The triterpenes α-amyrin (3) and β-amyrin (5) were isolated from copal and nanche bark in 7.0% and 2.0%, respectively [17]. Because in preliminary studies a greater activity was observed with α-amyrin than with β-amyrin, we decided to prepare some esters of α-amyrin to assay their activity against cariogenic microorganisms.

Three esters of α-amyrin 4a–c were synthesized in good to excellent yields (70–98%) (Scheme 1). The spectroscopic data of 4a and 4b are in agreement with those reported in the literature [6,18]. To the best of our knowledge, α-amyrin phenylacetate (4c) is reported and completely characterized for the first time. Its IR spectra showed an absorption band at 1724 cm⁻¹ assigned to C=O of the ester group, and absorption bands between 1404–1583 of (C=C) for the aromatic ring. In the ¹H-NMR spectrum of 4c, signals at 0.75–2.03 ppm showed the characteristic terpene profile. The protons alpha to the C=O group of the phenylacetate fragment appeared as a singlet at 3.72 ppm; H-3 appeared as a double-doublet at 4.48 ppm with a J = 9.2, 5.6 Hz; and H-12 as a triplet at 5.11 ppm, with a J = 3.6 Hz. The five protons corresponding to the aromatic fragment appeared as multiplets at 7.24–7.33 ppm. In the ¹³C-NMR the signal for C-5 was at 55.0 ppm, C-3 at 81.2 ppm, C-12 at 123.9 ppm, C-13 at 139.3 ppm, and the C=O appeared at 170.9 ppm.

In this work, five compounds (namely 3, 4a–c, 5) were screened for antimicrobial activity using the macrodilution broth method, which is commonly use in the susceptibility assay for the antimicrobial evaluation of organic compounds [19]. Two concentrations of S. mutans were used, a lower (1 × 10³ cfu/mL) and a higher concentration (1 × 10⁶ cfu/mL), because a low concentration of S. mutans is associated with caries-free subjects, and the tooth surfaces susceptible to becoming carious are associated with a higher concentration [20].

During the experiments, chlorhexidine dihydrochloride (CHX) was used as a positive control, since produced 0% cell viability for all the assayed strains. The negative control consisted of a broth solution with 3.6 µL of Tween 80, which resulted in 100% cell viability for all strains tested.

To obtain information about the dosis-response relationship, S. mutans (at 10⁶ cfu/mL) was exposed to the five compounds α-amyrin (3), β-amyrin (5), α-amyrin palmitate (4a), α-amyrin octanoate (4b) and α-amyrin phenylacetate (4c) (Scheme 1), at the following concentrations: 10, 53.33, 100, 533.33, 1,000 μg/mL. The results shown in Table 1 indicate that the palmitate (83–43% viability) and the octanoate esters were less active (89–39% viability), the phenylacetate (19–9% viability) being the most active, followed by α-amyrin (3) and β-amyrin (5). It is worth mentioning that the effect of the highest concentration of α-amyrin (16% viability) was comparable with the effect produced by the lowest concentration of α-amyrin phenylacetate (19% viability), which proved to be about 100 times more active than α-amyrin (3). The results showed statistically significant differences between concentrations according the Kruskal-Wallis test, at α = 0.05.

Continuing the study, activity against S. salivarius, S. sanguinis, S. oralis (at 1 × 10⁶ cfu/mL) was tested, but only with the three most active compounds, using the same range of concentrations, and the results are shown in Table 2.

The range of cell development was related to the concentration of the three terpenes used for the experiments. In general, α-amyrin phenylacetate (4c) was the most active, especially at higher concentrations, except against S. sanguinis, for which α-amyrin was slightly more active at all the concentrations tested. For S. oralis, the inhibition activity of 4c was outstanding at all concentrations, showing a maximum of cell growth of 3% at the lowest concentration (10 µg/mL).

The high bacteriostatic effect displayed by α-amyrin phenylacetate (4c) against microorganisms responsible for dental caries at an initial bacteria concentration of 1 × 10⁶ cfu/mL prompted us to test its activity at a lower bacteria concentration (1 × 10³ cfu/mL) with the same concentrations of phenylacetate as before, and the results for Streptococcus mutans are shown in Figure 1. The cell viability was similar for all concentrations (around 60%), except for the 1,000 µg/mL, where the viability was 33%.

All reagents were from Aldrich and used without further purification; chlorhexidine gluconate (CHX) was from Sigma. ¹H-NMR and ¹³C-NMR spectra were recorded on a 400 MHz Varian instrument in CDCl₃, with tetramethylsilane (TMS) as the internal standard. FAB mass spectra were recorded on Thermo-Electron DFS. For preparative TLC, silica Gel 60, 20 × 20 cm plates of 0.25 mm thickness (Merck) were used. The triterpenoids were detected by spraying the plates with anisaldehyde-sulphuric acid (AS) reagent, and heating at 80–100 °C for 3 min.

The following microorganisms were used: Streptococcus mutans (ATCC 10449), S. salivarius (field strain), S. sanguinis (field strain), S. oralis (field strain), which are all associated with the oral cavity. The field strains were collected from the Medicine School of Universidad Nacional Autónoma de México (UNAM). All microorganisms were stored at −65 °C in 20% glycerol.

Mexican copal resin was purchased in Tepoztlan, Morelos, and nanche bark was collected in the state of Veracruz, México. α-Amyrin (3) and β-amyrin (5) were isolated from Mexican copal resin and barks of nanche by a reported extraction procedure in 7.0% and 2.0% yield, respectively [14]. Briefly, the crude Mexican copal Tepoztlan resin (3 g) was dissolved in n-hexane (volume, time, temperature), and the insoluble fraction (200 mg) was used for the extraction of α-amyrin, being dissolved in dichloromethane (0.5 mL) and applied to the preparative TLC plates. After developing the plate twice in hexane-dichloromethane-methanol (10:3:0.5), the reference band was exposed to the developer (anisaldehyde-sulphuric acid), while the sample area was covered with a glass plate. Two zones (α-amyrin and a mixture of 3-epi-lupeol and another unidentified compound) were scraped off and each fraction was extracted three times with ethyl acetate (20 mL). The resulting solution was filtered and the solvent evaporated, yielding 160 mg (80%) of amyrin [17]. The air-dried and powdered stem barks of nance residues (1 kg) were extracted with n-hexane (2 L × 2) at room temperature for 48 h and 20.6 g (2.06% dry weight) of a solid white extract was obtained after filtration and evaporation of the solvent under reduced pressure. Crystallization of β-amyrin was performed by dissolving the solid white extract (4 g) in dichloromethane (20 mL) at room temperature. After dissolution, 15–20 mL of methanol was added, and the mixture was kept at room temperature for 12 h before isolation from the mother liquor by suction filtration [17].

1.21 mmol of each acid 1a–c were dissolved in n-hexane (20 mL), and oxalyl chloride (3.63 mmol) was added. The mixture was refluxed for 24 h, and then the excess of reagents was removed by evaporation under vacuum, yielding the corresponding acid chlorides 2a–c, which were used without any purification. To a solution of the acid chloride in dry dichloromethane (20 mL) were added α-amyrin (0.5 mmol), pyridine (1.0 mmol) and DMAP (0.5 mmol). The resulting mixture was refluxed and monitored by TLC (Scheme 1). The reaction mixture was purified by column chromatography using different proportions of hexane-ethyl acetate, yielding the palmitate 4a, 71%, the octanoate 4b, 80%, and the phenylacetate 4c, 98% isolated yields. The spectroscopic data of 4a–b are consistent with those reported in the literature [18,22].

α-Amyrin phenylacetate (4c). The purified product was obtained as a white solid (98%). Rf: 0.8 hexane-ethyl acetate (90:10). IR (film) ν 3024, 2945, 2906, 1724 cm⁻¹. ¹H-NMR: δ (ppm) 0.75 (3H, s), 0.79 (3H, s), 0.90 (3H, s), 0.95 (3H, s), 1.05 (3H, s), 3.72 (2H, s, -CH₂-Ph), 4.48 (1H, dd, J = 9.2, 5.6, H-3), 5.11 (1H, t, J = 3.6, H-12), 7.24–7.33 (5H, m). ¹³C-NMR: δ (ppm) 38.2 (C-1), 23.4 (C-2), 81.2 (C-3), 37.6 (C-4), 55.0 (C-5), 18.1 (C-6), 32.7 (C-7), 39.8 (C-8), 47.4 (C-9), 36.6 (C-10), 23.3 (C-11), 123.9 (C-12), 139.3 (C-13), 41.9 (C-14), 26.5 (C-15), 28.0 8 (C-16), 33.7 (C-17), 58.9 (C-18), 39.5 (C-19), 39.9 (C-20). MS calculated for C₃₈H₅₇O₂: 545.4353; Found: 545.4336.

Stock solutions (1.25 and 12.5 mg/mL) of each compound 3, 4a–c, 5 were prepared in BHI broth supplemented with Tween 80 (3.6 µL/mL of broth) and sonicated before use. A series of dilutions was prepared from 10–1,000 µg/mL of α-amyrin (3), β-amyrin (5), α-amyrin phenylacetate (4c), α-amyrin octanoate (4b), and α-amyrin palmitate (4a). Each vial contained the Streptococcus strains with concentrations of 1 × 10³ or 1 × 10⁶ cfu/mL.

Control reactions were included consisting of inoculated growth medium without the terpene compounds; for the antimicrobial positive control, chlorhexidine gluconate (0.05%) was used. After 24 h of incubation at 37 °C, the surviving microorganisms were determined by the viable count cells.

Viable count cells: appropriate 1:10 dilutions in 0.85% sodium chloride were prepared. Each dilution (0.1 mL) was aseptically transferred to nutrient agar and the inoculum was spread with a sterile bacteriological loop. The inoculated plates were incubated for 24–48 h at 37 °C and the colonies were counted. The experiment for each dilution was done in triplicate.