Antileishmanial activity of standardized fractions of Stryphnodendron obovatum (Barbatimão) extract and constituent compounds

Author's Accepted Manuscript

Antileishmanial activity of standardized fractions of Stryphnodendron obovatum (Barbatimão) extract and constituent compounds Tatiana G. Ribeiro, André M. Nascimento, Bárbara O. Henriques, Miguel A. ChávezFumagalli, Juçara R. França, Mariana C. Duarte, Paula S. Lage, Pedro H.R. Andrade, Daniela P. Lage, Lívia B. Rodrigues, Lourena E. Costa, Vivian T. Martins, André A.G. Faraco, Eduardo A.F. Coelho, Rachel O. Castilho

PII: DOI: Reference:

S0378-8741(15)00126-9 http://dx.doi.org/10.1016/j.jep.2015.02.047 JEP9351

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Journal of Ethnopharmacology

www.elsevier.com/locate/jep

Received date: 26 May 2014 Revised date: 18 December 2014 Accepted date: 21 February 2015 Cite this article as: Tatiana G. Ribeiro, André M. Nascimento, Bárbara O. Henriques, Miguel A. Chávez-Fumagalli, Juçara R. França, Mariana C. Duarte, Paula S. Lage, Pedro H.R. Andrade, Daniela P. Lage, Lívia B. Rodrigues, Lourena E. Costa, Vivian T. Martins, André A.G. Faraco, Eduardo A.F. Coelho, Rachel O. Castilho, Antileishmanial activity of standardized fractions of Stryphnodendron obovatum (Barbatimão) extract and constituent compounds, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.02.047 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title Antileishmanial

activity

of

standardized

fractions

of

Stryphnodendron

obovatum

(Barbatimão) extract and constituent compounds

Author names Tatiana G. Ribeiroa, André M. Nascimentoa, Bárbara O. Henriquesa, Miguel A. ChávezFumagallib, Juçara R. Françaa, Mariana C. Duarteb, Paula S. Lageb, Pedro H.R. Andradec, Daniela P. Lagec, Lívia B. Rodriguesa, Lourena E. Costab, Vivian T. Martinsd, André A.G. Faracoa, e, Eduardo A.F. Coelhob, c, Rachel O. Castilhoa, e*

Author affiliations and addresses a

Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia,

Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil b

Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical,

Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil c

Departamento de Patologia Clínica, COLTEC, Universidade Federal de Minas Gerais, Belo

Horizonte, Minas Gerais, Brazil d

Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade

Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil e

Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de

Minas Gerais, Belo Horizonte, Minas Gerais, Brazil *Corresponding author. Tel.: +553134096936; fax.: +553134096935 E-mail address: [email protected]

1

ABSTRACT

Ethnopharmacological relevance: Stryphnodendron obovatum Benth. is a Brazilian tree used to treat skin ulceration, promote wound healing, and inhibit the growth of protozoa, including Trypanosoma and Leishmania species. Bioguided fractionation of the ethanol extract of S. obovatum stem bark was performed, and antileishmanial and antioxidant activities of the standardized fractions were analyzed. Materials and methods: Stationary-phase Leishmania amazonensis promastigotes, murine macrophages, and human red blood cells (RBCs) were exposed to plant extract, standardized fractions or isolated compounds for 48 h at 37°C to evaluate their antiparasitic activity and cytotoxicity. The 2,2-diphenyl-1-picryl-hidrazyl assay was used to evaluate antioxidant activity. Results: The S. obovatum extract and fractions showed antileishmanial and antioxidant activity; however, the organic fraction (OF) showed the best efficacy. We identified gallic acid, gallocatechin, epigallocatechin, catechin, and epigallocatechin gallate in the OF fraction. These compounds effectively inhibited L. amazonensis activity, with gallic acid, gallocatechin, and epigallocatechin gallate showing the highest selectivity. Furthermore, the evaluated compounds had no significant effect on murine macrophages and human RBCs. Conclusions: The compounds present in the S. obovatum plant bark ethanol extract may provide an alternative therapeutic approach for L. amazonensis treatment. Keywords: Cerrado, tannins, gallic acid, gallocatechin, antioxidant, Leishmania amazonensis

Abbreviations: RBCs, Red blood cells; OF, organic fraction; AF, aqueous fraction; GA, gallic acid; EGC, epigallocatechin; C, catechin; GC, gallocatechin, EGCG, epigallocatechin gallate.

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1. Introduction

Leishmaniasis is a protozoal disease with high morbidity and mortality worldwide. Over 350 million people in 98 countries are at risk of contracting leishmaniasis (WHOE, 2010), in addition to approximately 700,000 to 1.2 million cases of tegumentary leishmaniasis registered annually worldwide (Alvar et al., 2012). Leishmaniasis has a wide spectrum of clinical manifestations, attributable to the different protozoa species belonging to the Leishmania genus (Desjeux, 2004). Cutaneous leishmaniasis first develops as a localized papule, which evolves into an ulcer upon the loss of the epidermis, resulting in skin barrier impairment. While parenteral administration of pentavalent antimony organic compounds remains the first-line therapy for all forms of leishmaniasis, increased drug resistance and adverse side effects, including arthralgia, myalgia, pancreatitis, leukopenia, and cardiotoxicity, have been reported (Oliveira et al., 2011). Additionally, as leishmaniasis has emerged as an opportunistic infection in human immunodeficiency virus (HIV)-infected patients, the development of new and cost-effective alternative therapeutic strategies has become a priority (Kedzierski et al., 2009). In recent years, researchers have sought to identify plant-derived compounds for use as novel antileishmanial drugs (Ribeiro et al., 2014a). A wide variety of separation techniques have been developed to identify active molecules in plants, thereby supporting the development of new therapeutics. Although numerous studies have identified plant extracts and/or purified compounds with antileishmanial activity, effective alternative therapeutics for leishmania have not yet been developed (Lage et al., 2013). The genus Stryphnodendron, belonging to the Fabaceae family, consists of approximately 48 species, including Stryphnodendron obovatum Benth., called “barbatimao” (Costa et al., 2013). S. obovatum is a tree found in the Cerrado, a savannah region of Brazil (Sanches et al.,

3

2007). It has long been used in Brazil as an antiseptic and antiprotozoal agent, showing efficacy against Trypanosoma and Leishmania. Furthermore, S. obovatum has been used to treat skin ulceration and induce wound healing (Nascimento et al., 2013; Oliveira et al. 2014). Studies of Stryphnodendron species have identified anti-ulcerogenic, antioxidant, woundhealing, antimicrobial, and antileishmanial activity (Lopes et al., 2005; Luize et al. 2005; Souza et al., 2007). Stryphnodendron species contain tannins, including prodelphinidins and prorobinetinidins (Mello et al., 1999). Tannins are phenolic compounds that exhibit a remarkably wide range of bioactivities (Okuda, 2005). They are thought to mediate the curative and palliative efficacy, and health benefits of many traditional herbal medicines and foods (Kolodziej and Kiderlen, 2005). Owing to the beneficial effects S. obovatum, we sought to evaluate the antileishmanial and antioxidant activity of the ethanol extract (EE) of this plant. After bioguided fractionation, the organic fraction (OF) was analyzed and standardized to identify and quantify the compounds responsible for the antileishmanial activity. We identified five tannins that effectively inhibited L. amazonensis. Further we established the 50% inhibitory concentrations (IC50), the concentrations required to achieve 50% of the antioxidant effect (EC50), and the concentrations required to achieve 50% cytotoxicity in murine macrophages (CC50), and O+ human red blood cells (RBC50) of these compounds.

4

2. Materials and methods

2.1. Chemicals and reagents

High-performance liquid chromatography (HPLC)-grade acetonitrile and methanol were purchased from Tedia (Fairfield, OH, USA) and J.T. Baker (Phillipsburg, NJ, USA), respectively. Concentrated phosphoric acid (85% w/v, Merck, Darmstadt, Germany) and commercial ethanol (96% v/v) were used. Ultrapure water was obtained using a Milli-Q plus system from Millipore (Milford, MA, USA). The HPLC-grade reference substances used were as follows: gallic acid (GA, 98%, Acrós Organics, Geel, Belgium), epigallocatechin (EGC; 90%, Fluka, Milwaukee, WI, USA), catechin (C; 98%, Sigma, Milwaukee, WI, USA), gallocatechin (GC, 98%, Sigma), and epigallocatechin gallate (EGCG, 95%, Sigma). Schneider's medium, RPMI 1640 medium, fetal bovine serum (FBS), L-glutamine, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and penicillin/streptomycin solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Amphotericin B (AmpB) was provided by Cristália Produtos Químicos Farmacêuticos Ltd. (São Paulo, Brazil).

2.2. Plant material

The stem bark from S. obovatum Benth. was collected in Mato Grosso do Sul (2006), in Campo Grande City (S20°24ƍ37.4ƍƍ and WO54°36ƍ52.5ƍƍ), Brazil. The species was identified by Prof. Arnildo Pott at the Universidade Federal de Mato Grosso do Sul (UFMS), Brazil, and a voucher specimen (32997) was deposited at the CGMS herbarium, UFMS.

5

2.3. Preparation of the extract and fractions

The stem bark of S. obovatum was dried at 40 °C to constant weight. After grinding, stem bark (170 g) was extracted with 200 ml of 96% v/v commercial ethanol by percolation for two weeks at room temperature, changing the solvent every 24 h. The solvent was then removed in a rotary evaporator at 40 °C, yielding a dark brown solid (EE), representing 45.5% dry mass. The S. obovatum EE was fractionated by liquid-liquid extraction using ethyl acetate:butanol:2-propanol:water (3.5:0.5:1.0:4.5), yielding an organic fraction (OF and an aqueous fraction (AF). The OF and AF were separately evaporated under a warm air stream (40 °C) to obtain dry residues. The OF was standardized by reversed phase (RP)-HPLC as described by Nascimento et al. (2013).

2.4. Chromatographic analysis (RP-HPLC)

The EE, OF, and reference compounds (GA, EGC, C, GC, and EGCG) were dissolved in methanol to concentrations of 10.0, 5.0, and 1.0 mg/ml, respectively. Chromatographic analyses were performed using an HP1100 system (Agilent, Santa Clara, CA, USA) coupled to a quaternary pump, an auto sampler, and a programmable ultraviolet photodiode array detector (UV/DAD). An HPChemStation for LC3D systems software (Rev. B.02.01-SR2 [260] 2001-2006) was used to evaluate the data. An RP C18 pre-column (XDB Zorbax, 4 × 4 mm internal diameter [I.D.]; 5 µm, Agilent) was attached to a C18 column (LiChrospher100, 250 × 4 mm I.D.; 5 µm, Merck) at 40 °C. After filtration using a 0.45 µm polytetrafluoroethylene (PTFE) membrane, 10 µl solutions were automatically injected into the system at a flow rate of 1 ml/min, with detection at λ 210 nm. UV/DAD spectra were recorded on-line for peak purity and identification from Ȝ 190 to 400 nm, and compound 6

identities were confirmed by co-elution of standards, using 100 µl of the standard solutions. The degassed mobile phase was composed of aqueous 0.1% phosphoric acid (solution A), and 0.1% phosphoric acid in acetonitrile (solution B), with a linear gradient from A-B (95:5, v/v) to A-B (60:40, v/v) over 60 min. Cleaning and reconditioning of the column was performed for 15 min.

2.5. Free radical scavenging activity

Free radicals were formed using 2,2-diphenyl-1-picryl-hidrazyl (DPPH) from Sigma. Samples (100 µl; 0.43 to 71.40 µg/ml) were added to each well of a 96-well culture plate (Nunc, Nunclon, Roskilde, Denmark), using methanol as the diluent control and rutin (Sigma) as a positive control. The culture plate was shaken for 1 min, and then incubated for 30 min at 37 °C. Absorbance was measured using a multi-well scanning spectrophotometer (Tecan Infinity M200) at a wavelength of 517 nm. The results were presented as the EC50 of the radical scavenging capacity, corresponding to the concentration of sample required to decrease the initial DPPH absorbance by 50%. The data are representative of three independent experiments, performed in triplicate (Farias et al. 2013).

2.6. Parasites and mice

L. amazonensis (IFLA/BR/1967/PH-8) parasites were grown at 24 °C in Schneider’s medium (Sigma-Aldrich), supplemented with 20% heat-inactivated FBS (Sigma), 20 mM L-glutamine, 200 U/ml penicillin, and 100 Ɋg/ml streptomycin at pH 7.4. Stationary-phase promastigotes were prepared as described previously (Coelho et al., 2003).

7

Murine peritoneal macrophages were obtained from female BALB/c mice (aged 8 weeks), purchased from the Institute of Biological Sciences of the Universidade Federal de Minas Gerais (UFMG). The Animal Use Committee at UFMG approved the experimental protocols (code 136/2012).

2.7. Antileishmanial activity

Leishmania growth inhibition was assessed in vitro by cultivating stationary-phase L. amazonensis promastigotes (1 × 106 cells) in the presence of 0.78–100 Ɋg/ml EE, OF, AF, GA, EGC, C, GC, or EGCG in 96-well culture plates for 48 h at 24 °C. A control experiment was performed to determine the optimal incubation time to evaluate L. amazonensis growth inhibition (data not shown). Cell viability was assessed by measuring MTT (2 mg/ml; Sigma) cleavage. Absorbance was measured using a multi-well scanning spectrophotometer (LABTRADE, model 660) at a wavelength of 570 nm. AmpB (0.78–100 Ɋg/ml) was used as a positive control (Ribeiro et al., 2014b). The sample concentrations required to inhibit 50% Leishmania viability (IC50) were determined by applying a sigmoidal regression to the doseresponse curves. Data represent three independent experiments, performed in triplicate.

2.8. Cytotoxicity assay and hemolytic activity

The inhibition of macrophage viability by 50% (CC50) was investigated by cultivating macrophages (5 × 105 cells) in the presence of 0.78–100 Ɋg/ml EE, OF, AF, GA, EGC, C, GC, or EGCG in 96-well culture plates for 48 h at 37 °C. A titration curve was prepared to determine the optimal incubation time to evaluate viability (data not shown). An MTT assay was used to assess cell viability, using AmpB as a positive control (Coelho et al., 2003). The

8

CC50 was determined by applying a sigmoidal regression to the dose-response curves. The selectivity indices (SI) of the samples were calculated by determining the ratio between the CC50 and IC50. Hemolytic activity was investigated by incubating the same sample concentrations with a 5% RBC (human O+) suspension for 1 h at 37 °C. Briefly, the RBC suspension was centrifuged (1000 × g for 10 min), and cell lysis was determined spectrophotometrically (λ = 570 nm) as described previously (Coelho et al. 2003; Ribeiro et al., 2014). The absence of (negative control) or 100% presence of hemolysis (positive control) was determined by replacing the samples with an equal volume of phosphate-buffered saline (PBS) or distilled water, respectively. The percent hemolysis was calculated versus the negative and positive controls. The results were expressed as the mean percent hemolysis, compared to non-treated control wells, and the 50% hemolytic concentration (RBC50) was calculated for each sample. Data represent three independent experiments, performed in triplicate.

2.9. Statistical analyses

Data were analyzed using GraphPad Prism software (version 5.0 for Windows). The difference between groups was evaluated by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc multiple-comparison test. Differences were considered significant when the ’ value was less than 0.05 (Coelho et al., 2003).

3. Results and Discussion

In the current study, the EE, fractions, and compounds obtained from the S. obovatum stem bark displayed antioxidant and antileishmanial activity, as determined by DPPH assay and L.

9

amazonensis viability assays, respectively. Table 1 shows the results of the in vitro antioxidant activity (EC50), antileishmanial activity (IC50), and cytotoxicity (CC50 and RBC50) evaluations. Because the EE derived from the S. obovatum stem bark displayed antioxidant (EC50 = 6.3 µg/ml) and antileishmanial activity (IC50 = 60.0 µg/ml), the presence of phenols in the EE was evaluated and demonstrated using thin-layer chromatography with select reagents (Wagner et al., 1984) (supplementary data). The RP-HPLC fingerprint indicated the presence of proanthocyanidins and hydrolyzable tannins in the main peaks (wavelength absorptions around 260 and 280 nm, respectively) as previously described (Tarascou et al., 2010). This extract was fractionated by liquid-liquid extraction using a solvent system to separate tannins with a high molecular weight (AF) from those with a low molecular weight (OF), and to eliminate the cluster of polar compounds in the matrix, which are considered necessary for standardize vegetal complex matrices. The AF (IC50 = 26.2 µg/ml) and OF (IC50 = 24.5 µg/ml) were 2-fold more active against L. amazonensis than the EE (Table 1), although both fractions had similar antiprotozoal activity. Since the OF (EC50 = 5.9 µg/ml) had greater antioxidant activity than the AF (EC50 = 21.9 µg/ml), this fraction was chosen for further evaluation (Table 1). Table 1 Several tannins were identified in the OF, including GA (1), GC (2), EGC (3), C (4), and EGCG (5) (Fig. 1). The presence of these substances in the OF was confirmed by retention time (tR) co-elution experiments using RP-HPLC (Fig. 2). In a previous study, an analytical method was developed and validated to standardize the OF. The selected markers, GC (2) and EGCG (5) (Fig. 1), in S. obovatum and S. adstringens were quantified by chromatography. Quantification of the tannins present in the S. obovatum EE-derived OF revealed an average

10

of 12.2 µg/ml (1.2% w/w) GC and 14.2 µg/ml (1.4%, w/w) EGCG in the extracts (Nascimento et al., 2013). Figs. 1 and 2 GA (1) and EGCG (5) showed the most potent antioxidant activity, with EC50 values of 0.9 and 1.5 µg/ml, respectively. These substances also showed the best antileishmanial activity, with IC50 values of 1.7 and 16.3 µg/ml, respectively. C (4), GC (2) and EGC (3) also showed effective antioxidant activity (EC50 values < 5 µg/ml), and were more potent than the positive control, rutin (EC50 = 8.58 µg/ml). Furthermore, these compounds also showed antileishmanial activity, with IC50 values of 43, 30 and 86 µg/ml, respectively (Table 1). The antileishmanial activities of GA, GC, EGC, C, and EGCG in this study were comparable to those reported by Tasdemir et al. (2006), who evaluated the antileishmanial activities of GA, GC, EGC, and C (IC50s >30 µg/ml), and EGCG (IC50 of 19.1 µg/ml) against the amastigote form L. donovani. GA was more potent in our study than in the study reported by Tasdemir et al. (2006) (IC50 >30 µg/ml); however, our findings were consistent with those of Kolodziej et al. (2001), who revealed that the IC50 of GA against the intracellular amastigote form of L. donovani was 4.4 µg/ml. Many parasites are susceptible to oxidative stress (Bocedi et al., 2010). Polyamine (PA) synthesis pathway enzymes are considered important targets for antileishmanial drug development (Heby et al., 2007) because they have a central role in Leishmania proliferation, differentiation, and antioxidant pathways (Colotti and Ilari, 2011). Trypanosomatids prevent oxidative stress via the PA spermidine, which synthesizes trypanothione. Trypanothione protects the parasite from oxidative stress by removing reactive oxygen and nitrogen species (Bocedi et al., 2010), and other reactive species produced by the host defense system. Because the redox system plays an important role in parasite survival within the host, targeting this system represents a valid parasite-killing strategy (Tepea et al., 2009). Arginase (ARG), an 11

enzyme in the PA pathway, also prevents oxidative stress in trypanosomatids, and is considered a therapeutic target to control Leishmania infection (Silva et al., 2012). Interestingly, C (4) is a competitive inhibitor, ECCG (5) is a mixed inhibitor, and GA (1) is a noncompetitive inhibitor of ARG (Reis et al., 2013). In L. amazonensis, EGCG also damages mitochondria, contributing to parasitic death (Inacio et al., 2012). In contrast, in vitro studies have demonstrated that infected macrophages can augment and prolong their activation of the host defense system after incubation with tannins. Furthermore, polyphenols may have beneficial effects in various infectious conditions, although in vivo experiments

are

essential

to

support

the

therapeutic

benefits

of

polyphenolic

immunomodulators (Kolodziej and Kiderlen, 2005). The cytotoxicity of the EE, its fractions, and the isolated compounds was investigated in murine macrophages. The in vitro assays revealed low cytotoxicity, even when high concentrations of the samples were used (CC50 values ranging from 100-300 µg/ml). In contrast, the positive control (AmpB) had a CC50 of 0.8 µg/ml. The hemolytic activity of the samples was also determined in O+ human RBCs. No significant damage was observed in human RBCs after incubation with high concentrations of the samples (Table 1). These findings suggest that the EE, its fractions, and the isolated compounds were safe for use in mammalian cells. In contrast, AmpB efficiently eliminated parasites at low doses, but was also cytotoxic in mammalian cells, as previously reported by Ribeiro et al., 2014. Evaluation of the compound SIs revealed that GA (1), GC (2), and EGCG (5) were the most selective for Leishmania versus murine macrophages. These results suggest that it would be worthwhile to conduct further in vivo studies using these compounds in order to better understand their potential antileishmanial activity in mammals. Current leishmaniasis treatments are inadequate, due to high toxicity and cost, and growing parasitic resistance (Oliveira et al., 2011). Compounds extracted from natural products

12

represent an important resource for the development of novel therapeutic agents (Saklani and Kutty, 2008). Plants of the genus Stryphnodendron are known for their antiseptic bioactivity in Brazilian folk medicine, and are used for the treatment of skin ulceration, to induce wound healing, and to inhibit Leishmania growth (Nascimento et al., 2013; Oliveira et al. 2014). Nevertheless, few studies have evaluated their biological activity, or identified and characterize the compounds responsible for this effect. The results presented here show that following bioactivity-guided fractionation of the S. obovatum stem bark EE, both OF and AF exhibited antileishmanial activity. GA, GC, EGC, C, and EGCG were identified in the OF, and effectively inhibited L. amazonensis growth. The redox system, and the PA pathway in particular, likely play an important role in the antileishmanial mechanism of action. Therefore, it was important to correlate the antileishmanial effect with antioxidant activity.

4. Conclusion

The standardized OF derived from the S. obovatum stem bark EE, and the isolated compounds, could represent an alternative therapeutic approach for leishmaniasis treatment. Further studies are in progress to evaluate the activity of these compounds in murine models experimentally infected with L. amazonensis. Therefore, investigation of the chemical constituents of S. obovatum and their bioactivities will yield valuable information, facilitating further studies on plants of the Stryphnodendron genus.

Acknowledgments

This work was supported by grants from Pró-Reitoria de Pesquisa from UFMG (Edital 01/2014). The authors thank the Instituto Nacional de Ciência e Tecnologia em Nano-

13

biofarmacêutica (INCT-Nanobiofar), FAPEMIG (CBB-APQ-02364-08 and CBB-APQ00496-11), FUNDECT (41/100172/05), and CNPq (APQ-471374/2004-0APQ-472090/20119 and APQ-482976/2012-8).

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FIGURE LEGENDS

Fig. 1. Substances identified in the organic fraction (OF) of Stryphnodendron obovatum: gallic acid (1), gallocatechin (2), epigallocatechin (3), catechin (4), and epigallocatechin gallate (5).

Fig. 2. Chromatograms of the Stryphnodendron obovatum stem bark organic fraction (OF) with the identified peaks (GA, gallic acid; GC, gallocatechin; EGC, epigallocatechin; C, catechin; and EGCG, epigallocatechin gallate). Separation was performed by RP-HPLC, as described in the Materials and Methods.

TABLE LEGENDS

Table 1 Antileishmanial activity, cytotoxicity, selectivity index, and antioxidant activity of Stryphnodendron obovatum stem bark ethanolic extract, its fractions, and the isolated compounds.

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Table 1 DPPH IC50

CC50

RBC50c

Samples a

(µg/ml)

b

SId

EC50e

(µg/ml)

(µg/ml) Ethanolic extract (EE)

60.0 ± 0.5

103.6 ± 3.8

12.3 ± 0.1

1.7

6.3 ± 0.2

Organic fraction (OF)

24.5 ± 0.5

156.9 ± 1.7

8.2 ± 0.6

6.3

5.9 ± 0.1 21.9 ±

Aqueous fraction (AF)

26.2 ± 0.4

140.0 ± 1.7

14.3 ± 0.1

5.4

0.4

Catechin (C)

43.2 ± 2.1

117.3 ± 0.9

5.3 ± 0.8

2.7

4.6 ± 0.1

Gallocatechin (GC)

30.2 ± 0.7

271.2 ± 0.9

8.2 ± 0.2

9.0

3.4 ± 0.1

Epigallocatechin (EGC)

86.4 ± 0.5

233.9 ± 1.0

3.8 ± 0.9

2.7

3.4 ± 0.1

(EGCTG)

16.3 ± 0.3

120.3 ± 1.6

7.2 ± 0.1

7.4

1.5 ± 0.1

Gallic acid (GA)

1.7 ± 0.7

26.6 ± 1.2

13.4 ± 0.7

15.7

0.9 ± 0.1

Amphotericin Bf

0.1 ± 0.1

0.8 ± 0.2

NDg

8.0

ND

ND

ND

ND

ND

8.6 ± 0.2

Epigallocatechin gallate

Rutinh

Data represent three independent experiments. The results are expressed as mean ± standard deviation. a

IC50 = concentration producing 50% inhibition of L. amazonensis promastigotes.

b

CC50 = concentration producing 50% inhibition of murine macrophages.

c

RBC50 = concentration producing 50% hemolysis of human red blood cells.

d

SI = Selectivity index (ratio between CC50 and IC50).

e

EC50: concentration producing 50% antioxidant activity

f

Amphotericin B was used as a positive control for L. amazonensis promastigotes inhibition.

g

ND: Not done.

h

Rutin was used as a positive control for free radical scavenging activity (DPPH).

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Figure 1

O

OH

OH

OH OH

HO

O OH

HO

OH HO

O OH

OH OH

OH

1

OH

OH

OH

2

3

OH

OH OH

HO

O OH

HO

O OH

OH

O

OH

4

OH

OH O

OH OH

5

Figure 2

Graphical Abstract (for review)