Growth inhibition of streptococcus from the oral cavity by α-Amyrin esters

Article abstract of DOI:10.3390/molecules171112603

Five terpenoids were tested by the macrodilution broth method to determine their inhibition activity on cariogenic bacterial growth. In general, α-, β-amyrin and α-amyrin phenylacetate proved to be active, reducing the bacterial viability to less than 20%.

Full text of DOI:10.3390/molecules171112603

Molecules 2012, 17, 12603-12611; doi:10.3390/molecules171112603
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Communication
Growth Inhibition of Streptococcus from the Oral Cavity by
α-Amyrin Esters
Gloria Díaz-Ruiz 1, Liliana Hernández-Vázquez 2, Héctor Luna 2,
María del Carmen Wacher-Rodarte 1 and Arturo Navarro-Ocaña 1,*
1
Departamento de Alimentos y Biotecnología, Facultad de Química, UNAM. Circuito Exterior,
Ciudad Universitaria 04510, Mexico; E-Mails: gloria_druiz@yahoo.com.mx (G.D.-R.);
wacher@unam.mx (M.C.W.-R.)
2
Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana-Xochimilco,
Calz. del Hueso 1100, Col. Villa Quietud 04960, Mexico;
E-Mails: lhernandez@correo.xoc.uam.mx (L.H.-V.); lchm1964@correo.xoc.uam.mx (H.L.)
* Author to whom correspondence should be addressed; E-Mail: arturono@unam.mx;
Tel.: +52-55-5622-5346; Fax: +52-55-5622-5349.
Received: 19 September 2012; in revised form: 19 October 2012 / Accepted: 22 October 2012 /
Published: 25 October 2012
Abstract: Five terpenoids were tested by the macrodilution broth method to determine their
inhibition activity on cariogenic bacterial growth. In general, α-, β-amyrin and α-amyrin
phenylacetate proved to be active, reducing the bacterial viability to less than 20%.
Keywords: cariogenic microorganism; amyrin esters; antimicrobial activity
1. Introduction
The terpenoids α-amyrin (3) and β-amyrin (5) are commonly found in medicinal plants and
oleo-resins obtained by bark incision of several species of Bursera or Protium spp. of the Burseraceae
family (Figure 1) [1]. They have shown various pharmacological activities in vitro and in vivo against
several health-related conditions, including microbial, fungal, and viral infections, cancer and
inflammation. In addition, there are abundant examples of α-amyrin-containing plants used in
folk medicine. For example Strobilanthes callosus, which has anti-inflammatory and antimicrobial
activities [2], and Decalepis hamiltonii, which is used as an appetite stimulant, blood purifier and food
preservative [3,4].

Molecules 2012, 17
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α/β-Amyrin modulate acute periodontal inflammation by reducing neutrophils infiltration, oxidative
stress, and the production of pro-inflammatory cytokine TNF-α, which suggests they might be useful
as therapeutic agents for the treatment of gingivitis and periodontitis [5]. Other studies have
demonstrated that the α/β-amyrin mixture also has anti-inflammatory, gastroprotective, antiallergenic
and antinociceptive activities [6].
The antifungal activity of amyrin and some 3-O-acyl derivatives has been evaluated against Candida
species. Additionally, inhibition of the adhesion of C. albicans to human epithelial cells in vitro was also
determined. Amyrin formate and acetate derivatives were the most active compounds, inhibiting all the
opportunistic Candida species when tested in concentrations from 30 to 250 mg/mL. The formate also
showed interesting inhibition of the adhesion ability of C. albicans [7]. β-Amyrin acetate isolated from
Heliotropium marifolum showed potent activity against Penicillum chrysogenum, Escherichia coli and
Klebsiella pneumonia [8].
Dental caries is a common bacterial pathology caused by a biofilm consisting of a multitude of
microorganisms present on the tooth surface [9], being streptococci the bacteria most frequently
associated with cariogenesis [10]. One method for caries prevention is the use of antimicrobial agents
to limit the growth of microorganisms such as Streptococcus mutans. Extracts of miswak, tea tree oil,
peppermint, green tea and manuka honey have all been recently incorporated into mouth rinses and
toothpastes [11–14].
Some compounds with potential activity against oral microorganisms have been found in Nature,
e.g., S. mutans was sensitive to totarol, a diterpenoid isolated from the bark of Podocarpus nagi with
MICs of 0.78 g/mL and MBC of 0.78 g/mL [15]. The methanolic extract from the bark of
B. crassifolia and the β-amyrin isolated therefrom inhibited the growth of bacteria, including
S. mutans, at concentrations ranging from 64 to 1088 g/mL; the minimum inhibitory concentration
(MIC) for β-amyrin was 128 g/mL [16].
The aim of this work was to isolate β-amyrin from nanche (Byrsonima crassifolia) and α-amyrin
from copal (Bursera spp) and prepare some esters for the evaluation of their capacity to inhibit the
growth of oral streptococci associated with dental caries.
2. Results and Discussion
2.1. Isolation of α- and β-Amyrin
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 -amyrin to assay their activity
against cariogenic microorganisms.
2.2. Esterification of α-Amyrin
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

Molecules 2012, 17
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1
group, and absorption bands between 1404–1583 of (C=C) for the aromatic ring. In the H-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.
Scheme 1. Structure of α and β-amyrin and esterification reactions of α-amyrin.
2.3. Antimicrobial Activity Using the Macrodilution Broth Method
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 g/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

Molecules 2012, 17
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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.
Table 1. Percentage of viability of Streptococcus mutans after 24 h exposure to the five
terpenes at the concentrations used in this study. The viability was adjusted using BHI
broth + Tween 80 as a control (100%).The initial cellular concentration was adjusted to
106 cfu/mL.
Terpene concentration (g/mL)
Compound
10
62
72
83
89
19
53.33
46
100
25
58
68
60
12
533.33
25
1000
16
37
43
39
9
α-amyrin (3)
β-amyrin (5)
61
46
α-amyrin palmitate (4a)
α-amyrin octanoate (4b)
α-amyrin phenylacetate (4c)
82
46
62
40
16
12
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.
Table 2. Percentage of viability, related to terpene concentration, of Streptococcus salivarius,
Streptococcus sanguinis and Streptococcus oralis after 24 h exposure to three selected
derivatives at the concentrations used in this study 10–1,000 (µg/mL). The viability has
been adjusted using BHI broth + Tween 80 as a control (100%).The initial cellular
concentration was adjusted to 106 cfu/mL.
Compound
S. salivarius
68–56%
S. sanguinis
56–20%
S. oralis
95–47%
100–46%
3–1%
α-amyrin (3)
β-amyrin (5)
68–57%
90–63%
α-amyrin phenylacetate (4c)
86–12%
83–48%
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%.

Molecules 2012, 17
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Figure 1. Viability of Streptococcus mutans after 24 h exposure to α-amyrin phenylacetate
at the concentrations used in this study. The viability was adjusted using BHI broth +
Tween 80 as a control (100%).The initial cellular concentration was adjusted to
abc
103 cfu/mL.
indicates if there is a statistically significant difference between
concentrations by derivate (Kruskal-Wallis test, α = 0.05). A different letter indicates there
is a statistically significant difference.
100
90
80
70
a
b
b
bc
60
50
40
30
20
10
0
c
α‐amyrin phenylacetate
10 μg/mL 53.33 μg/mL 100 μg/mL 533.3 μg/mL 1000 μg/mL
3. Experimental
3.1. General
All reagents were from Aldrich and used without further purification; chlorhexidine gluconate
1
(CHX) was from Sigma. H-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.
3.2. Microorganisms
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.
3.2.1. Inoculum Preparation
Bacteria were refreshed in Brain Heart Infusion (BHI) broth (Difco) at 37 °C for 17 h, and later
incubated at the same conditions for an additional 4 h. The optical density (OD600) of the Streptococcus
culture was measured at 600 nm; an OD = 0.01 corresponds to approximately 106 cells/mL.

Molecules 2012, 17
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3.2.2. Statistical Analysis
The Kruskal-Wallis analysis of variance test was used for the determination of significant
differences in the study [21].
3.3. Isolation of Triterpenes
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].
3.4. Esterification of α-Amyrin
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)  3024, 2945, 2906, 1724 cm−1. 1H-NMR:  (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,
13
H-3), 5.11 (1H, t, J = 3.6, H-12), 7.24–7.33 (5H, m). C-NMR:  (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),

Molecules 2012, 17
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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.
3.5. Macrodilution Broth Method for Growth Inhibition Determination
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.
4. Conclusions
α- and β-Amyrin were isolated from commercial crude copal resin and nanche bark, respectively.
Three esters 4a–c of α-amyrin (3) were synthesized in good to excellent yields (70–98%). To the best
of our knowledge, α-amyrin phenylacetate (4c) is reported for the first time. Regarding biological
activity, S. mutans was the most sensitive strain to the effect of the antimicrobial compounds evaluated
in this work. α-amyrin phenylacetate showed the greatest bacteriostatic activity on an initial
concentration of 103 and 106 cfu/mL of S. mutans. This property makes it a very promising compound
for caries prevention.
Acknowledgments
The authors are grateful for the financial support of Instituto de Ciencia y Tecnología del D.F. Grant
number: PICS08-40-ICYTDF.
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© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).