Chemicals
The following compounds were used for the synthesis of eugenol esters: chloroacetic acid (Sigma-Aldrich, 99%), dichloroacetic acid (Sigma-Aldrich, ≥ 99%), trichloroacetic acid (Chempur, 99%), oxallyl chloride (Alfa Aesar, 98%), eugenol (Keten, p.a.), pyridine (AR Ubichem), dimethylformamide (DMF, Acros, 99%), chloroform (Chempur, p.a.), and deionized water.
The amounts of these compounds were selected to maintain the molar ratio of carboxylic acid:oxalyl chloride:eugenol:amines as 81: 71: 51:123. For the determination of antioxidant activity and to assess the lipophilicity of the esters obtained, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and sodium lauryl sulfate were purchased from Sigma Aldrich (USA); Folin–Ciocalteu reagent, disodium phosphate (p.a.), and potassium dihydrogen phosphate (p.a.) were purchased from Merck, Darmstadt, Germany; ethanol (96% v/v), methanol (50% v/v, concentrated) (all of the analytical grade) sodium chloride, potassium chloride, and gallic acid (GA) were purchased from Chempur, Piekary Śląskie (Poland); and acetonitrile for HPLC was purchased from J.T. Baker, PBS.
Synthesis and characterization of new esters
First, EChA, EDChA, and ETChA were obtained by the method described in our previous publication (Makuch et al. 2019). After synthesis, the obtained products were identified by mass spectrometry coupled with gas chromatography (GC-MS). Chromatographic analyses were performed with a TRACE GC series apparatus with a VOYAGER mass detector using a DB5 capillary column (30m × 0.25μm × 0.5μm). The following separation parameters were used for the analysis: helium flow of 1.0 ml/min, sample chamber temperature of 240 ˚C, and detector voltage of 350 V. The thermostat temperature increased according to the following program: isothermal at 50 ˚C for 1 minute, increase at a rate of 8 ˚C/min, isothermal at 260˚C for 5 minutes, and then cooled to 50 ˚C. The sample partition coefficient in the dispenser was 20, the volume of dispensed sample was 0.1 µl, and the ion mass range was 25–350 mV/z.
The degree of conversion of eugenol and the purity of the obtained products were determined by gas chromatography (GC). Chromatographic analyses were performed on a FOCUS apparatus (Thermo Electron) with a flame ionization detector (FID) and a RESTEK RTX-5 capillary column (0.53 mm x 30 m x 1.5 μm). Infrared spectroscopy (FTIR/ATR) was characterized by functional groups present in the structures of the products obtained. Analyses were performed on a THERMO NICOLED 380 apparatus using a measuring range of 4000–400 cm-1. The same method also used eugenol for comparative purposes.
The structures of the obtained esters (dissolved in deuterated chloroform (CDCl3)) were confirmed based on the analysis of nuclear magnetic resonance (NMR) spectroscopy spectra, which was performed with a Bruker DPX-400 spectrometer. The conditions for recording 13C-NMR spectra were as follows: 100.62 MHz, a spectrum width of 24 kHz, 65.5 K data points, a resolution of 1.46 Hz/point, a data acquisition time of 1.37 s, a repetition time of 1 s, a pulse width of 9.2 μs, and 1–8 scans. In addition, 1H-NMR spectra were recorded under the following conditions: 400.13 MHz, a 12 kHz spectrum width, 65.5 K data points, a 0.488 Hz/point resolution, a data acquisition time of 4.09 s, a repetition time of 1 s, a 7.8 μs pulse width, and 16–32 scans.
On the 1H-NMR spectrum, which is presented in Fig. 3, the presence of clear peaks indicating the presence of certain groups of protons in the structure of the tested ester was observed. The proton signals recorded as intense peaks corresponded to hydrogen atoms from groups belonging to hydrocarbon chains, indicating the presence of an aromatic system in the molecule of the compound tested. The analysis of the 13C-NMR spectrum presented in Fig. 4 supplemented the information obtained based on the interpretation of the 1H-NMR spectrum and additionally confirmed the structure of the obtained ester. The chemical shift values of carbon atoms and protons as well as the signals assigned to atoms 13C and 1H were as follows:
13C-NMR (101 MHz, CDCl3) δ 160.24, 150.51, 140.43, 137.71, 136.75, 121.45, 120.77, 116.50, 116.46, 89.54, 56.04, 40.09;
1H NMR (400 MHz, CDCl3) δ 6.98 (d, J = 8.0 Hz, 1H), 6.80 – 6.67 (m, 2H), 5.97 – 5.81 (m, 1H), 5.04 (dtd, J = 14.7, 3.4, 1.7 Hz, 2H), 3.76 (s, 3H), 3.32 (dt, J = 6.7, 1.5 Hz, 2H).
For the remaining esters, the chemical shift values of carbon atoms and protons and the signals assigned to atoms 13C and 1H were as follows:
13C-NMR (101 MHz, CDCl3) δ 165.62, 150.59, 139.66, 137.55, 136.91, 122.13, 120.72, 116.34, 112.82, 55.86, 40.71, 40.10 - (EChA);
1H NMR (400 MHz, CDCl3) δ 6.90 (d, J = 7.9 Hz, 1H), 6.80 - 6.65 (m, 2H), 5.88 (ddt, J = 17.0, 10.3, 6.8 Hz, 1H), 5.11 - 4.97 (m, 2H), 4.25 (s, 2H), 3.73 (s, 3H), 3.30 (dt, J = 6.8, 1.5 Hz, 2H) - (EChA);
13C-NMR (101 MHz, CDCl3) δ 162.84, 150.65, 140.24, 137.42, 136.91, 121.81, 120.86, 116.55, 113.12, 64.14, 56.08, 40.20 - (EDChA);
1H NMR (400 MHz, CDCl3) δ 7.02 (d, J = 8.0 Hz, 1H), 6.83–6.80 (m, 1H), 6.78 (q, J = 1.9 Hz, 1H), 6.19 (s, 1H), 5.96 (ddt, J = 9.4, 17.6 Hz, 1H), 5.11 (dt, 2H), 3.83 (s, 3H), 3.39 (dd, J = 1.6, 6.8 Hz, 2H) - (EDChA).
Measurement of lipophilicity of eugenol and new esters
To determine the lipophilicity of eugenol and its ester derivatives, the values of the n-octanol/water partition coefficient (P) were examined. Determining the lipophilicity of a substance involves determining its partition coefficient between two immiscible liquids: n-octanol and water (which model the properties of cell structures well). The partition coefficient was expressed as the logarithm ratio of substance concentrations in both phases (Piwowarczyk et al. 2017):
where P is the partition coefficient, Cn-octanol is the concentration of the compound in the octanol phase, and Cwater is the concentration of the compound in the aqueous phase.
N-octanol was mixed with water in a 1:1 ratio containing the test compound at a concentration of 32.00–178.00 mg/100 ml solution. The mixture was then shaken on a shaker (TS-2 Orbital Shaker) for 24 hours at a constant temperature of 25 °C, which was controlled by an immersion thermostat. Concentrations of substances in the analyzed samples were determined by spectrophotometry at the following wavelengths: 279 nm (in the case of eugenol), 272 nm (in the case of EChA), 277 nm (in the case of EDChA), and 280 nm (in the case of ETChA). For comparison purposes, log P values for DChAA and TChAA were also determined by potentiometric method. Furthermore, blanks were performed for each compound tested. For this purpose, n-octanol was mixed with water in a 1:1 ratio, and then the mixture was shaken for 24 hours under constant temperature conditions, after which the aqueous layers were analyzed by a spectrophotometric method using appropriate wavelengths λ (Piwowarczyk et al. 2017).
Furthermore, compounds contained in aqueous solutions were identified by thin-layer chromatography (TLC). Based on the obtained chromatograms, the retention factor (Rf) values for the corresponding ester and pure eugenol were determined. The absence of eugenol in aqueous solutions of the esters tested indicated a lack of the hydrolysis of these compounds (Waszczuk et al. 2020).
Measurement of the antioxidant capacity using DPPH, ABTS and Folin–Ciocalteu methods
Studies on the antioxidant activity of eugenol and its esters were carried out by free radical reduction (DPPH) (Brand-Williams et al. 1995; Nowak et al. 2017), ABTS (Nowak et al. 2017), and the Folin–Ciocalteu method (Nowak et al. 2019). The ABTS assay is based on the generation of a blue/green ABTS, which is applicable to both hydrophilic and lipophilic antioxidant systems; whereas DPPH assay uses a radical dissolved in organic media and is, therefore, applicable to hydrophobic systems (Floegel et al. 2011).
The analyses were performed on a Merck Spectroquant Pharo 300 apparatus at the following wavelengths λ: 517 nm (in the case of the DPPH method), 734 nm (in the case of the ABTS method), and 765 nm (in the case of the Folin–Ciocalteu method). Trolox was used as a reference substance in the DPPH and ABTS methods, while gallic acid was used as a standard to assess the polyphenol contents in the sample tested by the Folin–Ciocalteu method. The antioxidant activity results obtained by these methods are expressed in mmol/dm3.
The antioxidant activity of eugenol and its ester derivatives was measured as follows: 2850 μl of an ethanol solution with the DPPH radical was introduced into the tube, and its absorbance at λ = 517 nm was about 1.000 ± 0.020 with 150 μl of the ethanol solution containing the tested antioxidant. The tube was wrapped in aluminum foil and its contents were sealed with a stopper and then incubated for 10 minutes at room temperature. After this time, spectrophotometric measurements were carried out at the appropriate wavelength and in triplicate.
First, an aqueous solution of potassium persulfate (2.45 mM) was prepared, in which an appropriate amount of ABTS reagent was introduced to obtain a 7mM solution of ABTS in an aqueous solution of potassium persulfate. The solution prepared in this way was incubated at 4 °C for 24 hours and then diluted with methanol (50% v/v) to obtain an absorbance of approximately 1.000 ± 0.020.
The antioxidant activity of eugenol and its ester derivatives was measured as follows: 2500 μl of prepared ABTS solution and 25 μl of an ethanol solution with the tested antioxidant were introduced into the spectrophotometric cuvette. The cuvette was sealed with a stopper and then incubated for 6 minutes at room temperature. After this time, the spectrophotometric measurement was carried out at the appropriate wavelength and in triplicate.
This method is based on the use of the Folin–Ciocalteu reagent (takes place in an alkaline medium), which is used to determine the total content of phenolic compounds found in the tested samples. The reaction is based on the spectrophotometrically recorded color change of the test solution from yellow to blue.
Two hundred microliters of Folin–Ciocalteu reagent in 1800 μl of water was dissolved in a dark bottle. The solution prepared in this way was incubated at room temperature for 60 minutes. The antioxidant activity of eugenol and its ester derivatives was measured as follows: 1350 μl of distilled water and 1350 μl of sodium carbonate solution (0.01 mol/dm3) were introduced into the spectrophotometric cuvette with 150 μl of the prepared Folin–Ciocalteu solution and 150 μl of an ethanol solution containing the tested antioxidant. The cuvette was sealed with a stopper and then incubated for 15 minutes at room temperature. After this time, spectrophotometric measurements were carried out at the appropriate wavelength and in triplicate.
Skin permeation studies of eugenol and new esters
Porcine skin was used for the study due to its similar properties permeability to human skin (Čuříková et al. 2017; Janus et al. 2020). The skin came from a local slaughterhouse. A fresh portion of skin from the abdomen was washed several times with a solution of PBS at pH 7.4. Skin with a thickness of 0.5 mm was cut with a dermatome, and then it was wrapped in aluminum foil and frozen at –20 °C for a maximum of 3 months. This freezing time ensured the stability of the skin barrier properties (Badran et al. 2009). Before the examination, the skin was thawed at room temperature for about 30 minutes, and then it was soaked in a PBS solution for 15 minutes to hydrate it (Haq et al. 2018; Kuntsche et al. 2008; Simon et al. 2016). In the next stage, the skin was mounted in Franz diffusion cells.
The integrity of skin was checked one hour after its installation in the Franz diffusion chamber (SES GmbH Analyze Systeme, Germany). Skin impedance was measured using an LCR 4080 meter (Conrad Electronic, Germany) operating in parallel mode at 120 Hz (kΩ error < 0.5%). To make the measurement, the tips of the probes were immersed in the donor and acceptor chambers filled with the PBS solution (Kopečná et al. 2017). Membranes with an electrical resistance of >3 kΩ, corresponding to the resistance measured for human skin, were used in the study (Davies et al. 2004).
The penetration of eugenol and its ester derivatives was assessed in a Franz diffusion chamber consisting of a 2 ml donor chamber and an 8 ml acceptor chamber. The area through which the tested active ingredients permeated was 1 cm2. The acceptor fluid, mixed with a magnetic stirrer, was a PBS solution that maintained the physiological pH. The acceptor chamber was kept at a constant temperature of 37 ± 0.5 °C with the VEB MLW Prüfgeräte-Werk type 3280 thermostat. Before starting the test, Franz diffusion cells were allowed to equilibrate at 37 °C for 15 minutes. After this time, ethanol solutions of test compounds (at a concentration of 1% w/v) were placed in the donor chamber. For the study, 500 µL of the ethanol solution of the test compound (at a concentration of 1% w/v) was applied to the outer layer of the skin in each donor chamber to ensure continuous delivery of the active substance during the experiment. All donor chambers were closed with a plastic stopper to prevent excessive evaporation of the solution. The described tests were carried out for 24 hours, while 0.3 ml of the solution located in the acceptor chamber was taken at specified intervals (30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 8 h and 24 h), and then supplemented with a fresh portion buffer at the same pH (Kopečná et al. 2017). The samples were analyzed by high-performance liquid chromatography (HPLC) with a UV spectrophotometric detector (Knauer, Berlin, Germany). The components tested were separated on a 125 x 4 mm column containing Hyperisil ODS; particle size 5 μm. The flow rate of the mobile phase, consisted of acetonitrile, water, and MeOH (28:64:8, by vol) was 1 ml/min. Twenty microliters of each analyzed sample was injected onto the column.
After the experiment was carried out, the skin was extracted to estimate the residual volume of tested active ingredients accumulated in it. The antioxidant activity of the obtained extracts was also tested using modified methods described in (Brand-Williams et al. 1995; Nowak et al. 2017; Nowak et al. 2019). Extraction was carried out as follows: after the experiment was completed, the Franz diffusion chambers were dismantled, while the skin surface was washed three times with an aqueous solution of sodium lauryl sulfate (at a concentration of 0.5% w/w) to elute the ethanol solution of the test compound. A patch (1 cm2 diffusion surface) was cut from the skin prepared in this way, dried at room temperature, and then weighed and cut into smaller pieces. Then, 2 ml of concentrated methanol was added to it, and extraction was carried out for 24 hours at 4 °C. After 24 hours of incubation, the skin was homogenized (for 3 minutes) using a homogenizer (IKA®T18 digital ULTRA TURRAX, Germany). The extracts obtained were then centrifuged at 3500 rpm for 5 minutes. The supernatant was analyzed by HPLC to determine the content of active ingredients, while to evaluate the antioxidant activity of the obtained extracts the DPPH, Folin–Ciocalteau, and ABTS methods were applied.
The cumulative mass of active substance (µg) permeating into the receptor chamber was calculated based on the concentrations of compounds determined by HPLC. The permeation rate was determined based on the amount of permeation of a compound over a given period (μg/cm2/h). The accumulation of compounds in the skin was calculated by determination of remaining ingredients in the liquid obtained after skin extraction; the results are given in μg/cm2 of skin.
The antioxidant activity of solutions of test compounds applied to the skin, acceptor fluid taken after 24 hours of penetration, and solutions obtained after skin extraction taken at the end of the experiment was determined using the DPPH, Folin–Ciocalteau, and ABTS methods, as described in sections: measurement of the antioxidant capacity of eugenol and new esters using the DPPH, the ABTS and the Folin–Ciocalteu methods.
Statistical calculations were done using Statistica 13 PL software (StatSoft, Polska). The results were evaluated using one-way analysis of variance (ANOVA). Significant differences between the permeation of individual compounds were evaluated using Tukey post-hoc test. Probabilities < 0.05 were considered to be statistically significant. Results are presented as the mean ± standard deviation (SD).