New group of azastilbene analogs of resveratrol: Synthesis, anticandidal activity and toxicity evaluation

2.1 Synthesis of Schiff bases

The four resveratrol analogs, designated A, B, C and D, were synthesized and characterized in the chemical laboratory of the Federal University of Juiz de Fora, as described by and in accordance with other data in the literature (Lima et al., 2013).

Resveratrol analog D was synthesized by means of condensation between 2-hydroxyaniline with a variety of aromatic aldehydes in ethanol. This compound was characterized by ¹H and ¹³C nuclear magnetic resonance (NMR), infrared (I.R.), and melting point (M.P).

2.2 Antifungal activity

The Candida albicans American Type Culture Collection (ATCC) 10,231 species was used in this study, obtained from the André Tosello Foundation (Campinas, São Paulo, Brazil). The procedures were performed according to the M27-A2 protocol from the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2002). The fungal suspension was prepared in sterile saline (0.85% NaCl w/v) and then it was diluted in RPMI 1640 culture medium, buffered with 3-(N-morpholino)-propanesulphonic acid (MOPS) and the pH was adjusted to 7.0 ± 0.1, so as to obtain from 5 × 10² to 2.5 × 10³ colony forming units (CFU) per mL. The analogs were diluted in RPMI 1640 medium buffered with MOPS and 20 µL mL⁻¹ Tween-80/Dimethyl Sulfoxide (DMSO) (1:1, v/v) in a concentration range of 10–5000 µg mL⁻¹. The assay was performed in 96-well sterile microplates (Sarstedt, Germany), to which 100 µL of analogs dilutions and 100 µL RPMI 1640 were added, buffered with MOPS and inoculated with a suitable number of the microorganism’s colony forming units. The growth control consisted of 100 µL of the same inoculated culture medium and 20 µL mL⁻¹ Tween 80/DMSO (1:1, v/v) and a sufficient quantity of the uninoculated medium to add up to 200 µL. The negative control was prepared by adding 200 µL of uninoculated medium. The drug Amphotericin B (Cristália, Brazil) was used as positive control at concentrations from 0.0313 to 16.0 µg mL⁻¹. The microplates were incubated at 35 °C/48 h. The minimum inhibitory concentration (MIC) was established as the lowest concentration at which no turbidity was observed in the culture medium. After determining the MIC, an aliquot of 20 µL was retained from those wells which showed no visible growth and re-incubated with 4 mL of Tryptic Soy Broth (TSB) without the addition of an antifungal agent, for another 48 h at 35 °C. The lowest concentration at which no turbidity was noticed after this period was considered to be the Minimum Fungicidal Concentration (MFC). Results were expressed in µg mL⁻¹.

2.3 Scanning electron microscopy

The material used for observation by scanning electron microscopy was formed by colonies of fungal species that were not subjected to treatment (negative control), and colonies exposed to the tested compounds and Amphotericin B (reference drug).

The sample preparation was performed according to the method described by Gao et al. (2011) with some modifications. The same antifungal activity standardization procedure was used in the preparation of the fungal suspension, in order to obtain a suspension between 5 × 10² and 2.5 × 10³ CFU mL⁻¹.

The suspensions were transferred to a microplate with 100 μL inoculated medium, and, after incubation in an oven at 35 °C for 48 h, both the compounds and the reference drug were transferred to their respective wells. After this procedure, the microplate was re-incubated. In this study, the concentrations used for the compounds and reference drug were the MFC values found. After that, the samples were then centrifuged at 5000 rpm for 10 min (Eppendorf, 5417R, Germany) and the pellets formed were transferred to glass slides and fixed with 2.5% glutaraldehyde solution for 12 h. After this period, coverslips were washed with 0.1 M phosphate buffer (pH = 7.4) and dehydrated with increasing concentrations of ethanol (50% to 100%), with an interval of 20 min between each exchange. Then the coverslips were dried at room temperature and fixed in stubs with double-sided carbon tape. Subsequently, the coverslips were placed in the metallizer (Balzers Union FL – 9496, Balzers, Germany) for 5 min, resulting in a layer of gold–palladium thereon. Samples were observed by scanning electron microscope (JSM 5310, Jeol, Japan), at 25 kV power and 17 mm work distance.

2.4 Brine shrimp lethality test

The assay was carried out according to Meyer et al. (1982) Artemia salina Leach eggs were cultured in artificial sea water: NaCl 24.0 g L⁻¹, CaCl₂·2H₂O 1.5 g L⁻¹, KBr 0.1 g L⁻¹, KCl 0.7 g L⁻¹, Na₂SO₄ 4.0 g L⁻¹, NaHCO₃ 0.3 g L⁻¹, MgCl₂.6H₂O 11.0 g L⁻¹. The resveratrol and analogs were dissolved in Tween 80 and dimethyl sulfoxide (DMSO) (1:2, v/v), and then in artificial sea water. Ten brine shrimps (A. salina) were transferred to test tubes containing the following concentrations of the compounds: 10, 50, 100, 250, 500 and 1000 μg mL⁻¹ (n = 5). The tubes were kept in constant illumination and thymol was used as standard control. Living nauplii were counted after 24 h of exposition to the compounds tested. The final volume used of the solution of Tween 80 and DMSO (1:2, v/v) was also tested.

For the LC₅₀ calculations, the probit analysis method was used, which is defined by the necessary concentration to cause death of 50% of the A. salina larvae in a 24 h period. The calculations were performed in the statistical computer program “MICRO PROBIT”.

The compounds were considered highly toxic when the LC₅₀ was lower than 80 µg mL⁻¹, moderately toxic with LC₅₀ between 80 µg mL⁻¹ and 250 µg mL⁻¹ and with low toxicity or not toxic with LC₅₀ higher than 250 µg mL⁻¹ (Pompilho et al., 2014).

2.5 Evaluation of cellular viability using MTT assays

Cell viability of human keratinocytes (HaCaT) was evaluated after culture in Dulbecco's Modified Eagle Medium (DMEM) (Nutricell, Brazil) supplemented with 10 % fetal bovine serum (SFB) (Invitrogen, USA), 100 U mL⁻¹ penicillin, 100 U mL⁻¹ streptomycin, and 10 mM 4-(2-HidroxiEthil)-1-PiperazinEthanolSulfonic buffer (HEPES) (Mosmann, 1983). Cells were cultured in sterile 96-well plates with level depths (Sarstedt, Germany) at a density of 5 × 10³ cells per well and were then incubated in an oven at 37 ± 2 °C in an atmosphere containing 5 % CO₂ for 48 h. Subsequently, the culture media were replaced with sample solutions (resveratrol and analogs) at concentrations from 10 to 100 µM. Plates were, then incubated at 37 ± 2 °C in an atmosphere containing 5% CO₂ for 48 h. Control was performed under the same conditions, but without adding the tested substance. After 48 h, culture media were removed and 100 µL aliquots of 10 % [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT; 5 mg/mL) in DMEM were added to all wells and the plates were immediately incubated at 37 ± 2 °C in an atmosphere containing 5 % CO₂ for 3 h. Finally, the resulting formazan crystals were dissolved in DMSO and the absorbance was evaluated using a spectrophotometer (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA) at 570 nm (Twentyman and Luscombe, 1987).

2.6 Statistical analysis

Data were expressed as means ± standard errors of the mean (SEM) and are representative of 5 replicates. Differences were identified using analyses of variance (ANOVA) followed by Bonferroni’s test with the help of the Statistical Package for Social Sciences software, version 21.0 (IBM SPSS Statistics 21) and were considered significant when p < 0.05.

3.1 Antifungal activity

The results obtained can be observed in Table 1. The capacity to inhibit fungal growth was decreasing in the following order: A, B/C and D (p < 0.05). The assessed fungicide potential was the same for A, B and D (p > 0.05), followed by C (p < 0.05) (Table 1). Resveratrol was not active in the experimental conditions. The microorganism tested was considered sensitive to amphotericin B, since the MIC value was lower than 1.0 µg mL⁻¹ (CLSI, 2002).

Table 1 Minimal inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of resveratrol analogs, resveratrol and reference drug against Candida albicans ATCC 10,231.

Compound	Chemical structure	MIC (µg mL−1)	MFC (µg mL−1)
Analog A		156.3	1250
Analog B		312.5	1250
Analog C		312.5	2500
Analog D		625.0	1250
Resveratrol		>5000	>5000
Amphotericin B		0.13	0.50

All analogs were active under the experimental conditions. It is noteworthy that the use of Amphotericin B is restricted to invasive and disseminated fungal infections and severe adverse reactions, such as nephrotoxicity, limit its application (Karimzadeh et al., 2015; Rocha et al. 2015). Furthermore, cases of cardiotoxicity, hepatotoxicity, neurotoxicity, phlebitis, anemia and others have also been reported due to its use (Karimzadeh et al., 2015).

The A, B, C and D analogs differ from each other due to the group linked to the second benzene ring. According to Anthony et al. (2014), the groups attached to the aromatic ring influence the antifungal activity.

The analysis of the structure–activity relationship of resveratrol analogs showed that the presence of nitro and metoxi group in the para position, favored antifungal activity. In particular, the analog with the nitro group (A) in this position was the most promising antifungal agent.

Anitha et al. (2012) pointed out that the presence of nitrogen in the molecule is important for antifungal activity, as hydrogen bonds may form between cell components and this atom, consequently, interfering in the cell metabolism. The fact that resveratrol does not have such an atom can justify its inactivity in this experiment. Kingsbury et al. (2012) found no resveratrol activity against C. albicans SC5314 in their experiments. On the other hand, Lee and Lee (2015) have observed that at low concentrations, resveratrol was able to permeate the cell membrane without causing damage, and induce apoptosis via metacaspase activation and release of cytochrome c.

Lakum et al. (2014) synthesized Schiff bases and chalcone derivatives and found MICs ranging between 0.78 and 200 μg mL⁻¹ against C. albicans MTCC 183. These studies showed that the compounds presenting halogenated groups were more potent against the specific fungal strain and promoted a selective action. For C. albicans MTCC 183 (MIC = 0.78 µg mL⁻¹), the 2,5-dichlorophenyl group was responsible for the highest activity observed when connected to the triazinyl chalcone derivative.

On the other hand, Patil et al. (2012) found that the Schiff bases presented higher fungistatic activity than the parent compounds against Aspergillus niger MTCC 1881, A. flavus MTCC 1883 and Cladosporium MTCC 1777 when complexed with Co (II), Ni (II) or Cu (II). In this case, the authors attributed the increased activity to the presence of metals, with possible interference in the microorganisms’ membrane permeability and alteration of parameters such as solubility, conductivity and dipole moment. Futhermore, the Cu (II) complexes were more active, followed by Ni (II) and Co (II).

These results are promising, since the analogs in the present study can be complexed with metals, which can lead to increased antifungal activity. That occurs because coordination and chelating change the structure of the molecule, producing more potent compounds. Additionally, there may be a reduction in the polarity of the metal compound, favoring penetration in the fungal cell and the action of the compound (Anitha et al., 2012; Dhanaraj and Johnson, 2014).

3.2 Scanning electron microscopy

The electron micrographs of the active analogs and amphotericin B against C. albicans ATCC 10,231, as well as the one for microorganism that was not subjected to the pharmacological treatment, can be observed in Fig. 1. In all of the pharmacological tests performed, invaginations were observed on the plasma membrane (Fig. 1 - II, III, IV, V and VI).

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Fig. 1

Electron micrographs of Candida albicans ATCC 10,231 not subjected to pharmacological treatment and submitted to treatment with the resveratrol analogs and the reference drug. I: C. albicans not subjected to pharmacological treatment. II: C. albicans subjected to treatment with amphotericin B. III: C. albicans subjected to treatment with analog A. IV: C. albicans subjected to treatment with analog B. V: C. albicans subjected to treatment with analog C. VI: C. albicans subjected to treatment with analog D. a: invaginations in the plasma membrane. b: pseudo-hyphae. c: irregular plasma membrane. d: leakage of intracellular material.

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In Fig. 1 - I, it was possible to notice that the fungal structure presented a well-defined, smooth and regular surface. Treatment with analogs B, C and D (Fig. 1 - IV, V e VI) led to the appearance of roughness on the membrane and cells with leakage of intracellular material. In the treatment with analog A (Fig. 1 - III) pseudohyphae were verified, which may be related to the virulence of the fungus’ mechanisms (Grow et al., 2012).

3.3 Brine shrimp lethality test

For all the tested compounds, a moderate toxicity was observed against the microcrustacean A. salina (Table 2). The analog A, considered in this study as the most promising compound regarding antifungal activity (MIC = 156 µg mL⁻¹), presented the most significant lethality values (LC₅₀ = 112.07 µg mL⁻¹), followed by resveratrol (LC₅₀ = 145.11 µg mL⁻¹). In regard to the reference drug (LC₅₀ = 14.22 µg mL⁻¹), the analog A was 8 times less toxic.

Data are expressed as means ± standard errors of the mean (SEM) and are representative of 5 replicates. Data marked with an asterisk are significantly different (p < 0.05) with respect to the positive control (Thymol). Differences were identified using analyses of variance (ANOVA) (IBMS SPSS Statistical 21).

Among the bioassays that aim to guide the drug discovery process, the A. salina assay is considered efficient, quick, cheap and require only from 2 to 20 mg of the compounds (Pimenta et al., 2003; Ajaiyeoba et al., 2006; Dutra et al., 2012). This method has a high correlation with the acute toxicity recorded in rodents and can be predictive to the cytotoxicity in human cell cultures (Hartl and Humpf, 2000). Besides that, it correlates with numerous biological activities (Pompilho et al., 2014; Ngoumfo et al., 2010; El- Menshawi and Linder, 2010) such as antitumoral activity (McLaughlin et al., 1993), activity against Trypanosoma cruzi (Zani et al. 1995), antibacterial activity (Brasileiro et al., 2006) and antifungal activity (Niño et al., 2006). It is commonly considered that compounds which present toxicity to A. salina (LC₅₀ < 250 μg mL⁻¹) also present high potential of having the biological activities cited (Costa et al., 2009). Thus, it is suggested that the compounds hereby studied present this potential.

3.4 Evaluation of cellular viability using MTT assays

Resveratrol and its analogs were evaluated between 10 µM and 100 µM and the results can be observed below (Fig. 2).

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Fig. 2

Human keratinocytes viability after treatment with resveratrol or analogs for 48 h. Non-treated group viability (control) was set as 100%. Values represent mean ± SEM (n = 6). Data marked with an asterisk are significantly different with respect to the non-treated group (p < 0.05). Data were statistically analyzed by variance analysis followed by Bonferroni post hoc test (IBMS SPSS Statistical 21).

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Resveratrol is a polyphenol that possesses great biological properties (Baur and Sinclair, 2006). Concerning the keratinocyte cells (HaCaT) exposed in this study to resveratrol and analogs for 48 h with increasing concentrations (10 to 100 µM), the results demonstrate a dose-depending increase in cell mortality (Fig. 2). Comparing the groups treated with resveratrol and the analogs A, B, C and D, statistical differences were verified in all concentrations used in the test (p < 0.05). The cell viability of resveratrol was 60.7% and 68.1% at 100 and 75 µM concentrations, respectively. At 50 µM, the analog B presented a better result than resveratrol (77.4% cell viability). In comparison to the untreated group of cells, there was a 7% drop in the viability with the resveratrol treatment at 10 µM. Analog A presented cell viability in a range from 50.6 (100 μM = 2.28 µg m ⁻¹) to 90% (10 μM = 228 µg mL⁻¹). The results obtained in the MTT cytotoxicity assay suggest that all compounds tested had low cytotoxic activity in keratinocyte cell (HaCaT) cultures (Fig. 2). These results are important since both resveratrol and its derivatives can be used in dermal preparations, being effective in wound healing (Chedea et al., 2017; Tsai et al., 2013). According to Almeida et al. (2014), the compounds can be classified as not presenting cytotoxic activity (NA), as presenting low activity (LA), when the cell growth inhibition ranges from 0 to 50%, as presenting moderate activity (MA), when the inhibition ranges from 50 to 75% and as presenting high activity (HA), when the cytotoxicity is higher than 75%.

An in vitro study conducted by Clement et al. (1998) showed resveratrol as a potent natural chemopreventive agent in the HL60 cell line. This happened due to a better expression of the CD95L in HL60 cells treated with resveratrol. On the other hand, resveratrol presented low cytotoxicity to normal human lymphocyte cell cultures (PBLs) in concentrations up to 32 µmol L⁻¹, suggesting CD95L specificity in resveratrol antitumoral activity, as well as its therapeutic efficacy.

Thus, these results are promising for the development of new drugs since other modifications in the structures of the analogs can be performed in order to enhance their fungicidal activity. More studies are needed to establish the mechanism of action of the new compounds.