Antitrypanosomal Activity of a Semi-purified Subfraction Rich in Labdane Sesquiterpenes, Obtained from Flowers of Anthemis Tinctoria, against Trypanosoma Cruzi

In Brazil and several other Latin American countries, Chagas' disease still constitutes a serious medical and social problem, and there is a need to develop new, more-potent drugs with fewer side effects to effectively treat this disease. We investigated the antitrypanosomal effect of a crude extract, fractions, and a semi-purified subfraction rich in a mixture of isomeric labdane sesquiterpenes, obtained from flowers of Anthemis tinctoria, against Trypanosoma cruzi. In epimastigote forms, the aqueous crude extract, dichloromethane fraction, and semi-purified subfraction showed a dose-dependent inhibitory activity, with IC 50 of 2.3 μg/ml, 1.8 μg/ml, and 0.2 μg/ml, respectively. In the interaction index , the semi-purified subfraction showed a reduction in both the percentage of infected LLCMK 2 cells and the mean number of amastigotes per infected cell. The cytotoxicity evaluation demonstrated that the cytotoxic concentrations of the semi-purified subfraction were higher for LLCMK 2 cells than for the protozoans, with a selectivity index of 35.0. Epimastigote forms treated with the semi-purified subfraction showed ultrastructural and morphological alterations such as rounding of the cells and bleb formation in the flagellum and cytoplasmic membrane. These results show that the flowers from A. tinctoria may be a source of new drugs with antiprotozoal activity. However, additional in vitro and in vivo studies are needed to validate the use of A. tinctoria in the treatment of Chagas' disease.


Introduction
About 65% -80% of the population in developing countries essentially depends on plants for primary health care.Some 25% of all modern medicines are derived directly or indirectly from plants [1].Many plants from Brazilian ecosystems such as the savanna, and the Atlantic and Amazon forests are used in traditional medicine [2].Also, many exotic plants that were introduced into Brazil and incorporated into traditional medicine display curative properties [3].Various studies have demonstrated a strong correlation between popular use and experimenttally demonstrated pharmacological activity.Many plant extracts and essential oils have been shown to exert in vitro and in vivo activity, which justifies research on plants used in traditional medicine [4].
Plants produce a variety of compounds with antimicrobial properties, which have led to the development of new drugs for treatment of infectious diseases [5].
The family Compositae is one of the most species-rich among the flowering plants.Anthemis L. is the secondlargest genus in this family with approximately 25 000 species, widely distributed in subtropical and temperate areas.Species of Anthemis are widely used in the pharmaceutical, cosmetic and food industries.Anthemis tinctoria L. is a perennial herb cultivated in Mediterranean countries, and several secondary metabolites have been identified in this species, such as volatile oils, triterpenes, polyacetylenes, and flavonoids [6].In traditional medicine, this plant is used to treat liver problems and jaundice [7].Its flowers have well-known antiseptic and medicinal properties, derived from flavonoids as well as es-from Flowers of Anthemis tinctoria, against Trypanosoma cruzi sential oils [8].In Europe, extracts, dyes, teas, and ointments are used as anti-inflammatory, antibacterial, antispasmodic, and sedative agents [9].The antimicrobial activity of essential oils and extracts from different species of Anthemis has been studied previously [10,11].
Trypanosoma cruzi is the etiologic agent of Chagas' disease, which in Brazil and several other Latin American countries constitutes, a serious social and medical problem [12].Transmission to vertebrates occurs through feces of hemipteran insects contaminated with metacyclic trypomastigotes, the infective stage of the parasite [13].The acute phase of Chagas' disease is frequently asymptomatic, and the chronic phase usually develops 10 to 20 years after the infection, affecting about 10% to 30% of infected individuals [14].
In spite of the impressive progress in the understanding of the biology of T. cruzi, the drugs available (nifurtimox and benzonidazole) are active on the acute stage of Chagas' disease, with about 80% effectiveness; but have limited utility against the established chronic disease [15].The side effects of both compounds can be quite severe [16].Therefore, trypanocidal drugs with less-serious side effects are necessary.In this context, plants are a reservoir of chemical and biological diversity that has led to the development of hundreds of pharmaceutical drugs [17].The objective of the present study was to investigate the activity of the extracts, fractions, and a sequiterpene-rich semi-purified subfraction, obtained from flowers of A. tinctoria, against T. cruzi.

Collections of the Plant
Flowers of Anthemis tinctoria were collected in November 2004 at the "Profa.Irenice Silva" Garden of Medicinal Plants of the State University of Maringá.The plant was identified through authentic comparison by Dr. Cirino Correia Júnior, and a voucher specimen (No.HUM 1133) is deposited at the Herbarium of the State University of Maringá, Paraná, Brazil.

Parasites
Epimastigote forms of T. cruzi Y strain were cultured in LIT (Liver Infusion Triptone broth) [18] with 10% fetal bovine serum (SFB) (Gibco Invitrogen Corporation, New York, USA) at 28˚C for 96 h.Amastigote and trypomastigote forms were grown in monolayers of LLCMK 2 cells in DMEM medium (Gibco Invitrogen) with 10% SFB in 5% CO 2 at 37˚C.

Antiproliferative Activity against Epimastigote Forms
The aqueous and ethyl-acetate crude extracts, fractions (hexane, dichloromethane, ethyl acetate, methanol, and methanol/water ) and the semi-purified subfraction were dissolved in dimethyl sulphoxide (DMSO, Sigma Chemical Co., St. Louis, Missouri, USA) at a final concentration not exceeding 1% [19] and assayed against the epimastigote form of T. cruzi.The experiments were performed on 24-well polystyrene plates containing 1 ml of diluted compound at different concentrations (from 1.0 to 1,000 μg/ml).The starting inoculum consisted of 10 6 parasites in logarithmic growth phase per well.The cells were incubated at 28˚C and the growth was determined by counting the parasites with a Neubauer hemocytometer (Improved Double Neubauer Ruling) every 24 h over a 7-day period.Benznidazole (N-benzyl-2-nitro-1-imidazolacetamide, Roche Pharmaceuticals, Rio de Janeiro,

Growth Inhibition Assay of Mammalian-Stage Amastigote Form of T. cruzi
LLCMK 2 cells (kidney cells from Mulatto monkey) were plated on glass coverslips (diameter 13 mm) into 24-multiwell tissue culture plates and maintained in a humidified 5% CO 2 atmosphere at 37˚C.The cells were infected with trypomastigote forms at a parasite/cell ratio of 10:1.After 24 h, non-adhered parasites were washed out, and fresh DMEM medium, and the semi-purified subfraction containing sesquiterpene in various concentrations, was added.The cells were incubated in a humidified 5% CO 2 atmosphere at 37˚C for 96 h.The coverslips were washed 3 times with PBS, fixed with methanol and stained with Giemsa.Next, the coverslips were mounted on glass slides using Entellan (Merck), and the percentage of LLCMK 2 cells with internalised parasites (1) and the number of internalised parasites per LLCMK 2 cell (2) were determined by counting at least 200 cells per sample under a microscope (Olympus CX 31).The product of 1 and 2 determined the survival index.

Cytotoxicity Activity
LLCMK 2 cells were seeded onto 24-well microtitre plates at a concentration of 2.0 × 10 5 cells/ml and allowed to proliferate for 48 h in DMEM containing 5% FBS at 37˚C with 5% CO 2 to form a cell monolayer.Different concentrations of the semi-purified subfraction containing sesquiterpene were applied to the monolayer and incubated at 37˚C with 5% CO 2 for 72 h.Next, the cells were treated with 10% trichloroacetic acid at 4˚C for one hour, gently washed in tap water, and allowed to dry at room temperature.A solution of 0.4% sulforhodamine B (in 1% acetic acid) was added to each well, and the plate was kept protected from light for 30 min at 4˚C.The wells were washed four times with 1% acetic acid, and 150 µl of 10 mM Tris-base was added and homogenised for 15 min.The absorbance was read at 530 nm in a microplate spectrophotometer (Bio Tek-Power Wave XS).The CC 50 (concentration that lysed 50% of cells) of the subfraction was calculated.
Antitrypanosomal Activity of a Semi-Purified Subfraction Rich in Labdane Sesquiterpenes, Obtained 50 from Flowers of Anthemis tinctoria, against Trypanosoma cruzi

Evaluation of Morphological Alterations by Scanning Electron Microscopy
Epimastigote forms treated with IC 50 (0.2 µg/ml) or IC 90 (1.0 µg/ml) of the semi-purified sesquiterpene subfraction for 96 h were collected by centrifugation, washed in PBS and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, containing 1.0 mM CaCl 2 at 4˚C.After fixation, small drops of the sample were placed on a specimen support with poly-L-lysine.Subsequently, the samples were dehydrated in graded ethanol, critical-point dried in CO 2 , sputter-coated with gold and observed in a SHIMADZU SS-550 Scanning electron microscope.

Evaluation of the Ultrastructural Alterations in T. cruzi by Transmission Electron Microscopy
After treatment with IC 50 or IC 90 of the semi-purified sesquiterpene subfraction for 96 h, epimastigote forms were washed in 0.01 M phosphate-buffered saline and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2.The cells were postfixed in a solution containing 1% OsO 4 and 0.8% potassium ferrocyanide in 0.1 M cacodylate buffer, dehydrated in acetone, and embedded in Epon.Thin sections were collected on a copper grid (300 mesh), stained with uranyl acetate and lead citrate, and observed in a Zeiss 900 transmission electron microscope.

Statistical Analysis
Statistical analysis was performed with the program GraphPad Prism 4 (GraphPad Software, San Diego, California, USA).One-Way Anova was applied, and a pvalue less than 0.05 was regarded as significant.

Results and Discussion
In the attempt to develop new drugs for treatment of infectious diseases, studies are carried out with compounds of both natural and synthetic origin.Many studies have demonstrated that crude extracts, fractions, and compounds isolated from medicinal plants exhibit antiprotonzoal activity [20][21][22][23][24][25][26].In this study, we evaluated the antitrypanosomal activity of the crude extract, fractions, and a subfraction rich in a mixture of isomeric labdane sesquiterpenes, obtained from flowers of A. tinctoria, against epimastigote, and amastigote forms of T. cruzi.
The analyses of the 1 H and 13 C NMR spectrum of the semi-purified subfraction showed a signal of a mixture of isomers of cross-conjugated terpenoid ketones derived from labdane sesquiterpenes that are frequently found in the family Asteraceae [27].Three types of classes of secondary metabolites have been detected in Anthemis: polyacetylenes [28], flavonoids [29], and sesquiterpene lactones [30].Previous chemical studies of species of Anthemis have shown the presence of sesquiterpene lactones [31][32][33][34][35][36][37].The three major types of sesquiterpene lactones are germacrolides, eudesmanolides, and guaianolides [38].Recently it was demonstrated that anthecularin, a sesquiterpene lactone isolated from Anthemis auriculata, shows antimalarial activity, and also antitrypanosomal activity against Trypanosoma brucei [39].
The hydroalcoholic crude extracts, fractions, and semipurified subfraction obtained from A. tinctoria flowers were used in order to investigate the antiprotozoal activity of this plant against T. cruzi.A progressive increase in the antitrypanosomal effect was observed in the course of the purification process.Figure 1(a) shows the percentage of growth inhibition of the epimastigote form treated with the aqueous phase of the crude extract for 96 h of incubation at 28˚C.This extract showed a dose-dependent inhibitory activity of 77.4% and 91.9% at 5 and 10 μg/ml, respectively.In the same concentrations, the ethyl-acetate extract showed an inhibitory activity of 31.7% and 76.8%.The 50% inhibitory concentration (IC 50 ) of the crude extract aqueous phase was 2.3 g/ml.On the basis of this finding, the crude aqueous extract was fractionated on silica gel into five fractions: hexane (F1), dichloromethane (F2), ethyl-acetate (F3), methanol (F4), and methanol:water (F5).The F2 fraction showed an IC 50 of 1.8 g/ml (Figure 1(b)), and the F3 fraction an IC 50 of 5.0 g/ml (data not shown).The other fractions (F1, F4, and F5) showed lower inhibitory activity, with IC 50 of 26.8, 23.6, and 145 g/ml, respectively.Subsequently, the F2 fraction was submitted to the Sephadex column, yielding 8 subfractions: F2a, F2b, F2c, F2d, F2e, F2f, F2g, and F2h. Figure 1(c) illustrates the inhibitory activeity of the F2g subfraction, with a percentage of inhibition of growth of the epimastigote form above 90% for all concentrations used (1 -1000 µg/ml).The F2g subfraction, which was rich in a mixture of isomeric labdane sesquiterpenes, showed an IC 50 at 0.2 g/ml.In comparison, Luize et al. [40] investigated the in vitro antiproliferative effect of eupomatenoid-5, a sesquiterpene isolated from leaves of Piper regnellii var.pallecens, a plant of the same family (Compositae) as A. tinctoria, against T. cruzi.Our results obtained with a mixture of sesquiterpenes also showed that this mixture was more active than Benzonidazole.
The effect of the semi-purified subfraction obtained from flowers of A. tinctoria on the interaction of T. cruzi with LLCMK 2 cells was evaluated.The treatment of LLCMK 2 cells infected with the amastigote form showed that the semi-purified subfraction had a dose-dependent from Flowers of Anthemis tinctoria, against Trypanosoma cruzi antitrypanosomal activity (Figure 2), leading to considerable reduction in both the percentage of infected cells and the mean number of parasites per infected cell.After 96 h of incubation, the percentage of LLCMK 2 cells with internalised parasites was higher for the control than for cells treated with the semi-purified subfraction.At that time, the control showed a mean of 34.1 amastigotes per cell, with 40.6% of cells infected.Cells treated with 1.0 g/ml showed a mean of 18.1 internalised amastigotes per cell, with 22.7% of cells infected.Treatment of the cells with 5.0 g/ml resulted in only 4.7% infected cells and 4.2 parasites per cell.
Important criteria for the investigation of compounds with activity against T. cruzi are both their therapeutic potential, and the lack of a cytotoxic effect on mammalian cells.We evaluated the cytotoxicity in LLCMK 2 cells treated with the crude extract aqueous phase, di-chloromethane fraction, and the semi-purified subfraction (Figure 3).The 50% cytotoxicity concentration (CC 50 ) in LLCMK 2 cells treated with the crude extract aqueous phase, dichloromethane fraction, and semi-purified subfraction were 17.3 μg/ml, 4.0 μg/ml, and 7.0 μg/ml, respectively.The best selectivity index (SI) ratio (CC 50 for LLCMK 2 cells/IC 50 for antiprotozoa) was obtained by the semi-purified subfraction, with SI of 35.0.The dichloromethane fraction and crude extract showed SI of 2.2 and 7.5, respectively (Table 1).
Morphological alterations in epimastigote forms treated

Conclusions
In conclusion, this study showed that extracts, fractions, and a semi-purified subfraction containing sesquiterpene obtained from flowers of A. tinctoria have potential anti-from Flowers of Anthemis tinctoria, against Trypanosoma cruzi proliferative activity in vitro against T. cruzi.Therefore, natural products may be a source of new drugs with antiprotozoal activity that could be used to treat parasitic infectious diseases such as Chagas' disease.

Figure 2 .
Figure 2. Growth inhibition assay of mammalian-stage amastigote form of Trypanosoma cruzi treated with a semipurified subfraction obtained from Anthemis tinctoria.
with the semi-purified subfraction were observed by scanning electron microscopy.Epimastigote forms in the presence of the semi-purified subfraction at IC 50 (0.2 g/ml) showed distortions of the parasite cell body, and the cell shape was severely affected.Increase of the cell volume and rounding of the cell body were observed (Figures4(b), (c), and (d)).At IC 90 (1.0 g/ml), alterations were more evident (Figures4 (e) and (f)).Figure4(a) shows the characteristic elongated shape of an untreated protozoan, or treated only with 1% DMSO (control), with a terminal flagellum that emerges from the flagellar pocket and remains tightly attached to the cell body along its length, typical of the epimastigote form.Ultrastructural alterations of the epimastigote form treated with the semi-purified subfraction were observed by transmission electron microscopy.Untreated T. cruzi or epimastigotes treated with 1% DMSO showed the typical ultrastructure (Figure5(a)).Epimastigotes treated with the semi-purified sesquiterpene-rich subfraction at concentrations of IC 50 (Figures5(b), (c), and (d)) and IC 90 (Figures 5(e) and (f)) showed ultrastructural alterations.One important change took place in the membrane lining the cell body and flagellum, with portions of the membrane detached from the body, forming blebs at the membrane flagellar (Figures 5(b) and (d)).The formation of blebs was also observed in T. cruzi treated with com-

Figure 4 .
Figure 4. Morphological alterations of Trypanosoma cruzi epimastigote form cultured at 28˚C for 96 h, observed by scanning electron microscopy, in the absence (a) and presence of the semi-purified subfraction at concentrations of IC 50 (b), (c), and (d) and IC 90 (e) and (f).Bar = 1 μm.

Figure 5 .
Figure 5. Ultrastructural alterations of Trypanosoma cruzi epimastigote forms cultured at 28˚C for 96 h, observed by transmission electron microscopy, in the absence (a) and presence of semi-purified subfraction at concentrations of IC 50 (b), (c) and (d) and IC 90 (e) and (f) er-endoplasmic reticulum; f-flagellum; m-mitochondrion; n-nucleus; v-vacuole.Bar = 1 μm.poundsER27856 and BPQ-OH (3-biphenyl-4yl)-3-hydroxy quinuclidine BPQ-OH)[41].These compounds are inhibitors of scalene synthase, the enzyme involved in the biosynthesis of ergosterol.The change in the chemical composition of the membrane alters the ratio of phospholipids and sterols, affecting the stability of the membranes, and especially the integrity of the cell body[42].This may explain the morphological changes observed in epimastigotes treated with the sesquiterpene-rich subfraction of the flower of A. tinctoria.Pedroso et al.[43] also showed the presence of blebs in cell and flagellar-pocket membranes in Crithidia deanei treated with essential oil from Cymbopogons citratus.

Table 1 . Citotoxicity, growth inhibition and selectivity index of the crude extract aqueous phase, dichlorometane fraction, and semi-purified subfraction obtained of Anthemis tinctoria.
Antitrypanosomal Activity of a Semi-Purified Subfraction Rich in Labdane Sesquiterpenes, Obtained 52 from Flowers of Anthemis tinctoria, against Trypanosoma cruzi