G. brasiliensis Mart. epicarp (EEE) and other fractions obtained by additional extractions. The evaluation of the hexane fractions (HF), ethyl acetate fraction (EAF), and aqueous fraction (AF) enables the isolation of molecules with a potential therapeutic use in the treatment of schistosomiasis, as substances with potential antiparasitic activity have been isolated from plants in this family in recent years [24].
Results and Discussion !
Recently, the development of new drugs from medicinal plants for the treatment of neglected diseases, including schistosomiasis, has attracted the attention of many scientific research centers around the world. For example, plants of the genus Artemisia [25], extracts of Zanthoxylum naranjillo [26], and crude aqueous extracts of Zingiber officinale [27], among others, have been effective against Schsitosoma mansoni. With this in mind, the present study was devised to evaluate the schistosomicidal potential of EEE and the partition-obtained fractions (HF, EAF, FA) and compounds isolated from the fractions, the benzophenone 7-epi and the bioflavonoid fuk, by means of a bioguided test. The total amount of flavonoid found in the ethanolic epicarp extract of G. brasiliensis was 22.52 µg in quercetin equivalents/g of the extract. The flavonoids were known for their beneficial effects on health. An important effect of flavonoids is the scavenging of oxygen-derived free radicals. Moreover, in vitro experimental systems also showed that flavonoids possess anti-inflammatory, antiallergic, antiviral, and anticarcinogenic properties [28]. Many plants rich in flavonoids have demonstrated activity against S. mansoni such as Baccharis trimera, popularly known as carqueja [29], Allium sativum and Allium cepa, popularly known as garlic and onion, respectively [30], and Zingiber officinale, popularly known as ginger [27]. The yields of the EEE and the partitioned fractions HF, EAF, and AF " Fig. 2, the were 10.0 %, 39.0 %, 42.0 %, and 14.5 %, respectively. In l retention times, areas, and quantification of the isolated compounds in the extract and the fractions are shown. The analytical curves consisted of six data points, and three replicate injections at each concentration level were performed. The statistical analysis of the data revealed excellent linearity (r > 0.99) over a concentration range from 10.0 to 300.0 mg/mL.
The limits of detection (LOD) and quantitation (LOQ) were 1.5 and 4.5 mg/mL for fuk (y = 77 296 x-269 178/SD = 173 217), and 0.87 and 2.6 mg/mL for 7-epiclusianone (y = 38 539 x-84 655/ SD = 145 667), respectively. Chemical analysis of the EEE identified 7-epi as the major constit" Fig. 2 A). In uent (140.02 mg/g), followed by fuk (35.86 mg/g) (l the HF, 7-epi was identified as the major constituent (278, " Fig. 2 B). A 74 mg/g), whereas the fuk peak was not identified (l chemical analysis of the EAF identified 7-epi as the major constit" Fig. 2 C). uent (209.04 mg/g [15]), followed by fuk (24.19 mg/g) (l Finally, in the AF, neither 7-epi nor fuk peaks were identified " Fig. 2 D). These peaks may be compared with the standards of (l " Fig. 2 E, F). these compounds (l Those results are in accord with previous results that demonstrated four phenolic components, identified as fukugiside andguttiferone A; besides fuk and 7-epi in EEE [10]. These compounds were characterized by comparison to the standards previously isolated [31, 32]. The 7-epi, a natural polyisoprenylated benzophenone, was first isolated from the fruits of G. gardneriana [12] and has shown activity against trypomastigotes of Trypanosoma cruzi in vitro [13], a potent endothelium vasodilator effect [14], and anti-anaphylactic [15], anti-HIV [16], antimicrobial [17], antispasmodic [11], antiproliferative [19], and leishmanicidal activities [20]. Various parameters, including changes in the integrity of the integument and motility as well as reductions in mating and oviposition, are often evaluated as indicators of biological activity and used in in vitro toxicity studies against adult worms of Schistosoma species [4–6, 33, 34]. The schistosomicidal effect of the EEE and its fractions and isolated compounds in vitro was analyzed with respect to the time of incubation and concentration. All adult worms were dead after 24 h exposure to 100.0 µg/mL of EEE, 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 (the highest dose tested). In contrast, the worms remained viable in the negative control group (RPMI-1640 supplemented with RPMI and methanol). The use of PZQ at 2.0 µg/mL as a positive control resulted in the death of all of the parasites within 24 h. Mating and egg laying of S. mansoni females werenʼt observed at doses greater than 50.0 µg/mL and 4.0 µg/mL for the EEE (and their fractions) and 7-epi, respectively. Adult male and female worms remained separate and no oviposition was observed. Regarding the reproductive aptitude of the adult worms of S. mansoni, the components of other plants have shown effects on the oviposition of the worm. For example, curcumin, which was extracted from the rhizomes of Curcuma longa [4] and some phlor-
Castro AP et al. Potent Schistosomicidal Constituents … Planta Med 2015; 81: 733–741
Downloaded by: University of Illinois. Copyrighted material.
734
Original Papers
735
oglucinol derivatives that were obtained from the rhizomes of the Dryopteris species [5], demonstrated in vitro effects on egg laying. A decrease in oviposition was also observed using Nigella sativa [35] and a ginger rhizome (Z. officinale) extract [36]. Similarly, severe tegumental morphological changes were also observed after the incubating of adult S. mansoni worms with the extracts and fractions at concentrations above 50.0 µg/mL and with 7-epi at concentrations greater than 10.0 µg/mL. However, no abnormality was observed in worms in the control group (RPMI-1640 supplemented with RPMI and methanol). The damage to the tegument of adult S. mansoni worms was confirmed using a Hoechst 33 258 probe, which is a specific marker for cellular DNA. To assess the damage of the integument, the worms exposed to 2.0 µg/mL of PZQ and 14.0 µg/mL (ED100) of 7-epi were incubated and subsequently placed in contact with the Hoechst 33 258 probe. Analysis by microscopy showed progressive damage on the surface, causing blisters and the detachment " Fig. 3). PZQ and 7-epi were able to induce leof the tegument (l sions in the tegument of parasites, and the lesions in S. mansoni " Fig. 3). Changes males were more pronounced than in females (l in the surface of the tegument showed a dose-dependent effect. The Hoechst 33 258 probe is very sensitive in marking the regions where damage is present, and any damage or injury to the integument of the worm is precisely marked by this probe [37]. This soft tissue damage was similar to the damage reported in studies with PZQ [38]. Data obtained in this study demonstrated that the EEE, HF, EAF, and 7-epi exhibited greater activity against adult male S. mansoni worms. Indeed, 7-epi was more effective, which demonstrated an " Fig. 4). The lethality was greater in male ED100 of 14.0 µg/mL (l worms after 24 h of incubation with 10.0 µg/mL of 7-epi. There-
fore, this compound paralyzes the excretory activity in concentrations more than 12 and 14 µg/mL of 7-epi in males and females, respectively. In several studies, based on the in vitro activity, disparities in drug susceptibility between males and females of S. mansoni have been observed. S. mansoni males are often more susceptible than female worms in studies on resistance to oxamniquine [39] and in studies regarding the bioactivity of ginger extract [36]. In contrast, the results with other compounds, such as fatty aminoalkanethiosulfuric [34] and 2-[butylamino]-1-phenyl-1-ethanethiosulfuric acid [6], revealed higher survival rates for males than females. Notably, in vitro data obtained in this study demonstrated that the EEE, HF, EAF, and 7-epi exhibited greater activity against adult male S. mansoni worms. Serious injury was reported in the integument of female adult S. mansoni worms when treated with artemether [33, 40]. The seed tegument is extremely important for successful infection and survival in the host and has been a major target for antischistosomal drug development. Therefore, most of the drugs that are currently used against schistosomiasis, including PZQ [38], mefloquine [40], and artemether [41], act by damaging the integument of the worm. The results of these experiments revealed that 7-epi was able to paralyze the excretory activity of the adult worms of S. mansoni. This was observed by labeling the parasites with resorufin after a previous exposure to 7-epi, which prevented the output of the probe [substrate for P-glycoprotein (Pgp)] to the external environment. The secretory activity of worms initially exposed to resorufin and subsequently incubated with 14.0 µg/mL (ED100) of 7-epi was completely inhibited, as characterized by the accumulation of the probe in tissues other than the excretory system
Castro AP et al. Potent Schistosomicidal Constituents …
Planta Med 2015; 81: 733–741
Downloaded by: University of Illinois. Copyrighted material.
Fig. 2 Chromatograms obtained by high-performance 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 components found in the extract and the fractions identified by HPLC.
Original Papers
Fig. 3 In vitro evaluation of the effect of 14.0 µg/ mL of 7-epi on the tegument of S. mansoni adult worms. A, B S. mansoni LE not exposed to 7-epi and labelled with the probe Hoechst 33 258. C, D S. mansoni exposed to 7-epi and labelled with the probe Hoechst 33 258. The fluorescent areas indicate intense lesions. Each scale bar represents 100 µm. E, F S. mansoni exposed to 2 µg/ml PZQ and labelled with the probe Hoechst 33 258. The fluorescent areas indicate intense lesions. (Color figure available online only.)
Fig. 4 In vitro effects of 7-epi and fuk against adult S. mansoni; *** p < 0.001.
" Fig. 5). This compound paralyzed the excretory activity, too, as (l observed in other concentrations of 12, 16 and 18 µg/mL. How-
ever, with 12 µg/mL, there was no change observed in the female worms. The modulatory effect of some drugs has previously been shown through interactions with the cellular transport proteins of the excretory system in male adult S. mansoni worms [42]. Thus, the results from present work suggest, for the first time, that 7-epi acted in the excretory system and interfered with the activity of Pgp in an unknown manner. A similar result was observed in the presence of amiloride (an inhibitor of Na+ and Na+/H+ ATPase) [42]. Some hypotheses can be considered to explain the results obtained in this study, such as (i) it is possible that 7-epi depletes energy by increasing the calcium influx into tissues, leading to an excessive 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 Resorufin; (iv) 7-epi can act indirectly on the excretion of resorufin, affecting tubular membrane phospholipids and causing the probe to diffuse to places other than the excretory system [43], or (v) a simple contraction induced by 7-epi could cause a delay in the excretion of the probe due to a lack of a constant intestinal motility from the excretory organ. Thus, the probe would have a greater opportunity to diffuse to the surrounding tissues. In the present study, we used the loss of motility and tail as measures to evaluate the sensitivity of cercariae when incubated with 7-epi. At a concentration of 12.5 µg/mL of 7-epi, the cercariae were immobile after 2 h and 8 h of incubation, and the separation
Castro AP et al. Potent Schistosomicidal Constituents … Planta Med 2015; 81: 733–741
Downloaded by: University of Illinois. Copyrighted material.
736
Original Papers
737
Downloaded by: University of Illinois. Copyrighted material.
Fig. 5 In vitro evaluation of the effect of 14.0 µg/ mL of 7-epi on the excretory system of S. mansoni adult worms. A,B S. mansoni labelled with the probe resorufin and not exposed to 7-epi. C,D S. mansoni labelled with the probe resorufin and exposed to 14.0 µg/mL of 7-epi. Arrow: Main tubule (MT), nephridiopore (N), and ramifications of the excretory system. E,F S. mansoni labelled with the probe resorufin and exposed to 2 µg/ml of PZQ. (Color figure available online only.)
Fig. 6 Effect of different concentrations of 7-epi on the viability of cercariae, based on the rate (%) of loss of tail. Data are presented as the average of three experiments; ***p < 0.0001.
Fig. 7 Effect of various concentrations of 7-epi on the viability of schistosomules, based on the rate (%) of loss of movement. Data are presented as average of three experiments; ***p < 0.0001.
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" Fig. 6). The exact ence tail loss) were observed for up to 8 h (l mechanism of induction of tail loss is not fully understood. It is believed that the process relates to a particular structure between the body and the tail of cercariae. The rearward end of the body of cercariae is folded and tapered like a collar on a narrow structure that connects the body to the tail. This connective structure is delicate and can be easily broken [44].
In the absence of 7-epi, schistosomula viability appeared normal, without any morphological alteration for up to 48 h. Slight morphological changes, such as granularity, shortening body with a sickle shape, and low activity, were observed after 24 h of incubation 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, and " Fig. 7). However, at all of these 48 h of incubation, respectively (l doses, severe morphological changes, such as black granules, and an intense vacuolization around the body associated with a re-
Castro AP et al. Potent Schistosomicidal Constituents …
Planta Med 2015; 81: 733–741
Original Papers
duction in length, were observed in the first two hours of incubation. Concentrations from 7-epi 25.0 µg/mL resulted in a complete lack of mobility and intense vacuolization after 24 h incubation. Schistosomula belonging to the negative control group (RPMI or RPMI methanol 0.1 %) demonstrated viability for a standard 48 h. A complete lack of mobility and severe vacuolation were also observed after PZQ treatment. The effects observed in this study were similar to those observed in schistosomula incubated with 15.0 µg/mL of mefloquine and 100.0 µg/mL of the essential oil of Piper cubeta [45]. The generalized use of folk medicine suggests that natural products are harmless; however, the traditional use of these natural products is not satisfactory proof of their efficacy. In this context, the efficacy and safety of natural products require further study because the toxicity of the extract and the isolated molecules, 7epi and fuk, have been demonstrated on murine peritoneal macrophages. However, no significant toxicity was observed in mammalian cell extracts when the isolated molecules were used [20]. We demonstrated that 7-epi is an effective compound for use against S. mansoni adult worms, cercarie, and schistosomula in vitro. With its extensive effects on the mortality rate, reproductive fitness, parasite tegument morphology, and adult S. mansoni worm excretory activity, 7-epi is a promising composite schistosomicidal. However, further studies are needed to elucidate the mechanism(s) of toxicity of 7-epi and evaluate the in vivo activity of this compound.
Materials and Methods !
Plant material and extract preparation The fruits of G. brasiliensis (Mart.) were collected on the campus of the Federal University of Viçosa-MG, Brazil in February (summer) of 2011. Botanical identification was performed in the Horto Botânico of the Federal University of Viçosa by Dr. João Augusto Alves Meira Neto. A voucher specimen (number VIC2604) was deposited at the herbarium of the Federal University of Viçosa. Epicarps of G. brasiliensis were dried in an oven with a circulating air temperature of 40 °C for eight days. Dried and powdered G. brasiliensis fruit pericarps (1000.0 g) were extracted by maceration with 3.0 L of ethanol at room temperature, filtered, and then dried using a rotary evaporator under reduced pressure at 45 °C. This procedure was repeated five times yielding an amount of 80.0 g of fruit pericarp of the ethanolic extract (EEE).
Isolation and characterization A portion of the EEE (1.0 g) was then subjected to a liquid/liquid partition as follows: the sample was dissolved into an ethanol : water solution (1 : 4, 50.0 mL), and after the addition of hexane (50.0 mL), the mixture was vigorously stirred. The organic layer was removed to give the HF (this procedure was performed five times). Some insoluble compounds were retained in the aqueous phase, to which ethyl acetate (50.0 mL) was added and vigorously stirred. Similarly, the organic layer was removed to produce the EAF (this procedure was performed five times). The HF, EAF, and AF were concentrated at a reduced pressure using a rotary evaporator and were then dried in a lyophilizer and stored in a freezer [46]. To isolate the bioactive compounds, the HF and EAF were generated from the partition of the EEE and chromatographed on a silica gel (230–400 mesh) column (8 × 100 cm). Samples were eluted with increasingly polar mixtures of hexane/ethyl
acetate and ethyl acetate/ethanol to give 50 fractions, and the fractions (200.0 mL) were concentrated on a rotavapor at a reduced pressure. These fractions were pooled in three groups according to their similarities in thin-layer chromatography (TLC). When necessary, the samples were rechromatographed on a silica gel column as reported above to give 26 fractions (10.0 mL) and were then pooled into five groups according to similarities in TLC, and recrystallized several times using methanol to obtain pure compounds. 7-Epi was isolated from the HF, and both 7-epi and fuk were isolated from the EAF. Previously, isolated 7-epi and fuk from the Laboratory of Phytochemistry and Medicinal Chemistry of UNIFAL‑MG were used as standards for identification as well as for spectral data [10, 11]. The purity of 7-epi and fuk (99.0 and 98.0%, respectively) was evaluated by chromatographic analysis. The procedure adopted for the preparation of the ex" Fig. 8. tract, fractions, and isolated compounds is outlined in l
Chromatographic analysis and quantification The chromatographic analysis of the extracts and fractions, and the quantification of isolated molecules present in the extract and fractions were performed on a liquid chromatography device (Shimadzu HPLC) using an NST column (Nano Separation Technologies) C18-154 605 (150 × 4.6 mm; 5.0 mm in particle size) [20]. The mobile phase consisted of a mixture of acetic acid (5 mM/L) (eluent A) and methanol/acetic acid (0.1 % v/v) (eluent B). The injection volume was 20.0 µL at a flow rate of 1.0 mL/ min. During the initial 10 min, an analysis was performed using 50.0 % of eluent B followed by an increase in the concentration of eluent B to 100.0 % over 20 min. The analysis continued for 30 min at a concentration of 100.0 % eluent B. After the analysis with eluent B was concluded, the column was prepared for the next analysis. Chromatograms were obtained at 254 nm and the peaks were compared with the peaks of the compounds that had been previously isolated in the laboratory. For the analytical curves, aliquots of 0.100, 0.300, 0.500, 1.00, 2.00, and 3.00 mL, from the standard stock solutions of fuk and 7-epi (1.00 mg/mL) were transferred to separate 10.0 mL volumetric flasks. The volumes were completed with deionized water prior to injection. Concentration ranges from 10.0 to 300.0 µg/mL of fuk and 7-epi were obtained. Each solution was analyzed in triplicate, and the resulting peak areas were plotted against the respective concentrations. The criteria used to determine the LOD and LOQ were based on the determination of the slope (S) of the analytical curve and the standard deviation of responses (SD) in accordance with the formulas LOD = 3.3 SD/S and LOQ = 10 SD/S. The SD was determined from the standard error estimated by the regression line [47]. Extracts samples were assayed against reference standards and each concentration of fuk and 7-epi was calculated using an analytical curve.
Determination of total flavonoids In a 10.0-mL test tube, 0.5 mL of 20.0 % (w/v) EEE, 1.5 mL of ethanol, 0.1 mL of 10.0 % (w/v) AlCl3 · 6H2O, and 0.1 mL of 1 M potassium acetate were combined and mixed. The volume was then brought up to a final volume of 5.0 mL with H2O. After 30 min, the mixture was measured at 425 nm. The standard curve for total flavonoids was generated with a quercetin [Quercetin dehydrate (≥ 99.0% HPLC) was purchased from Sigma-Aldrich Co.] standard solution (25–120 g/mL) using the same procedure as above. Total flavonoids were determined as quercetin equivalents
Castro AP et al. Potent Schistosomicidal Constituents … Planta Med 2015; 81: 733–741
Downloaded by: University of Illinois. Copyrighted material.
738
Original Papers
739
(mg quercetin/g extract), and the values are presented as the means of triplicate analyses [48].
Parasite and experimental animals The LE strain of S. mansoni used in this study was routinely maintained by serial passage in Biomphalaria glabrata and Swiss mouse models. The LE strain has been maintained for more than 30 years at the Research Center René Rachou/Oswaldo Cruz Foundation. All experiments were authorized by the Ethical Committee for Animal Care of the Federal University of Alfenas (approval number: 534/2013 January 20, 2014) in accordance with the national and international accepted principles for laboratory animal use and care.
In vitro evaluation of the effect of ethanolic extract of Garcinia brasiliensis Mart. epicarp, its partition-obtained fractions (hexane fractions, ethyl acetate fraction, and aqueous fraction) and the isolated molecules fukugetin and 7-epiclusianone on adult Schistosoma mansoni worms Mice infected with cercariae of S. mansoni (LE strain) were sacrificed 45 days post-infection using 3.0 % pentobarbital sodium, which was administered intraperitoneally (± 0.3 mL/mouse) and perfused, according to the method previously described [49]. The recovered worms were cultured in 6-well culture dishes (four pairs per well) with a final volume of 4.0 mL of RPMI-1640 medium supplemented with 5.0 % heat-inactivated fetal bovine serum and 1.0 % penicillin (10 000 IU/mL) and streptomycin (10.0 mg/mL) (Sigma). The samples, including EEE, fractions (HF, EAF, and AF), and the isolated compounds 7-epi and fuk were prepared with methanol in a concentration of 2.0 mg/mL. These
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 at 50.0, 100.0, and 200.0 µg/mL; 7-epi at 5.0, 10.0, 14.0, 15.0, and 25.0 µg/mL) and maintained at 37 °C with 5.0 % CO2. The cultures were analyzed at 2 h and 24 h after addition to the samples. In the control groups, the worms were kept under the same conditions, except for the presence of G. brasiliensis-derived samples. After 24 h, the worms were washed with culture medium and maintained under the same conditions, but without the presence of the sample, for the remainder of the experiment. Under an inverted microscope, observations about the worms were documented at 2 and 24 h after the addition of the samples (extract, fraction, and compounds). In addition, the samples were removed and the worms were analyzed at 24, 48, 72, and 96 h after the first rinsing. A final time point was analyzed eight days after the initiation of the experiment.
Evaluation of the excretory system activity of the adult Schistosoma mansoni worm after exposure to 7-epiclusianone via resorufin labeling After recovery and washing, four couple worms were maintained in 4.0 mL of RPMI-1640 culture medium supplemented with 5.0 % (v/v) BFS and 1.0 % penicillin (10 000 IU/mL) and streptomycin (10.0 mg/mL). Then, 10.0 µL of resorufin (stock solution 10.0 mg/ mL in medium) was added to each well and incubated at 37 °C under 5.0 % CO2 for 30 min. Afterwards, the worms were washed five times with 2.0 mL of RPMI to remove the excess of probe. Subsequently, (i) 4.0 mL of RPMI-1640 supplemented medium (medium control), (ii) 40.0 µL of methanol (solvent control), (iii) 2.5 µL (2.0 µg/mL) of a PZQ (Cestox, MERCK, 100.01 % purity) stock solution at 0.8 mg/
Castro AP et al. Potent Schistosomicidal Constituents …
Planta Med 2015; 81: 733–741
Downloaded by: University of Illinois. Copyrighted material.
Fig. 8 Bioassay scheme used for the extraction and purification of the constituents from G. brasiliensis epicarp.
Original Papers
mL (compound control), and (iiii) 7-epi at concentrations of 12.0 µg/mL, 14.0 µg/mL, and 20.0 µg/mL from a 2.0-mg/mL stock solution were added into each well. Finally, the worms were incubated at 37 °C and 5.0 % CO2 for 15 min. They were once again washed (five times with culture medium) and transferred to glass slides delimited with vaseline, to prevent leakage, using a small quantity of culture medium supplemented with 1.0 % sodium pentobarbital at 3.0 % (Hypnol, Fontoveter) to inhibit the parasites movements. These tests were performed in triplicate. Afterwards, the tests were analyzed with a fluorescence microscope (Nikon, Eclipse 80i) using a rhodamine filter for resorufin (maximum excitation/emission of resorufin at 571/585 nm).
Statistical analyses Statistical evaluation of the results was performed using the SISVAR 5.3 software using analysis of variance (ANOVA) and the test of SNK was applied to observe significant differences between mean values (p < 0.05).
Acknowledgements The authors thank FAPEMIG, CNPq, CAPES, and FINEP for financial support and scholarships, and Dr. Paulo Marcos Zech Coelho for his help and support.
Conflict of Interest !
Evaluation of damage to the tegument of the adult worm of Schistosoma mansoni exposed to a 7-epiclusianone probe after labeling with Hoechst 33 258 The methods utilized to detect tegument damage were described by Lima et al. [50]. Briefly, the worms were washed five times with 2.0 mL of RPMI to remove the excess of probe. Subsequently, (i) 4.0 mL of RPMI-1640 supplemented medium (medium control), (ii) 40.0 µL of methanol (solvent control), (iii) 2.5 µL (2.0 µg/ mL) of a PZQ stock solution at 0.8 mg/mL (compound control), and (iiii) 7-epi at concentrations of 12.0 µg/mL, 14.0 µg/mL, and 20.0 µg/mL from a 2.0-mg/mL stock solution were added into each well and incubated for 24 hours at 37 °C with 5.0 % CO2. At the end of this period, the worms were washed and incubated for 15 min with 10.0 µL of Hoechst 33 258 (stock solution 10.0 mg/ml). Damage was evaluated by fluorescence microscopy after washing to remove excess probe from the worms. Parasites were put onto the slides with a small amount of culture medium supplemented with 1.0% to 3.0% pentobarbital sodium (Hypnol, Fontoveter) to inhibit parasite movement. The slides were then observed with a fluorescence microscope (Nikon, Eclipse 80i) using 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-epiclusianone in cercariae Infected B. glabrata were induced to eliminate cercariae by exposing them to light for 1 h in dechlorinated water. Approximately 100 cercariae (100.0 uL) were placed into a 24-well tissue culture plate with 900.0 µL of dechlorinated water, and exposed to 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 performed in triplicate and repeated at least three times using methanol and pure water as negative controls. PZQ up to 12.5 µg/mL was used as a positive control.
In vitro studies of 7-epiclusianone in schistosomula Cercariae were transformed into schistosomula by mechanical transformation using a Vortex mixer [51]. Schistosomula were cultured in RPMI-1640 supplemented medium (RPMI-1640 + 5.0 % BFS + 100.0 µg/mL of penicillin and streptomycin) and exposed to 8.0, 12.0, 24.0, 25.0, 50.0, and 100.0 µg/mL of 7-epi. at 37 °C under 5.0 % CO2. The schistosomula were analyzed after 2, 24, and 48 h using inverted microscopy. All experiments were performed in triplicate and repeated at least three times using a methanol solution with supplemented RPMI-1640 medium as a negative control and PZQ at 12.5 mg/mL as a positive control.
The authors declare no conflict of interest.
Affiliations 1
2
3
4
Department of Pharmacy, Laboratory of Phytochemistry and Medicinal Chemistry, Federal University of Alfenas, Alfenas, Minas Gerais, Brazil Laboratory of Schistosomiasis, Institute René Rachou-Fiocruz, Belo Horizonte, Brazil Department of Pathology and Parasitology, Laboratory of Parasitology, Institute of Biomedical Sciences, Federal University of Alfenas, Alfenas, Minas Gerais, Brazil Departament of Chemistry, Federal University of Viçosa, Viçosa-MG, Brazil
References 1 King CH. Parasites and poverty: the case of schistosomiasis. Acta Trop 2010; 113: 95–104 2 Steinmann P, Keiser J, Bos R, Tanner M, Utzinger J. Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. Lancet Infect Dis 2006; 6: 411–425 3 King CH, Dickman K, Tisch DJ. Reassessment of the cost of chronic helmintic infection: a meta-analysis of disability-related outcomes in endemic schistosomiasis. Lancet 2005; 365: 1561–1569 4 Magalhães LG, Machado CB, Morais ER, Moreira EBDC, Soares CS, da Silva SH, Da Silva Filho AA, Rodrigues V. In vitro schistosomicidal activity of curcumin against Schistosoma mansoni adult worms. Parasitol Res 2009; 104: 1197–1201 5 Magalhães LG, Kapadia GJ, da Silva Tonuci LR, Caixeta SC, Parreira NA, Rodrigues V, Da Silva Filho AA. In vitro schistosomicidal effects of some phloroglucinol derivatives from Dryopteris species against Schistosoma mansoni adult worms. Parasitol Res 2010; 106: 395–401 6 de Araújo SC, de Mattos ACA, Teixeira HF, Coelho PMZ, Nelson DL, de Oliveira MC. Improvement of in vitro efficacy of a novel schistosomicidal drug by incorporation into nanoemulsions. Int J Pharm 2007; 337: 307–315 7 Tonuci LRS, de Melo NI, Dias HJ, Wakabayashi KAL, Aguiar GP, Aguiar DP, Mantovani ALL, Ramos RC, Groppo M, Rodrigues V, Veneziani RCS, Cunha WR, da Silva Filho AA, Magalhães LG, Crotti AEM. In vitro schistosomicidal effects of the essential oil of Tagetes erecta. Rev Bras Farmacogn 2012; 22: 88–93 8 Derogis PBMC, Martins FT, de Souza TC, de C Moreira ME, Souza Filho JD, Doriguetto AC, de Souza KRD, Veloso MP, Dos Santos MH. Complete assignment of the 1H and 13C NMR spectra of garciniaphenone and keto-enol equilibrium statements for prenylated benzophenones. Magn Reson Chem 2008; 46: 278–282 9 Santa-Cecília FV, Freitas LA, Vilela FC, Veloso CDC, da Rocha CQ, Moreira MEC, Dias DF, Giusti-Paiva A, dos Santos MH. Antinociceptive and antiinflammatory properties of 7-epiclusianone, a prenylated benzophenone from Garcinia brasiliensis. Eur J Pharmacol 2011; 670: 280–285 10 Santa-Cecília FV, Vilela FC, da Rocha CQ, Dias DF, Cavalcante GP, Freitas LA, dos Santos MH, Giusti-Paiva A. Anti-inflammatory and antinociceptive effects of Garcinia brasiliensis. J Ethnopharmacol 2011; 133: 467– 473 11 Coelho LP, Serra MF, Pires AL, Cordeiro RSB, Rodrigues e Silva PM, dos Santos MH, Martins MA. 7-Epiclusianone, a tetraprenylated benzophenone, relaxes airway smooth muscle through activation of the nitric oxide-cGMP pathway. J Pharmacol Exp Ther 2008; 327: 206–214 12 dos Santos MH, Nagem TJ, de Oliveira TT, Braz-Filho R. 7-Epiclusianona, a nova benzofenona tetraprenilada e outros constituintes químicos dos frutos de Rheedia gardneriana. Quim Nova 1999; 22: 654–660
Castro AP et al. Potent Schistosomicidal Constituents … Planta Med 2015; 81: 733–741
Downloaded by: University of Illinois. Copyrighted material.
740
13 Alves TM, Alves R, Romanha AJ, Zani CL, dos Santos MH, Nagem TJ. Biological activities of 7-epiclusianone. J Nat Prod 1999; 62: 369–371 14 Cruz AJ, Lemos VS, dos Santos MH, Nagem TJ, Cortes SF. Vascular effects of 7-epiclusianone, a prenylated benzophenone from Rheedia gardneriana, on the rat aorta. Phytomedicine 2006; 13: 442–445 15 Neves JS, Coelho LP, Cordeiro RSB, Veloso MP, Rodrigues e Silva PM, dos Santos MH, Martins MA. Antianaphylactic properties of 7-epiclusianone, a tetraprenylated benzophenone isolated from Garcinia brasiliensis. Planta Med 2007; 73: 644–649 16 Piccinelli AL, Cuesta-Rubio O, Chica MB, Mahmood N, Pagano B, Pavone M, Barone V, Rastrelli L. Structural revision of clusianone and 7-epi-clusianone and anti-HIV activity of polyisoprenylated benzophenones. Tetrahedron 2005; 61: 8206–8211 17 Almeida LSB, Murata RM, Yatsuda R, Dos Santos MH, Nagem TJ, Alencar SM, Koo H, Rosalen PL. Antimicrobial activity of Rheedia brasiliensis and 7-epiclusianone against Streptococcus mutans. Phytomedicine 2008; 15: 886–891 18 Murata RM, Branco-de-Almeida LS, Franco EM, Yatsuda R, dos Santos MH, de Alencar SM, Koo H, Rosalen PL. Inhibition of Streptococcus mutans biofilm accumulation and development of dental caries in vivo by 7-epiclusianone and fluoride. Biofouling 2010; 26: 865–872 19 Murata RM, Yatsuda R, dos Santos MH, Kohn LK, Martins FT, Nagem TJ, Alencar SM, de Carvalho JE, Rosalen PL. Antiproliferative effect of benzophenones and their influence on cathepsin activity. Phytother Res 2010; 24: 379–383 20 Pereira IO, Marques MJ, Pavan AL, Codonho BS, Barbiéri CL, Beijo LA, Doriguetto AC, DʼMartin EC, dos Santos MH. Leishmanicidal activity of benzophenones and extracts from Garcinia brasiliensis Mart. fruits. Phytomedicine 2010; 17: 339–345 21 Lim H, Son KH, Chang HW, Kang SS, Kim HP. Effects of anti-inflammatory biflavonoid, ginkgetin, on chronic skin inflammation. Biol Pharm Bull 2006; 29: 1046–1049 22 Lin YM, Anderson H, Flavin MT, Pai YH, Mata-Greenwood E, Pengsuparp T, Pezzuto JM, Schinazi RF, Hughes SH, Chen FC. In vitro anti-HIV activity of biflavonoids isolated from Rhus succedanea and Garcinia multiflora. J Nat Prod 1997; 60: 884–888 23 Pang X, Yi T, Yi Z, Cho SG, Qu W, Pinkaew D, Fujise K, Liu M. Morelloflavone, a biflavonoid, inhibits tumor angiogenesis by targeting rho GTPases and extracellular signal-regulated kinase signaling pathways. Cancer Res 2009; 69: 518–525 24 Hay AE, Merza J, Landreau A, Litaudon M, Pagniez F, Le Pape P, Richomme P. Antileishmanial polyphenols from Garcinia vieillardii. Fitoterapia 2008; 79: 42–46 25 Ferreira JFS, Peaden P, Keiser J. In vitro trematocidal effects of crude alcoholic extracts of Artemisia annua, A. absinthium, Asimina triloba, and Fumaria officinalis: trematocidal plant alcoholic extracts. Parasitol Res 2011; 109: 1585–1592 26 Braguine CG, Costa ES, Magalhães LG, Rodrigues V, da Silva Filho AA, Bastos JK, Silva MLA, Cunha WR, Januário AH, Pauletti PM. Schistosomicidal evaluation of Zanthoxylum naranjillo and its isolated compounds against Schistosoma mansoni adult worms. Z Naturforsch C 2009; 64: 793–797 27 Mostafa OMS, Eid RA, Adly MA. Antischistosomal activity of ginger (Zingiber officinale) against Schistosoma mansoni harbored in C57 mice. Parasitol Res 2011; 109: 395–403 28 Nijveldt RJ, van Nood E, van Hoorn DE, Boelens PG, van Norren K, van Leeuwen PA. Flavonoids: a review of probable mechanisms of action and potential applications. Am J Clin Nutr 2001; 74: 418–425 29 de Oliveira RN, Rehder VLG, Santos Oliveira AS, Júnior ÍM, de Carvalho JE, de Ruiz ALTG, Jeraldo Vde L, Linhares AX, Allegretti SM. Schistosoma mansoni: in vitro schistosomicidal activity of essential oil of Baccharis trimera (less) DC. Exp Parasitol 2012; 132: 135–143 30 Mantawy MM, Ali HF, Rizk MZ. Therapeutic effects of Allium sativum and Allium cepa in Schistosoma mansoni experimental infection. Rev Inst Med Trop Sao Paulo 2011; 53: 155–163 31 Martins FT, Assis DM, Dos Santos MH, Camps I, Veloso MP, Juliano MA, Alves LC, Doriguetto AC. Natural polyprenylated benzophenones inhibiting cysteine and serine proteases. Eur J Med Chem 2009; 44: 1230– 1239
32 Naldoni FJ, Claudino AL, Cruz JW, Chavasco JK, Faria e Silva PM, Veloso MP, Dos Santos MH. Antimicrobial activity of benzophenones and extracts from the fruits of Garcinia brasiliensis. J Med Food 2009; 12: 403–407 33 Xiao SH, Catto BA, Webster LT. Effects of praziquantel on different developmental stages of Schistosoma mansoni in vitro and in vivo. J Infect Dis 1985; 151: 1130–1137 34 de Oliveira Penido ML, Zech Coelho PM, de Mello RT, Piló-Veloso D, de Oliveira MC, Kusel JR, Nelson DL. Antischistosomal activity of aminoalkanethiols, aminoalkanethiosulfuric acids and the corresponding disulfides. Acta Trop 2008; 108: 249–255 35 Mohamed AM, Metwally NM, Mahmoud SS. Sativa seeds against Schistosoma mansoni different stages. Mem Inst Oswaldo Cruz 2005; 100: 205–211 36 Sanderson L, Bartlett A, Whitfield PJ. In vitro and in vivo studies on the bioactivity of a ginger (Zingiber officinale) extract towards adult schistosomes and their egg production. J Helminthol 2002; 76: 241–247 37 Oliveira FA, Kusel JR, Ribeiro F, Coelho PMZ. Responses of the surface membrane and excretory system of Schistosoma mansoni to damage and to treatment with praziquantel and other biomolecules. Parasitology 2006; 132: 321–330 38 William S, Botros S, Ismail M, Farghally A, Day TA, Bennett JL. Praziquantel-induced tegumental damage in vitro is diminished in schistosomes derived from praziquantel-resistant infections. Parasitology 2001; 122 Pt 1: 63–66 39 Cioli D, Pica-Mattoccia L, Archer S. Antischistosomal drugs: past, present … and future? Pharmacol Ther 1995; 68: 35–85 40 Manneck T, Haggenmüller Y, Keiser J. Morphological effects and tegumental alterations induced by mefloquine on schistosomula and adult flukes of Schistosoma mansoni. Parasitology 2010; 137: 85–98 41 Xiao SH, Booth M, Tanner M. The prophylactic effects of artemether against Schistosoma japonicum infections. Parasitol Today 2000; 16: 122–126 42 Sato H, Kusel JR, Thornhill J. Functional visualization of the excretory system of adult Schistosoma mansoni by the fluorescent marker resorufin. Parasitology 2002; 125: 527–535 43 Harder A, Goossens J, Andrews P. Influence of praziquantel and Ca2+ on the bilayer-isotropic-hexagonal transition of model membranes. Mol Biochem Parasitol 1988; 29: 55–59 44 Liang YS, Wang W, Dai JR, Li HJ, Tao YH, Zhang JF, Li W, Zhu YC, Coles GC, Doenhoff MJ. Susceptibility to praziquantel of male and female cercariae of praziquantel-resistant and susceptible isolates of Schistosoma mansoni. J Helminthol 2010; 84: 202–207 45 Magalhães LG, de Souza JM, Wakabayashi KA, Laurentiz RDS, Vinhólis AHC, Rezende KCS, Simaro GV, Bastos JK, Rodrigues V, Esperandim VR, Ferreira DS, Crotti AEM, Cunha WR, e Silva ML. In vitro efficacy of the essential oil of Piper cubeba L. (Piperaceae) against Schistosoma mansoni. Parasitol Res 2012; 110: 1747–1754 46 Scoparo CT, de Souza LM, Dartora N, Sassaki GL, Gorin PA, Iacomini M. Analysis of Camellia sinensis green and black teas via ultra high performance liquid chromatography assisted by liquid-liquid partition and two-dimensional liquid chromatography (size exclusion × reversed phase). J Chromatogr A 2012; 1222: 29–37 47 Swartz ME, Krull IS. Validation of chromatographic methods. Pharm Technol 1998; 22: 104–119 48 Kalia K, Sharma K, Singh HP, Singh B. Effects of extraction methods on phenolic contents and antioxidant activity in aerial parts of Potentilla atrosanguinea Lodd. and quantification of its phenolic constituents by RP-HPLC. J Agric Food Chem 2008; 56: 10129–10134 49 Smithers SR, Terry RJ. The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of the adult worms. Parasitology 1965; 55: 695–700 50 Lima SF, Vieira LQ, Harder A, Kusel JR. Altered behaviour of carbohydrate-bound molecules and lipids in areas of the tegument of adult Schistosoma mansoni worms damaged by praziquantel. Parasitology 1994; 109: 469–477 51 Ramalho-Pinto FJ, Gazzinelli G, Howells RE, Mota-Santos TA, Figueiredo EA, Pellegrino J. Schistosoma mansoni: defined system for stepwise transformation of cercaria to schistosomule in vitro. Exp Parasitol 1974; 36: 360–372
Castro AP et al. Potent Schistosomicidal Constituents …
Planta Med 2015; 81: 733–741
741
Downloaded by: University of Illinois. Copyrighted material.
Original Papers
733
Authors
Aline Pereira Castro 1, 3, Ana Carolina Alves de Mattos 2, Neusa Araújo Pereira 2, Naira Ferreira Anchieta 3, Matheus Siqueira Silva 1, Danielle Ferreira Dias 1, Claudinei Alves Silva 1, Giulliano Vilela Barros 3, Raquel Lopes Martins Souza 3, Marcelo Henrique Dos Santos 4, Marcos José Marques 3
Affiliations
The affiliations are listed at the end of the article
Key words " Clusiaceae l " Garcinia brasiliensis l " 7‑epiclusianone l " fukugetin l " Schistosoma mansoni l " Schistosomiasis l
Abstract !
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
Introduction !
received revised accepted
February 16, 2015 February 16, 2015 March 14, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1545927 Published online April 23, 2015 Planta Med 2015; 81: 733–741 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Marcos José Marques Laboratory of Parasitology Institute of Biomedical Sciences Federal University of Alfenas Department of Pathology and Parasitology Gabriel Monteiro da Silva – 700 Alfenas, Minas Gerais 37130000 Brazil Phone: + 55 35 32 99 10 67 [email protected]
Schistosomiasis is a neglected tropical disease that is caused by trematode flatworms of the genus Schistosoma. It is a disease that threatens millions of people, especially in poor regions [1]. The World Health Organization (WHO) estimated, in 2003, that approximately 207 million people were infected with Schistosoma. Moreover, 779 million people are at risk of contracting this disease [2], and at least 200 000 people die each year due to schistosomiasis [3]. Praziquantel (PZQ) is effective against all clinical forms of schistosomiasis, However, the emergence of resistant strains has spurred on the search for new active antihelminthic compounds, especially those derived from plants [4, 5]. The search for antiparasitic compounds from natural sources has increased over the last decade [4–6]. In this context, plants remain an important source of biologically active compounds, which can provide structures for the development of new drugs [7].
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.
Several chemical studies have shown that the genus Garcinia is an important source of natural products with a wide variety of biologically active metabolites, including polyisoprenylated benzophenones, flavonoids, xanthones, and proanthocyanidins, which have been proven to be effective against various diseases [8]. Many studies have demonstrated that Garcinia brasiliensis provides anti-inflammatory [9], antinociceptive [10], antioxidant, and antitumor activities [11]. The tetraprenylated benzophenone 7-epiclusianone (7" Fig. 1) was first isolated from the fruits of epi) (l G. brasiliensis [12]. Studies have demonstrated that the 7-epi extracted from the fruits of G. brasiliensis is effective against the trypomastigotes of Trypanosoma cruzi [13] and has vasodilatory [14], antianaphylactic [15], anti-HIV [16], antimicrobial [17, 18], antispasmodic [19], and antiproliferative effects [20]. Antileishmanial activity [21] and analgesic and anti-inflammatory [10] effects have also been assigned to this benzophe" Fig. 1) is a bioflavonoid none. Fukugetin (fuk) (l initially obtained from Garcinia spp, which also has demonstrated anti-inflammatory [21] and
Castro AP et al. Potent Schistosomicidal Constituents …
Planta Med 2015; 81: 733–741
Downloaded by: University of Illinois. Copyrighted material.
Potent Schistosomicidal Constituents from Garcinia brasiliensis
Original Papers
Fig. 1 A Chemical structure of 7-epi. B Chemical structure of fuk.
anti-HIV‑1 activities [22] as well as tumor angiogenesis inhibition [23]. Due to the difficulties in treating diseases caused by helminths, especially schistosomiasis, this study evaluated the schistosomicidal activity of both an ethanolic extract of G. brasiliensis Mart. epicarp (EEE) and other fractions obtained by additional extractions. The evaluation of the hexane fractions (HF), ethyl acetate fraction (EAF), and aqueous fraction (AF) enables the isolation of molecules with a potential therapeutic use in the treatment of schistosomiasis, as substances with potential antiparasitic activity have been isolated from plants in this family in recent years [24].
Results and Discussion !
Recently, the development of new drugs from medicinal plants for the treatment of neglected diseases, including schistosomiasis, has attracted the attention of many scientific research centers around the world. For example, plants of the genus Artemisia [25], extracts of Zanthoxylum naranjillo [26], and crude aqueous extracts of Zingiber officinale [27], among others, have been effective against Schsitosoma mansoni. With this in mind, the present study was devised to evaluate the schistosomicidal potential of EEE and the partition-obtained fractions (HF, EAF, FA) and compounds isolated from the fractions, the benzophenone 7-epi and the bioflavonoid fuk, by means of a bioguided test. The total amount of flavonoid found in the ethanolic epicarp extract of G. brasiliensis was 22.52 µg in quercetin equivalents/g of the extract. The flavonoids were known for their beneficial effects on health. An important effect of flavonoids is the scavenging of oxygen-derived free radicals. Moreover, in vitro experimental systems also showed that flavonoids possess anti-inflammatory, antiallergic, antiviral, and anticarcinogenic properties [28]. Many plants rich in flavonoids have demonstrated activity against S. mansoni such as Baccharis trimera, popularly known as carqueja [29], Allium sativum and Allium cepa, popularly known as garlic and onion, respectively [30], and Zingiber officinale, popularly known as ginger [27]. The yields of the EEE and the partitioned fractions HF, EAF, and AF " Fig. 2, the were 10.0 %, 39.0 %, 42.0 %, and 14.5 %, respectively. In l retention times, areas, and quantification of the isolated compounds in the extract and the fractions are shown. The analytical curves consisted of six data points, and three replicate injections at each concentration level were performed. The statistical analysis of the data revealed excellent linearity (r > 0.99) over a concentration range from 10.0 to 300.0 mg/mL.
The limits of detection (LOD) and quantitation (LOQ) were 1.5 and 4.5 mg/mL for fuk (y = 77 296 x-269 178/SD = 173 217), and 0.87 and 2.6 mg/mL for 7-epiclusianone (y = 38 539 x-84 655/ SD = 145 667), respectively. Chemical analysis of the EEE identified 7-epi as the major constit" Fig. 2 A). In uent (140.02 mg/g), followed by fuk (35.86 mg/g) (l the HF, 7-epi was identified as the major constituent (278, " Fig. 2 B). A 74 mg/g), whereas the fuk peak was not identified (l chemical analysis of the EAF identified 7-epi as the major constit" Fig. 2 C). uent (209.04 mg/g [15]), followed by fuk (24.19 mg/g) (l Finally, in the AF, neither 7-epi nor fuk peaks were identified " Fig. 2 D). These peaks may be compared with the standards of (l " Fig. 2 E, F). these compounds (l Those results are in accord with previous results that demonstrated four phenolic components, identified as fukugiside andguttiferone A; besides fuk and 7-epi in EEE [10]. These compounds were characterized by comparison to the standards previously isolated [31, 32]. The 7-epi, a natural polyisoprenylated benzophenone, was first isolated from the fruits of G. gardneriana [12] and has shown activity against trypomastigotes of Trypanosoma cruzi in vitro [13], a potent endothelium vasodilator effect [14], and anti-anaphylactic [15], anti-HIV [16], antimicrobial [17], antispasmodic [11], antiproliferative [19], and leishmanicidal activities [20]. Various parameters, including changes in the integrity of the integument and motility as well as reductions in mating and oviposition, are often evaluated as indicators of biological activity and used in in vitro toxicity studies against adult worms of Schistosoma species [4–6, 33, 34]. The schistosomicidal effect of the EEE and its fractions and isolated compounds in vitro was analyzed with respect to the time of incubation and concentration. All adult worms were dead after 24 h exposure to 100.0 µg/mL of EEE, 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 (the highest dose tested). In contrast, the worms remained viable in the negative control group (RPMI-1640 supplemented with RPMI and methanol). The use of PZQ at 2.0 µg/mL as a positive control resulted in the death of all of the parasites within 24 h. Mating and egg laying of S. mansoni females werenʼt observed at doses greater than 50.0 µg/mL and 4.0 µg/mL for the EEE (and their fractions) and 7-epi, respectively. Adult male and female worms remained separate and no oviposition was observed. Regarding the reproductive aptitude of the adult worms of S. mansoni, the components of other plants have shown effects on the oviposition of the worm. For example, curcumin, which was extracted from the rhizomes of Curcuma longa [4] and some phlor-
Castro AP et al. Potent Schistosomicidal Constituents … Planta Med 2015; 81: 733–741
Downloaded by: University of Illinois. Copyrighted material.
734
Original Papers
735
oglucinol derivatives that were obtained from the rhizomes of the Dryopteris species [5], demonstrated in vitro effects on egg laying. A decrease in oviposition was also observed using Nigella sativa [35] and a ginger rhizome (Z. officinale) extract [36]. Similarly, severe tegumental morphological changes were also observed after the incubating of adult S. mansoni worms with the extracts and fractions at concentrations above 50.0 µg/mL and with 7-epi at concentrations greater than 10.0 µg/mL. However, no abnormality was observed in worms in the control group (RPMI-1640 supplemented with RPMI and methanol). The damage to the tegument of adult S. mansoni worms was confirmed using a Hoechst 33 258 probe, which is a specific marker for cellular DNA. To assess the damage of the integument, the worms exposed to 2.0 µg/mL of PZQ and 14.0 µg/mL (ED100) of 7-epi were incubated and subsequently placed in contact with the Hoechst 33 258 probe. Analysis by microscopy showed progressive damage on the surface, causing blisters and the detachment " Fig. 3). PZQ and 7-epi were able to induce leof the tegument (l sions in the tegument of parasites, and the lesions in S. mansoni " Fig. 3). Changes males were more pronounced than in females (l in the surface of the tegument showed a dose-dependent effect. The Hoechst 33 258 probe is very sensitive in marking the regions where damage is present, and any damage or injury to the integument of the worm is precisely marked by this probe [37]. This soft tissue damage was similar to the damage reported in studies with PZQ [38]. Data obtained in this study demonstrated that the EEE, HF, EAF, and 7-epi exhibited greater activity against adult male S. mansoni worms. Indeed, 7-epi was more effective, which demonstrated an " Fig. 4). The lethality was greater in male ED100 of 14.0 µg/mL (l worms after 24 h of incubation with 10.0 µg/mL of 7-epi. There-
fore, this compound paralyzes the excretory activity in concentrations more than 12 and 14 µg/mL of 7-epi in males and females, respectively. In several studies, based on the in vitro activity, disparities in drug susceptibility between males and females of S. mansoni have been observed. S. mansoni males are often more susceptible than female worms in studies on resistance to oxamniquine [39] and in studies regarding the bioactivity of ginger extract [36]. In contrast, the results with other compounds, such as fatty aminoalkanethiosulfuric [34] and 2-[butylamino]-1-phenyl-1-ethanethiosulfuric acid [6], revealed higher survival rates for males than females. Notably, in vitro data obtained in this study demonstrated that the EEE, HF, EAF, and 7-epi exhibited greater activity against adult male S. mansoni worms. Serious injury was reported in the integument of female adult S. mansoni worms when treated with artemether [33, 40]. The seed tegument is extremely important for successful infection and survival in the host and has been a major target for antischistosomal drug development. Therefore, most of the drugs that are currently used against schistosomiasis, including PZQ [38], mefloquine [40], and artemether [41], act by damaging the integument of the worm. The results of these experiments revealed that 7-epi was able to paralyze the excretory activity of the adult worms of S. mansoni. This was observed by labeling the parasites with resorufin after a previous exposure to 7-epi, which prevented the output of the probe [substrate for P-glycoprotein (Pgp)] to the external environment. The secretory activity of worms initially exposed to resorufin and subsequently incubated with 14.0 µg/mL (ED100) of 7-epi was completely inhibited, as characterized by the accumulation of the probe in tissues other than the excretory system
Castro AP et al. Potent Schistosomicidal Constituents …
Planta Med 2015; 81: 733–741
Downloaded by: University of Illinois. Copyrighted material.
Fig. 2 Chromatograms obtained by high-performance 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 components found in the extract and the fractions identified by HPLC.
Original Papers
Fig. 3 In vitro evaluation of the effect of 14.0 µg/ mL of 7-epi on the tegument of S. mansoni adult worms. A, B S. mansoni LE not exposed to 7-epi and labelled with the probe Hoechst 33 258. C, D S. mansoni exposed to 7-epi and labelled with the probe Hoechst 33 258. The fluorescent areas indicate intense lesions. Each scale bar represents 100 µm. E, F S. mansoni exposed to 2 µg/ml PZQ and labelled with the probe Hoechst 33 258. The fluorescent areas indicate intense lesions. (Color figure available online only.)
Fig. 4 In vitro effects of 7-epi and fuk against adult S. mansoni; *** p < 0.001.
" Fig. 5). This compound paralyzed the excretory activity, too, as (l observed in other concentrations of 12, 16 and 18 µg/mL. How-
ever, with 12 µg/mL, there was no change observed in the female worms. The modulatory effect of some drugs has previously been shown through interactions with the cellular transport proteins of the excretory system in male adult S. mansoni worms [42]. Thus, the results from present work suggest, for the first time, that 7-epi acted in the excretory system and interfered with the activity of Pgp in an unknown manner. A similar result was observed in the presence of amiloride (an inhibitor of Na+ and Na+/H+ ATPase) [42]. Some hypotheses can be considered to explain the results obtained in this study, such as (i) it is possible that 7-epi depletes energy by increasing the calcium influx into tissues, leading to an excessive 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 Resorufin; (iv) 7-epi can act indirectly on the excretion of resorufin, affecting tubular membrane phospholipids and causing the probe to diffuse to places other than the excretory system [43], or (v) a simple contraction induced by 7-epi could cause a delay in the excretion of the probe due to a lack of a constant intestinal motility from the excretory organ. Thus, the probe would have a greater opportunity to diffuse to the surrounding tissues. In the present study, we used the loss of motility and tail as measures to evaluate the sensitivity of cercariae when incubated with 7-epi. At a concentration of 12.5 µg/mL of 7-epi, the cercariae were immobile after 2 h and 8 h of incubation, and the separation
Castro AP et al. Potent Schistosomicidal Constituents … Planta Med 2015; 81: 733–741
Downloaded by: University of Illinois. Copyrighted material.
736
Original Papers
737
Downloaded by: University of Illinois. Copyrighted material.
Fig. 5 In vitro evaluation of the effect of 14.0 µg/ mL of 7-epi on the excretory system of S. mansoni adult worms. A,B S. mansoni labelled with the probe resorufin and not exposed to 7-epi. C,D S. mansoni labelled with the probe resorufin and exposed to 14.0 µg/mL of 7-epi. Arrow: Main tubule (MT), nephridiopore (N), and ramifications of the excretory system. E,F S. mansoni labelled with the probe resorufin and exposed to 2 µg/ml of PZQ. (Color figure available online only.)
Fig. 6 Effect of different concentrations of 7-epi on the viability of cercariae, based on the rate (%) of loss of tail. Data are presented as the average of three experiments; ***p < 0.0001.
Fig. 7 Effect of various concentrations of 7-epi on the viability of schistosomules, based on the rate (%) of loss of movement. Data are presented as average of three experiments; ***p < 0.0001.
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" Fig. 6). The exact ence tail loss) were observed for up to 8 h (l mechanism of induction of tail loss is not fully understood. It is believed that the process relates to a particular structure between the body and the tail of cercariae. The rearward end of the body of cercariae is folded and tapered like a collar on a narrow structure that connects the body to the tail. This connective structure is delicate and can be easily broken [44].
In the absence of 7-epi, schistosomula viability appeared normal, without any morphological alteration for up to 48 h. Slight morphological changes, such as granularity, shortening body with a sickle shape, and low activity, were observed after 24 h of incubation 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, and " Fig. 7). However, at all of these 48 h of incubation, respectively (l doses, severe morphological changes, such as black granules, and an intense vacuolization around the body associated with a re-
Castro AP et al. Potent Schistosomicidal Constituents …
Planta Med 2015; 81: 733–741
Original Papers
duction in length, were observed in the first two hours of incubation. Concentrations from 7-epi 25.0 µg/mL resulted in a complete lack of mobility and intense vacuolization after 24 h incubation. Schistosomula belonging to the negative control group (RPMI or RPMI methanol 0.1 %) demonstrated viability for a standard 48 h. A complete lack of mobility and severe vacuolation were also observed after PZQ treatment. The effects observed in this study were similar to those observed in schistosomula incubated with 15.0 µg/mL of mefloquine and 100.0 µg/mL of the essential oil of Piper cubeta [45]. The generalized use of folk medicine suggests that natural products are harmless; however, the traditional use of these natural products is not satisfactory proof of their efficacy. In this context, the efficacy and safety of natural products require further study because the toxicity of the extract and the isolated molecules, 7epi and fuk, have been demonstrated on murine peritoneal macrophages. However, no significant toxicity was observed in mammalian cell extracts when the isolated molecules were used [20]. We demonstrated that 7-epi is an effective compound for use against S. mansoni adult worms, cercarie, and schistosomula in vitro. With its extensive effects on the mortality rate, reproductive fitness, parasite tegument morphology, and adult S. mansoni worm excretory activity, 7-epi is a promising composite schistosomicidal. However, further studies are needed to elucidate the mechanism(s) of toxicity of 7-epi and evaluate the in vivo activity of this compound.
Materials and Methods !
Plant material and extract preparation The fruits of G. brasiliensis (Mart.) were collected on the campus of the Federal University of Viçosa-MG, Brazil in February (summer) of 2011. Botanical identification was performed in the Horto Botânico of the Federal University of Viçosa by Dr. João Augusto Alves Meira Neto. A voucher specimen (number VIC2604) was deposited at the herbarium of the Federal University of Viçosa. Epicarps of G. brasiliensis were dried in an oven with a circulating air temperature of 40 °C for eight days. Dried and powdered G. brasiliensis fruit pericarps (1000.0 g) were extracted by maceration with 3.0 L of ethanol at room temperature, filtered, and then dried using a rotary evaporator under reduced pressure at 45 °C. This procedure was repeated five times yielding an amount of 80.0 g of fruit pericarp of the ethanolic extract (EEE).
Isolation and characterization A portion of the EEE (1.0 g) was then subjected to a liquid/liquid partition as follows: the sample was dissolved into an ethanol : water solution (1 : 4, 50.0 mL), and after the addition of hexane (50.0 mL), the mixture was vigorously stirred. The organic layer was removed to give the HF (this procedure was performed five times). Some insoluble compounds were retained in the aqueous phase, to which ethyl acetate (50.0 mL) was added and vigorously stirred. Similarly, the organic layer was removed to produce the EAF (this procedure was performed five times). The HF, EAF, and AF were concentrated at a reduced pressure using a rotary evaporator and were then dried in a lyophilizer and stored in a freezer [46]. To isolate the bioactive compounds, the HF and EAF were generated from the partition of the EEE and chromatographed on a silica gel (230–400 mesh) column (8 × 100 cm). Samples were eluted with increasingly polar mixtures of hexane/ethyl
acetate and ethyl acetate/ethanol to give 50 fractions, and the fractions (200.0 mL) were concentrated on a rotavapor at a reduced pressure. These fractions were pooled in three groups according to their similarities in thin-layer chromatography (TLC). When necessary, the samples were rechromatographed on a silica gel column as reported above to give 26 fractions (10.0 mL) and were then pooled into five groups according to similarities in TLC, and recrystallized several times using methanol to obtain pure compounds. 7-Epi was isolated from the HF, and both 7-epi and fuk were isolated from the EAF. Previously, isolated 7-epi and fuk from the Laboratory of Phytochemistry and Medicinal Chemistry of UNIFAL‑MG were used as standards for identification as well as for spectral data [10, 11]. The purity of 7-epi and fuk (99.0 and 98.0%, respectively) was evaluated by chromatographic analysis. The procedure adopted for the preparation of the ex" Fig. 8. tract, fractions, and isolated compounds is outlined in l
Chromatographic analysis and quantification The chromatographic analysis of the extracts and fractions, and the quantification of isolated molecules present in the extract and fractions were performed on a liquid chromatography device (Shimadzu HPLC) using an NST column (Nano Separation Technologies) C18-154 605 (150 × 4.6 mm; 5.0 mm in particle size) [20]. The mobile phase consisted of a mixture of acetic acid (5 mM/L) (eluent A) and methanol/acetic acid (0.1 % v/v) (eluent B). The injection volume was 20.0 µL at a flow rate of 1.0 mL/ min. During the initial 10 min, an analysis was performed using 50.0 % of eluent B followed by an increase in the concentration of eluent B to 100.0 % over 20 min. The analysis continued for 30 min at a concentration of 100.0 % eluent B. After the analysis with eluent B was concluded, the column was prepared for the next analysis. Chromatograms were obtained at 254 nm and the peaks were compared with the peaks of the compounds that had been previously isolated in the laboratory. For the analytical curves, aliquots of 0.100, 0.300, 0.500, 1.00, 2.00, and 3.00 mL, from the standard stock solutions of fuk and 7-epi (1.00 mg/mL) were transferred to separate 10.0 mL volumetric flasks. The volumes were completed with deionized water prior to injection. Concentration ranges from 10.0 to 300.0 µg/mL of fuk and 7-epi were obtained. Each solution was analyzed in triplicate, and the resulting peak areas were plotted against the respective concentrations. The criteria used to determine the LOD and LOQ were based on the determination of the slope (S) of the analytical curve and the standard deviation of responses (SD) in accordance with the formulas LOD = 3.3 SD/S and LOQ = 10 SD/S. The SD was determined from the standard error estimated by the regression line [47]. Extracts samples were assayed against reference standards and each concentration of fuk and 7-epi was calculated using an analytical curve.
Determination of total flavonoids In a 10.0-mL test tube, 0.5 mL of 20.0 % (w/v) EEE, 1.5 mL of ethanol, 0.1 mL of 10.0 % (w/v) AlCl3 · 6H2O, and 0.1 mL of 1 M potassium acetate were combined and mixed. The volume was then brought up to a final volume of 5.0 mL with H2O. After 30 min, the mixture was measured at 425 nm. The standard curve for total flavonoids was generated with a quercetin [Quercetin dehydrate (≥ 99.0% HPLC) was purchased from Sigma-Aldrich Co.] standard solution (25–120 g/mL) using the same procedure as above. Total flavonoids were determined as quercetin equivalents
Castro AP et al. Potent Schistosomicidal Constituents … Planta Med 2015; 81: 733–741
Downloaded by: University of Illinois. Copyrighted material.
738
Original Papers
739
(mg quercetin/g extract), and the values are presented as the means of triplicate analyses [48].
Parasite and experimental animals The LE strain of S. mansoni used in this study was routinely maintained by serial passage in Biomphalaria glabrata and Swiss mouse models. The LE strain has been maintained for more than 30 years at the Research Center René Rachou/Oswaldo Cruz Foundation. All experiments were authorized by the Ethical Committee for Animal Care of the Federal University of Alfenas (approval number: 534/2013 January 20, 2014) in accordance with the national and international accepted principles for laboratory animal use and care.
In vitro evaluation of the effect of ethanolic extract of Garcinia brasiliensis Mart. epicarp, its partition-obtained fractions (hexane fractions, ethyl acetate fraction, and aqueous fraction) and the isolated molecules fukugetin and 7-epiclusianone on adult Schistosoma mansoni worms Mice infected with cercariae of S. mansoni (LE strain) were sacrificed 45 days post-infection using 3.0 % pentobarbital sodium, which was administered intraperitoneally (± 0.3 mL/mouse) and perfused, according to the method previously described [49]. The recovered worms were cultured in 6-well culture dishes (four pairs per well) with a final volume of 4.0 mL of RPMI-1640 medium supplemented with 5.0 % heat-inactivated fetal bovine serum and 1.0 % penicillin (10 000 IU/mL) and streptomycin (10.0 mg/mL) (Sigma). The samples, including EEE, fractions (HF, EAF, and AF), and the isolated compounds 7-epi and fuk were prepared with methanol in a concentration of 2.0 mg/mL. These
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 at 50.0, 100.0, and 200.0 µg/mL; 7-epi at 5.0, 10.0, 14.0, 15.0, and 25.0 µg/mL) and maintained at 37 °C with 5.0 % CO2. The cultures were analyzed at 2 h and 24 h after addition to the samples. In the control groups, the worms were kept under the same conditions, except for the presence of G. brasiliensis-derived samples. After 24 h, the worms were washed with culture medium and maintained under the same conditions, but without the presence of the sample, for the remainder of the experiment. Under an inverted microscope, observations about the worms were documented at 2 and 24 h after the addition of the samples (extract, fraction, and compounds). In addition, the samples were removed and the worms were analyzed at 24, 48, 72, and 96 h after the first rinsing. A final time point was analyzed eight days after the initiation of the experiment.
Evaluation of the excretory system activity of the adult Schistosoma mansoni worm after exposure to 7-epiclusianone via resorufin labeling After recovery and washing, four couple worms were maintained in 4.0 mL of RPMI-1640 culture medium supplemented with 5.0 % (v/v) BFS and 1.0 % penicillin (10 000 IU/mL) and streptomycin (10.0 mg/mL). Then, 10.0 µL of resorufin (stock solution 10.0 mg/ mL in medium) was added to each well and incubated at 37 °C under 5.0 % CO2 for 30 min. Afterwards, the worms were washed five times with 2.0 mL of RPMI to remove the excess of probe. Subsequently, (i) 4.0 mL of RPMI-1640 supplemented medium (medium control), (ii) 40.0 µL of methanol (solvent control), (iii) 2.5 µL (2.0 µg/mL) of a PZQ (Cestox, MERCK, 100.01 % purity) stock solution at 0.8 mg/
Castro AP et al. Potent Schistosomicidal Constituents …
Planta Med 2015; 81: 733–741
Downloaded by: University of Illinois. Copyrighted material.
Fig. 8 Bioassay scheme used for the extraction and purification of the constituents from G. brasiliensis epicarp.
Original Papers
mL (compound control), and (iiii) 7-epi at concentrations of 12.0 µg/mL, 14.0 µg/mL, and 20.0 µg/mL from a 2.0-mg/mL stock solution were added into each well. Finally, the worms were incubated at 37 °C and 5.0 % CO2 for 15 min. They were once again washed (five times with culture medium) and transferred to glass slides delimited with vaseline, to prevent leakage, using a small quantity of culture medium supplemented with 1.0 % sodium pentobarbital at 3.0 % (Hypnol, Fontoveter) to inhibit the parasites movements. These tests were performed in triplicate. Afterwards, the tests were analyzed with a fluorescence microscope (Nikon, Eclipse 80i) using a rhodamine filter for resorufin (maximum excitation/emission of resorufin at 571/585 nm).
Statistical analyses Statistical evaluation of the results was performed using the SISVAR 5.3 software using analysis of variance (ANOVA) and the test of SNK was applied to observe significant differences between mean values (p < 0.05).
Acknowledgements The authors thank FAPEMIG, CNPq, CAPES, and FINEP for financial support and scholarships, and Dr. Paulo Marcos Zech Coelho for his help and support.
Conflict of Interest !
Evaluation of damage to the tegument of the adult worm of Schistosoma mansoni exposed to a 7-epiclusianone probe after labeling with Hoechst 33 258 The methods utilized to detect tegument damage were described by Lima et al. [50]. Briefly, the worms were washed five times with 2.0 mL of RPMI to remove the excess of probe. Subsequently, (i) 4.0 mL of RPMI-1640 supplemented medium (medium control), (ii) 40.0 µL of methanol (solvent control), (iii) 2.5 µL (2.0 µg/ mL) of a PZQ stock solution at 0.8 mg/mL (compound control), and (iiii) 7-epi at concentrations of 12.0 µg/mL, 14.0 µg/mL, and 20.0 µg/mL from a 2.0-mg/mL stock solution were added into each well and incubated for 24 hours at 37 °C with 5.0 % CO2. At the end of this period, the worms were washed and incubated for 15 min with 10.0 µL of Hoechst 33 258 (stock solution 10.0 mg/ml). Damage was evaluated by fluorescence microscopy after washing to remove excess probe from the worms. Parasites were put onto the slides with a small amount of culture medium supplemented with 1.0% to 3.0% pentobarbital sodium (Hypnol, Fontoveter) to inhibit parasite movement. The slides were then observed with a fluorescence microscope (Nikon, Eclipse 80i) using 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-epiclusianone in cercariae Infected B. glabrata were induced to eliminate cercariae by exposing them to light for 1 h in dechlorinated water. Approximately 100 cercariae (100.0 uL) were placed into a 24-well tissue culture plate with 900.0 µL of dechlorinated water, and exposed to 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 performed in triplicate and repeated at least three times using methanol and pure water as negative controls. PZQ up to 12.5 µg/mL was used as a positive control.
In vitro studies of 7-epiclusianone in schistosomula Cercariae were transformed into schistosomula by mechanical transformation using a Vortex mixer [51]. Schistosomula were cultured in RPMI-1640 supplemented medium (RPMI-1640 + 5.0 % BFS + 100.0 µg/mL of penicillin and streptomycin) and exposed to 8.0, 12.0, 24.0, 25.0, 50.0, and 100.0 µg/mL of 7-epi. at 37 °C under 5.0 % CO2. The schistosomula were analyzed after 2, 24, and 48 h using inverted microscopy. All experiments were performed in triplicate and repeated at least three times using a methanol solution with supplemented RPMI-1640 medium as a negative control and PZQ at 12.5 mg/mL as a positive control.
The authors declare no conflict of interest.
Affiliations 1
2
3
4
Department of Pharmacy, Laboratory of Phytochemistry and Medicinal Chemistry, Federal University of Alfenas, Alfenas, Minas Gerais, Brazil Laboratory of Schistosomiasis, Institute René Rachou-Fiocruz, Belo Horizonte, Brazil Department of Pathology and Parasitology, Laboratory of Parasitology, Institute of Biomedical Sciences, Federal University of Alfenas, Alfenas, Minas Gerais, Brazil Departament of Chemistry, Federal University of Viçosa, Viçosa-MG, Brazil
References 1 King CH. Parasites and poverty: the case of schistosomiasis. Acta Trop 2010; 113: 95–104 2 Steinmann P, Keiser J, Bos R, Tanner M, Utzinger J. Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. Lancet Infect Dis 2006; 6: 411–425 3 King CH, Dickman K, Tisch DJ. Reassessment of the cost of chronic helmintic infection: a meta-analysis of disability-related outcomes in endemic schistosomiasis. Lancet 2005; 365: 1561–1569 4 Magalhães LG, Machado CB, Morais ER, Moreira EBDC, Soares CS, da Silva SH, Da Silva Filho AA, Rodrigues V. In vitro schistosomicidal activity of curcumin against Schistosoma mansoni adult worms. Parasitol Res 2009; 104: 1197–1201 5 Magalhães LG, Kapadia GJ, da Silva Tonuci LR, Caixeta SC, Parreira NA, Rodrigues V, Da Silva Filho AA. In vitro schistosomicidal effects of some phloroglucinol derivatives from Dryopteris species against Schistosoma mansoni adult worms. Parasitol Res 2010; 106: 395–401 6 de Araújo SC, de Mattos ACA, Teixeira HF, Coelho PMZ, Nelson DL, de Oliveira MC. Improvement of in vitro efficacy of a novel schistosomicidal drug by incorporation into nanoemulsions. Int J Pharm 2007; 337: 307–315 7 Tonuci LRS, de Melo NI, Dias HJ, Wakabayashi KAL, Aguiar GP, Aguiar DP, Mantovani ALL, Ramos RC, Groppo M, Rodrigues V, Veneziani RCS, Cunha WR, da Silva Filho AA, Magalhães LG, Crotti AEM. In vitro schistosomicidal effects of the essential oil of Tagetes erecta. Rev Bras Farmacogn 2012; 22: 88–93 8 Derogis PBMC, Martins FT, de Souza TC, de C Moreira ME, Souza Filho JD, Doriguetto AC, de Souza KRD, Veloso MP, Dos Santos MH. Complete assignment of the 1H and 13C NMR spectra of garciniaphenone and keto-enol equilibrium statements for prenylated benzophenones. Magn Reson Chem 2008; 46: 278–282 9 Santa-Cecília FV, Freitas LA, Vilela FC, Veloso CDC, da Rocha CQ, Moreira MEC, Dias DF, Giusti-Paiva A, dos Santos MH. Antinociceptive and antiinflammatory properties of 7-epiclusianone, a prenylated benzophenone from Garcinia brasiliensis. Eur J Pharmacol 2011; 670: 280–285 10 Santa-Cecília FV, Vilela FC, da Rocha CQ, Dias DF, Cavalcante GP, Freitas LA, dos Santos MH, Giusti-Paiva A. Anti-inflammatory and antinociceptive effects of Garcinia brasiliensis. J Ethnopharmacol 2011; 133: 467– 473 11 Coelho LP, Serra MF, Pires AL, Cordeiro RSB, Rodrigues e Silva PM, dos Santos MH, Martins MA. 7-Epiclusianone, a tetraprenylated benzophenone, relaxes airway smooth muscle through activation of the nitric oxide-cGMP pathway. J Pharmacol Exp Ther 2008; 327: 206–214 12 dos Santos MH, Nagem TJ, de Oliveira TT, Braz-Filho R. 7-Epiclusianona, a nova benzofenona tetraprenilada e outros constituintes químicos dos frutos de Rheedia gardneriana. Quim Nova 1999; 22: 654–660
Castro AP et al. Potent Schistosomicidal Constituents … Planta Med 2015; 81: 733–741
Downloaded by: University of Illinois. Copyrighted material.
740
13 Alves TM, Alves R, Romanha AJ, Zani CL, dos Santos MH, Nagem TJ. Biological activities of 7-epiclusianone. J Nat Prod 1999; 62: 369–371 14 Cruz AJ, Lemos VS, dos Santos MH, Nagem TJ, Cortes SF. Vascular effects of 7-epiclusianone, a prenylated benzophenone from Rheedia gardneriana, on the rat aorta. Phytomedicine 2006; 13: 442–445 15 Neves JS, Coelho LP, Cordeiro RSB, Veloso MP, Rodrigues e Silva PM, dos Santos MH, Martins MA. Antianaphylactic properties of 7-epiclusianone, a tetraprenylated benzophenone isolated from Garcinia brasiliensis. Planta Med 2007; 73: 644–649 16 Piccinelli AL, Cuesta-Rubio O, Chica MB, Mahmood N, Pagano B, Pavone M, Barone V, Rastrelli L. Structural revision of clusianone and 7-epi-clusianone and anti-HIV activity of polyisoprenylated benzophenones. Tetrahedron 2005; 61: 8206–8211 17 Almeida LSB, Murata RM, Yatsuda R, Dos Santos MH, Nagem TJ, Alencar SM, Koo H, Rosalen PL. Antimicrobial activity of Rheedia brasiliensis and 7-epiclusianone against Streptococcus mutans. Phytomedicine 2008; 15: 886–891 18 Murata RM, Branco-de-Almeida LS, Franco EM, Yatsuda R, dos Santos MH, de Alencar SM, Koo H, Rosalen PL. Inhibition of Streptococcus mutans biofilm accumulation and development of dental caries in vivo by 7-epiclusianone and fluoride. Biofouling 2010; 26: 865–872 19 Murata RM, Yatsuda R, dos Santos MH, Kohn LK, Martins FT, Nagem TJ, Alencar SM, de Carvalho JE, Rosalen PL. Antiproliferative effect of benzophenones and their influence on cathepsin activity. Phytother Res 2010; 24: 379–383 20 Pereira IO, Marques MJ, Pavan AL, Codonho BS, Barbiéri CL, Beijo LA, Doriguetto AC, DʼMartin EC, dos Santos MH. Leishmanicidal activity of benzophenones and extracts from Garcinia brasiliensis Mart. fruits. Phytomedicine 2010; 17: 339–345 21 Lim H, Son KH, Chang HW, Kang SS, Kim HP. Effects of anti-inflammatory biflavonoid, ginkgetin, on chronic skin inflammation. Biol Pharm Bull 2006; 29: 1046–1049 22 Lin YM, Anderson H, Flavin MT, Pai YH, Mata-Greenwood E, Pengsuparp T, Pezzuto JM, Schinazi RF, Hughes SH, Chen FC. In vitro anti-HIV activity of biflavonoids isolated from Rhus succedanea and Garcinia multiflora. J Nat Prod 1997; 60: 884–888 23 Pang X, Yi T, Yi Z, Cho SG, Qu W, Pinkaew D, Fujise K, Liu M. Morelloflavone, a biflavonoid, inhibits tumor angiogenesis by targeting rho GTPases and extracellular signal-regulated kinase signaling pathways. Cancer Res 2009; 69: 518–525 24 Hay AE, Merza J, Landreau A, Litaudon M, Pagniez F, Le Pape P, Richomme P. Antileishmanial polyphenols from Garcinia vieillardii. Fitoterapia 2008; 79: 42–46 25 Ferreira JFS, Peaden P, Keiser J. In vitro trematocidal effects of crude alcoholic extracts of Artemisia annua, A. absinthium, Asimina triloba, and Fumaria officinalis: trematocidal plant alcoholic extracts. Parasitol Res 2011; 109: 1585–1592 26 Braguine CG, Costa ES, Magalhães LG, Rodrigues V, da Silva Filho AA, Bastos JK, Silva MLA, Cunha WR, Januário AH, Pauletti PM. Schistosomicidal evaluation of Zanthoxylum naranjillo and its isolated compounds against Schistosoma mansoni adult worms. Z Naturforsch C 2009; 64: 793–797 27 Mostafa OMS, Eid RA, Adly MA. Antischistosomal activity of ginger (Zingiber officinale) against Schistosoma mansoni harbored in C57 mice. Parasitol Res 2011; 109: 395–403 28 Nijveldt RJ, van Nood E, van Hoorn DE, Boelens PG, van Norren K, van Leeuwen PA. Flavonoids: a review of probable mechanisms of action and potential applications. Am J Clin Nutr 2001; 74: 418–425 29 de Oliveira RN, Rehder VLG, Santos Oliveira AS, Júnior ÍM, de Carvalho JE, de Ruiz ALTG, Jeraldo Vde L, Linhares AX, Allegretti SM. Schistosoma mansoni: in vitro schistosomicidal activity of essential oil of Baccharis trimera (less) DC. Exp Parasitol 2012; 132: 135–143 30 Mantawy MM, Ali HF, Rizk MZ. Therapeutic effects of Allium sativum and Allium cepa in Schistosoma mansoni experimental infection. Rev Inst Med Trop Sao Paulo 2011; 53: 155–163 31 Martins FT, Assis DM, Dos Santos MH, Camps I, Veloso MP, Juliano MA, Alves LC, Doriguetto AC. Natural polyprenylated benzophenones inhibiting cysteine and serine proteases. Eur J Med Chem 2009; 44: 1230– 1239
32 Naldoni FJ, Claudino AL, Cruz JW, Chavasco JK, Faria e Silva PM, Veloso MP, Dos Santos MH. Antimicrobial activity of benzophenones and extracts from the fruits of Garcinia brasiliensis. J Med Food 2009; 12: 403–407 33 Xiao SH, Catto BA, Webster LT. Effects of praziquantel on different developmental stages of Schistosoma mansoni in vitro and in vivo. J Infect Dis 1985; 151: 1130–1137 34 de Oliveira Penido ML, Zech Coelho PM, de Mello RT, Piló-Veloso D, de Oliveira MC, Kusel JR, Nelson DL. Antischistosomal activity of aminoalkanethiols, aminoalkanethiosulfuric acids and the corresponding disulfides. Acta Trop 2008; 108: 249–255 35 Mohamed AM, Metwally NM, Mahmoud SS. Sativa seeds against Schistosoma mansoni different stages. Mem Inst Oswaldo Cruz 2005; 100: 205–211 36 Sanderson L, Bartlett A, Whitfield PJ. In vitro and in vivo studies on the bioactivity of a ginger (Zingiber officinale) extract towards adult schistosomes and their egg production. J Helminthol 2002; 76: 241–247 37 Oliveira FA, Kusel JR, Ribeiro F, Coelho PMZ. Responses of the surface membrane and excretory system of Schistosoma mansoni to damage and to treatment with praziquantel and other biomolecules. Parasitology 2006; 132: 321–330 38 William S, Botros S, Ismail M, Farghally A, Day TA, Bennett JL. Praziquantel-induced tegumental damage in vitro is diminished in schistosomes derived from praziquantel-resistant infections. Parasitology 2001; 122 Pt 1: 63–66 39 Cioli D, Pica-Mattoccia L, Archer S. Antischistosomal drugs: past, present … and future? Pharmacol Ther 1995; 68: 35–85 40 Manneck T, Haggenmüller Y, Keiser J. Morphological effects and tegumental alterations induced by mefloquine on schistosomula and adult flukes of Schistosoma mansoni. Parasitology 2010; 137: 85–98 41 Xiao SH, Booth M, Tanner M. The prophylactic effects of artemether against Schistosoma japonicum infections. Parasitol Today 2000; 16: 122–126 42 Sato H, Kusel JR, Thornhill J. Functional visualization of the excretory system of adult Schistosoma mansoni by the fluorescent marker resorufin. Parasitology 2002; 125: 527–535 43 Harder A, Goossens J, Andrews P. Influence of praziquantel and Ca2+ on the bilayer-isotropic-hexagonal transition of model membranes. Mol Biochem Parasitol 1988; 29: 55–59 44 Liang YS, Wang W, Dai JR, Li HJ, Tao YH, Zhang JF, Li W, Zhu YC, Coles GC, Doenhoff MJ. Susceptibility to praziquantel of male and female cercariae of praziquantel-resistant and susceptible isolates of Schistosoma mansoni. J Helminthol 2010; 84: 202–207 45 Magalhães LG, de Souza JM, Wakabayashi KA, Laurentiz RDS, Vinhólis AHC, Rezende KCS, Simaro GV, Bastos JK, Rodrigues V, Esperandim VR, Ferreira DS, Crotti AEM, Cunha WR, e Silva ML. In vitro efficacy of the essential oil of Piper cubeba L. (Piperaceae) against Schistosoma mansoni. Parasitol Res 2012; 110: 1747–1754 46 Scoparo CT, de Souza LM, Dartora N, Sassaki GL, Gorin PA, Iacomini M. Analysis of Camellia sinensis green and black teas via ultra high performance liquid chromatography assisted by liquid-liquid partition and two-dimensional liquid chromatography (size exclusion × reversed phase). J Chromatogr A 2012; 1222: 29–37 47 Swartz ME, Krull IS. Validation of chromatographic methods. Pharm Technol 1998; 22: 104–119 48 Kalia K, Sharma K, Singh HP, Singh B. Effects of extraction methods on phenolic contents and antioxidant activity in aerial parts of Potentilla atrosanguinea Lodd. and quantification of its phenolic constituents by RP-HPLC. J Agric Food Chem 2008; 56: 10129–10134 49 Smithers SR, Terry RJ. The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of the adult worms. Parasitology 1965; 55: 695–700 50 Lima SF, Vieira LQ, Harder A, Kusel JR. Altered behaviour of carbohydrate-bound molecules and lipids in areas of the tegument of adult Schistosoma mansoni worms damaged by praziquantel. Parasitology 1994; 109: 469–477 51 Ramalho-Pinto FJ, Gazzinelli G, Howells RE, Mota-Santos TA, Figueiredo EA, Pellegrino J. Schistosoma mansoni: defined system for stepwise transformation of cercaria to schistosomule in vitro. Exp Parasitol 1974; 36: 360–372
Castro AP et al. Potent Schistosomicidal Constituents …
Planta Med 2015; 81: 733–741
741
Downloaded by: University of Illinois. Copyrighted material.
Original Papers
