The antimicrobial activities of methanol extract, fractions from the stem of E. caatingae Plowman were tested using the disc diffusion method at a concentration of 2000 μg/disc. The results are presented in Tables 1–3. All test fractions, except the hexane fractions, showed antimicrobial activity on gram-positive bacteria and fungi. The DMSO (control) did not produce inhibition haloes against the microorganisms studied, indicating that this solvent does not interfere with the antimicrobial activity results for the extract or fractions. The bacteria that were the most sensitive to the fractions tested were Micrococcus luteus and Mycobacterium smegmatis, with each showing inhibition zones of 13.5 ± 0.7 to 29.5 ± 0.7 mm and 14.0 ± 1.4 to 31.5 ± 1.4 mm, respectively. The AcOEt:MeOH (80:20), AcOEt:MeOH (90:10), AcOEt:MeOH (95:5) and the CHCl₃ phases showed significant inhibition zones of 18.0 ± 0.0 to 19.0 ± 1.4 mm for Candida albicans (Table 1). Evaluation of the minimum inhibitory concentration of the extract and fractions of E. caatingae (zone of inhibition > 12 mm) showed significant inhibitory concentrations for S. aureus, M. luteus, B. subtilis, M. smegmatis, E. faecalis and C. albicans. However, the lowest values of the minimum inhibitory concentration (MIC) and minimum bacteriostatic concentration (MBC) against M. luteus went to the AcOEt:MeOH (95:5), AcOEt:MeOH (90:10) and CHCl₃ fractions (Tables 1–3).

Plants are an important source of potentially useful structures for the development of new chemotherapeutic agents. The first step to identification of new chemotherapeutic agents is through trials of antibacterial activity in vitro. Many reports are available on the antiviral, antibacterial, antifungal, anthelmintic, antimolluskal and anti-inflammatory properties of plants. Some of these observations have proven helpful in the identification of the active principle compounds responsible for such activities and in the development of drugs for therapeutic use in humans [19].

There are a few reports of the antimicrobial activity of the genus Erythroxylum. Among them, we highlight the alkaline extract of the bark of Erythroxylum catuaba that has been shown to have a protective action against lethal infections of E. coli and S. aureus [20]. However, Rahman et al. (1998) [21] reported the absence of antibacterial activity against S. aureus, E. coli, B. subtilis and P. aeruginosa at the concentrations of 100 and 200 μg/mL and the presence of antifungal activity of the alcoholic extract of Erythroxylum moonii against C. albicans. In our study, the absence of any antimicrobial activity of E. caatingae against S. aureus, P. aeruginosa and E. coli was also verified, while the AcOEt:MeOH (80:20), AcOEt:MeOH (90:10), AcOEt:MeOH (95:5) and the CHCl₃ fractions inhibited the growth of C. albicans. However, the lowest values of MIC and MBC went to the AcOEt:MeOH (90:10), AcOEt:MeOH (95:5) and chloroform fractions against M. luteus. It was observed from the data that the most active fractions were more polar, probably due to the presence of phenolic compounds such as flavonoids or tannins. According Haslan (1996) [22], the antimicrobial activity exhibited by tannins is explained by the ability of these compounds present in complexing with macromolecules, such as polysaccharides and proteins present in bacteria.

The cytotoxicity of the 11 samples, including the extract, fractions and two isolated compounds on the human tumor cell lines were evaluated after 72 h using MTT, and the results are presented in Table 4. The AcOEt:MeOH (95:5) (5), AcOEt (6), CHCl₃ (7) and C₆H₆ (8) fractions were the most cytotoxic against the NCI-H292, HEp-2 and K562 cell lines. The most sensitive strains tested were the HEp-2 cell line, with an IC₅₀ value equal to 8.25 ± 0.36 μg/mL for the CHCl₃ fraction, and the K562 cell line, with an IC₅₀ value equal to 13.10 ± 0.63 μg/mL, 9.86 ± 0.56 μg/mL and 11.21 ± 0.46 μg/mL for the AcOEt:MeOH (95:5), AcOEt and CHCl₃ fractions, respectively. To verify whether the cytotoxicity observed was related to the injury of the cell plasma membrane, fractions 3–8 (Table 4) and the Catuabine B of E. caatingae were tested in the erythrocytes of mice (Mus musculus). The absence of hemolysis was observed in all substances.

The cytotoxic activity of Erytroxylum in carcinoma and adenocarcinoma cells was first described by Silva et al. (2001) [15]. This study showed that the methanolic extract and alkaloids of tropanes of Erytroxylum pervillei inhibited the growth of KB-V1 cells (human cervix carcinoma multidrug-resistant cells) in the presence of vinblastine and were much less cytotoxic to KB-V1 cells in the absence of vinblastine or KB cells (oral squamous cell carcinoma). The alkaloids of the species pervillene B and C showed cytotoxic activity in adenocarcinomas of ovary (SKOV3) cells and in adenocarcinomas of multidrug-resistant ovary (SKVLB) cells incubated with adriamycin. The following year, alkaloids isolated from the stem of Erytroxylum rotundifolium were also found to be cytotoxic in KB-V1 cells [23].

Extracts of Erytroxylum minutifolium and Erytroxylum confusum produced a significant cytotoxic effect in rat hepatocytes and displayed hepatoprotective, anti-inflammatory and antimicrobial activities [24,25].

Among the few reports of cytotoxicity of the genus Erythroxulum, our group found an absence of cytotoxicity in the methanol extract of E. caatingae stems and in the catuabine B against human cancer cell lines (HEp-2, NCI-H292 and KB) of cytotoxicity within in the tested concentration range of 50.0–1.56 μg/mL [26]. However, this cytotoxicity test showed that the AcOEt: MeOH (95:5), AcOEt, CHCl₃ and hexane fraction were the most active against the HEp-2, NCI-H292 and K562 cell lines. The extracts showed promising activity, with IC₅₀ values ranging between 8.2–40.5 μg/mL. Most of these values were within the cutoff point of the National Cancer Institute criteria for cytotoxicity (IC₅₀ < 30 μg/mL) in the screening of crude plant extracts, which indicates that these extracts are promising for further purification and study [27].

Although we observed cytotoxic activity, the E. caatingae extracts and fraction did not possess any activity against the mouse erythrocytes. These data suggest that the cytotoxic activity was not related to the lytic properties or the membrane instability induced by the extracts.

Phosphate buffered saline (PBS), penicillin-streptomycin liquid and DMEM (Dulbecco’s Modified Eagle’s Medium) were purchased from Gibco. MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) was purchased from Invitrogen. Eposide (Etoposide) was purchased from Blausiegel. Fetal bovine serum (FBS), glutamine, Triton X-100 and JC-1 (5,5′,6,6′-Tetrachloro-1,1′, 3,3′-tetraethylbenzimidazolcarbocyanine iodide) were purchased from Sigma-Aldrich Brazil. The Annexin V-FITC Apoptosis Detection Kit was purchased from Calbiochem. DMSO (Dimethyl sulfoxide), CaCl₂, NaCl, and paraformaldehyde were purchased from Vetec. Mueller Hinton Agar and Sabouraud were purchased from Difco, and kanamycin and ketoconazole were purchased from Cecon.

The stems of Erythroxylum caatingae Plowman were collected in Picuí, Paraíba, Brazil. The botanical material was identified by Maria de Fátima Agra of the Laboratory of Pharmaceutical Technology, Federal University of Paraíba. A dried specimen was deposited in the herbarium of Lauro Pires Xavier (JPB) of the Federal University of Paraíba under the identification label AGRA 5666.

The crude methanol extract (500 g) was dissolved in water and defatted with C₆H₆. The defatted aqueous extract was acidified with 3% HCl with mechanical mixing and filtered through Celite, which yielded a residue, which was discarded, and an acidic solution. The acidic solution was extracted with CHCl₃ (3 × 500 mL), which resulted in an acid chloroform phase and an aqueous phase that was neutralized to pH 7.0 with ammonium hydroxide. The aqueous phase, at pH 7.0, was extracted with CHCl₃ to yield an aqueous phase and the basic chloroform phase (4.0 g), which was submitted to column chromatography (CC) using a silica gel as the stationary phase and CHCl₃ and MeOH as the eluents, which were used either independently or in binary mixtures with increasing polarity. The result was 55 fractions of 100 mL. The 55 fractions were monitored by thin layer chromatography (TLC) and eluted with various solvent systems (CHCl₃ and/or CHCl₃-MeOH in order of increasing polarity) in chambers that were pre-saturated with NH₃ vapor. They were then revealed with Dragendorff’s reagent and placed in 21 groups based on their Rf. Fraction 25, 45 and 46 were submitted to repeated recrystallization with acetone and ether to obtain alkaloids. The elucidation of the chemical structures was described by Oliveira et al. (2011) [26]. 6β-Benzoyloxy-3α-(3,4,5-trimethoxybenzoyloxy) tropane (Catuabine B) and 3α,6β-dibenzoyloxytropane were also isolated. The extract and fractions obtained were evaporated until dry. The following extracts were all tested: the methanol extract of the stem of E. caatingae (MEEC) and the AcOEt:MeOH (60:40), AcOEt:MeOH (80:20), AcOEt:MeOH (90:10), AcOEt:MeOH (95:5), AcOEt, CHCl₃, and the hexane fractions, as well as the total alkaloids fraction.

The microorganisms, which were from the Antibiotics Department collection of the Federal University of Pernambuco were as follows: S. aureus, UFPEDA 01; Bacillus subtilis, UFPEDA 16; Enterococcus faecalis, UFPEDA 138; Micrococcus luteus, UFPEDA 06; Escherichia coli, UFPEDA 224; P. aeruginosa, UFPEDA 39; Serratia marcescens, UFPEDA 398; Mycobacterium smegmatis, UFPEDA 71; and Candida albicans, UFPEDA 1007, and these were used in the antimicrobial activity assays.

The cell lines used for the in vitro cytotoxicity tests were K562 (human chronic myelocytic leukemia), NCI-H292 (human lung mucoepidermoid carcinoma cells) and HEp-2 (human larynx epidermoid carcinoma cells), and these were obtained from the Adolph Lutz Institute (São Paulo, Brazil). The cells were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO₂.

Three Swiss mice (female, 25–30 g), obtained from the central animal house of the Universidade Federal de Pernambuco, Brazil, were used. The animals were housed in cages with free access to food and water. All animals were kept under a 12 h light:12 h dark cycle (lights on at 6:00 a.m.). The animals were treated according to the ethical principles of animal experimentation of the Brazilian College of Animal Experimentation (COBEA), Brazil. The Animal Studies Committee of the Universidade Federal do Pernambuco approved the experimental protocols (Number 23076.012173/2007-77).

Antimicrobial activity was verified in vitro by the method of diffusion in paper record [34]. The microorganisms were standardized with an optical density of McFarland 0.5 in physiological solution [35], which corresponds to a concentration of approximately 10⁷ UFC/mL for yeast and filamentous fungi and 10⁸ UFC/mL for bacteria. On the inoculated medium, Mueller Hinton Agar (S. aureus, B. subtilis, M. luteus, E. coli, P. aeruginosa and S. marcescens), glucose extracts of yeast (E. faecalis, M. smegmatis) and Sabouraud (C. albicans), and discs of barren paper (6 mm) were absorbed with 10 μL of the solution 200,000 μg/mL of the methanol extract from the stem of E. caatingae and its fractions and were placed on the agar. After placing the discs, the plates were incubated for 24 h and 48 h at 30 °C and 35 °C, respectively. The antibiotics kanamycin and ketoconazole were used as standards at 30 μg/disc and 300 μg/disc, respectively.

The Minimum Inhibitory Concentration (MIC) and Minimum Bacteriostatic Concentration (MBC) were applied to the substances that showed halos larger than 12 mm by serial dilutions in half-solids [36]. Aliquots of different volumes (0.03–1.0 mL) of the solutions at 20.000 μg/mL were placed in Petri dishes and homogenized with 10 mL of the appropriate culture medium. The microorganisms were streaked on the surface of the medium, and the plates were incubated at 35 °C and 30 °C for 24 h and 48 h, respectively. For values over 1000 μg/mL, the extract was considered inactive [37].

The cytotoxicity of the methanol extract of the stems of E. caatingae and its fractions were tested against the K562, NCI-H292 and HEp-2 tumor cell lines. For these experiments, the cells were plated in 96-well plates (10⁵ cells/mL for adherent cells or 0.3 × 10⁶ cells/mL for suspended cells). After 24 h, the extracts, fractions (6.25–50 μg/mL) and alkaloids (1.25–10 μg/mL) dissolved in DMSO were added to each well and incubated for 72 h. Control groups received DMSO (0.2%) in DMEN. Etoposide (1.25–20 μg/mL) was used as a positive control. The growth of the tumor cells was quantified by the ability of the living cells to reduce yellow tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5- diphenyltetrazolium bromide) to a blue formazan product [38,39]. At the end of the 72 h incubation period, MTT (5.0 mg/mL) was added to the plate. After three hours for the suspended cells or two hours for the adherent cells, the formazan product from the reduction of MTT was dissolved in DMSO, and the absorbance was measured using a multi-plate reader. The effect of the drug was quantified as the percentage of control absorbance the reduced dye at 450 nm (Multi-plate Reader Thermoplate).

The hemolytic assay was performed in 96-well plates, following the method previously described by Costa-Lotufo et al. (2005) [40]. Each well received 100 μL of a 0.85% NaCl solution containing 10 mM CaCl₂. The first well served as the negative control and only contained the vehicle (10% DMSO). The second well contained 100 μL of the test substance that was diluted 1:2. The extracts were tested at concentrations ranging from 15.62 to 2000 μg/mL. The serial dilution continued until the 11th well. The last well received 20 μL of 0.1% Triton X-100 (in 0.85% saline) to obtain 100% hemolysis (positive control). Each well received 100 μL of a 2% suspension of mouse erythrocytes in 0.85% saline containing 10 mM CaCl₂. After incubation at room temperature for 30 min and centrifugation, the supernatant was removed, and the released hemoglobin was measured by spectroscopic absorbance at 450 nm. Extracts with an EC₅₀ value lower than 200 μg/mL were considered to be active.

The following experiments were performed to elucidate the mechanisms involved in the cytotoxic activity of the fractions and alkaloids on K562 cells after 48 h of incubation.

Data are presented as the mean ± S.D. The IC₅₀ and EC₅₀ values and their 95% confidence intervals were obtained by nonlinear regression using SigmaPlot 11.0 (version 11.0.1; Systat Software Inc.: Chicago, IL, USA, 2011). The differences between the experimental groups were compared by one-way analysis of variance (ANOVA), followed by Newman-Keuls test; the significance level was set at 1%.