Antimicrobial activity of terpenoids from Copaifera langsdorffii Desf. against cariogenic bacteria

Article abstract of DOI:10.1002/ptr.3244

In the present work, the anticariogenic activities of nine labdane type-diterpenes and four sesquiterpenes were investigated. Among these metabolites, (-)-copalic acid (CA) was the most active compound displaying MIC values very promising (ranging from 2.0 to 6.0 μg/mL) against the main microorganisms responsible for dental caries: Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis and Lactobacillus casei. Time kill assays performed with CA against the primary causative agent (S. mutans) revealed that, in the first 12 h, this compound only inhibits the growth of the inoculum (bacteriostatic effect). However, its bactericidal effect is clearly noted thereafter (between 12 and 24 h). Also, CA did not show a synergistic effect when combined with the anticariogenic gold standard (chlorhexidine, CHD) in the checkerboard assays against S. mutans. In conclusion, the results points out CA as an important metabolite in the search for new effective anticariogenic agents.

Full text of DOI:10.1002/ptr.3244

PHYTOTHERAPY RESEARCH
Phytother. Res. 25: 215–220 (2011)
Published online 14 July 2010 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.3244
Antimicrobial Activity of Terpenoids from
Copaifera langsdorffii Desf. Against
Cariogenic Bacteriaptr_3244 215..220
Ariana B. Souza,1 Carlos H. G. Martins,1 Maria G. M. Souza,1 Niege A. J. C. Furtado,2
Vladimir C. G. Heleno,1 João P. B. de Sousa,2 Erilda M. P. Rocha,1 Jairo K. Bastos,2
Wilson R. Cunha,1 Rodrigo C. S. Veneziani1* and Sérgio R. Ambrósio1*
1Núcleo de Pesquisa em Ciências Exatas e Tecnológicas, Universidade de Franca, Franca, SP, Brazil
2Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo,
Ribeirão Preto, SP, Brazil
In the present work, the anticariogenic activities of nine labdane type-diterpenes and four sesquiterpenes were
investigated. Among these metabolites, (-)-copalic acid (CA) was the most active compound displaying MIC
values very promising (ranging from 2.0 to 6.0 mg/mL) against the main microorganisms responsible for dental
caries: Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis and Lactobacillus casei. Time kill
assays performed with CA against the primary causative agent (S. mutans) revealed that, in the first 12 h, this
compound only inhibits the growth of the inoculum (bacteriostatic effect). However, its bactericidal effect is
clearly noted thereafter (between 12 and 24 h). Also, CA did not show a synergistic effect when combined with
the anticariogenic gold standard (chlorhexidine, CHD) in the checkerboard assays against S. mutans. In
conclusion, the results points out CA as an important metabolite in the search for new effective anticariogenic
agents. Copyright © 2010 John Wiley & Sons, Ltd.
Keywords: dental caries; Copaifera langsdorffii Desf.; labdane-type diterpenes; cariogenic bacteria; Streptococcus mutans;
antimicrobial activity.
burning sensation at the tip of the tongue (Greenberg
INTRODUCTION
et al., 2008; More et al., 2008; Porto et al., 2009b). In addi-
tion, chlorhexidine is much less effective in reducing the
levels of Lactobacillus, which are strongly related to
caries evolution (Ambrósio et al., 2008). These prob-
lems, therefore, denote that finding new effective anti-
cariogenic compounds is essential.
Cariogenic bacteria are a group of microorganisms
responsible for dental caries, one of the main oral dis-
eases which affect humankind (More et al., 2008). This
pathology is caused by a biofilm on the tooth surface
(dental plaque) that is associated with the growth of
Streptococcus and Lactobacillus species (Chung et al.,
2006). Streptococcus mutans is considered one of the
main cariogenic microorganisms, since it is responsible
for the beginning of the caries process. Other aerobic
bacteria such as Enterococcus faecalis, Lactobacillus
casei, Streptococcus mitis, S. sanguinis, S. sobrinus and S.
salivarius are also important in the later formation of
the dental biofilm (Chung et al., 2006).
The mechanical removal of the dental plaque is the
most efficient procedure to prevent caries; however, the
use of chemicals as a complementary measure is also
necessary and has demonstrated to be of great value
with respect to decreasing the tooth surface biofilm
(Furiga et al., 2008). Nowadays, chlorhexidine is consid-
ered a gold standard anticariogenic and has received the
approval of the American Dental Association Council
on Dental Therapeutics (Ambrósio et al., 2008).
However, the regular use of oral care products contain-
ing this chemical are often associated with tooth and
restoration staining, changes in the taste of food, and a
Natural products have been a rich and promising
source for the discovery of novel biologically active
compounds (Newman, 2008). Several classes of second-
ary metabolites are synthesized by plants and, among
them, diterpenes are recognized as a class with a wide
spectrum of biological activities (Ambrósio et al., 2006),
including their significant antibacterial activity (Kúzma
et al., 2007; Almeida et al., 2008; Porto et al., 2009a).
Recently, our research group has demonstrated that
some ent-kaurane and ent-pimarane type diterpenes are
able to inhibit the growth of the main microorganisms
responsible for dental caries with very promising
minimal inhibitory concentration (MIC) values
(ranging from 2 to 10 mg/mL). In these previous works,
it was also pointed out that some of these metabolites
may be potentially useful in the further development of
natural anticaries agents (Ambrósio et al., 2008; Porto
et al., 2009b). These results allowed us to conclude that
this class of natural compounds is an important source
for the discovery of new efficient bioactive compounds
for dental application.
In agreement with our early findings and as part of
our ongoing efforts to explore the antimicrobial poten-
tial of diterpenes against the pathogens responsible for
dental caries, our research group has decided to inves-
tigate the activity of a different class of diterpenes.
* Correspondence to: Sérgio R. Ambrósio or Rodrigo C. S. Veneziani,
Núcleo de Pesquisa em Ciências Exatas e Tecnológicas, Universidade de
Franca, Franca, SP, Brazil.
E-mail: sergioambrosio@unifran.br; rcsvenez@unifran.br
Received 25 February 2010
Revised 11 May 2010
Copyright © 2010 John Wiley & Sons, Ltd.
Accepted 12 May 2010

216
A. B. SOUZA ET AL.
For this purpose, a commercially available and well
documented source of natural labdane-type diterpenes
(Veiga Júnior and Pinto, 2002), namely the oleoresin of
Copaifera langsdorffii, was chosen. Moreover, an anti-
microbial evaluation was also carried out of some semi-
synthetic diterpene derivatives and the main
sesquiterpenes present in this plant material.
using vacuum liquid chromatography (VLC) (Pelletier
et al., 1986) with increasing amounts of EtOAc in
n-hexane as eluent. This procedure furnished six frac-
tions (500 mL each) that were named F1 (4.9 g;
n-hexane), F2 (3.1 g; 20% EtOAc), F3 (3.6 g; 40%
EtOAc), F4 (2.2 g; 60% EtOAc), F5 (2.7 g; 80% EtOAc)
and F6 (2.1 g; EtOAc) after solvent evaporation.
Samples F1 and F2 were firstly analysed by GC and
GC/MS, in order to identify their main volatile
compounds. Fraction F3 (1.0 g) was fractionated over
silica gel 60 (Merck, art. 7734) using classic chromato-
graphy (isocratic, n-hexane: EtOAc 7 : 3), to furnish
compounds 1 (caryophyllene oxide; 117.0 mg) and 2
((-)-copalic acid; 450.0 mg).
MATERIALS AND METHODS
Plant material. Authentic oleoresin from Copaifera
langdsdorffii was kindly provided by the Brazilian
company Apis-Flora Comercial e Industrial.
Both F4 and F5 were initially chromatographed by
VLC over silica gel 60 H (Merck, art. 7736) as described
above, to give additional fractions (F4.1–F4.5 and F5.1–
F5.5). Compound 3 ((-)-acetoxycopalic acid; 230.0 mg)
was obtained from F4.3 (880.0 mg) through medium
pressure chromatography (flash chromatography)
using silica gel 60 (Merck, art. 9385), isocratic
n-hexane : EtOAc : CHCl3 (5 : 2 : 3) as mobile phase,
and a flow rate of 5 mL/min (Still et al., 1978). Subfrac-
tions F5.2 (350.0 mg) and 5.4 (270.0 mg) were also chro-
matographed by flash chromatography as described
above.These procedures led to the isolation of two com-
pounds which, after NMR analysis, were identified as
(–)-3-hydroxy- 14,15-dinorlabd-8(17)-en-13-one (4, 110.
0 mg), obtained from 5.2, and (-)-agathic acid (5, 150.
0 mg), originated from 5.4. Thin layer chromatography
(TLC) analysis of F5.2.5 showed a main spot, which was
later purified by preparative thin layer chromatography
using silica gel PF254 (Merck art. 9385; 1 mm thickness)
and isocratic n-hexane: EtOAc 1 : 1 as mobile phase.
Compound 6 ((-)-hydroxycopalic acid, 130.0 mg) was
obtained from this procedure and identified after NMR
analysis.
General. NMR spectra were recorded on a Bruker
1
Avance DRX 400 spectrometer (400 MHz for H and
100 MHz for 13C). Samples were dissolved in CDCl3,
and TMS was used as internal reference. High perfor-
mance liquid chromatography (HPLC) analyses were
performed using a Shimadzu CBM-20A liquid chroma-
tography controller, operating with the LC solution
software, equipped with a Shimadzu UV-DAD detec-
tor SPD-M20A and a Shimadzu ODS column (4.6 ¥
250 mm, 5 mm, 100 Å). Gas chromatography (GC)
analyses were carried out in Hewlett Packard GC
equipment, model 6890N, equipped with a split/
splitless injector inlet and a flame ionization detector
(FID). The output was recorded using the workstation.
An HP-5 capillary column (30 m of length ¥ 0.32 mm
of internal diameter ¥ 0.25 mm of film thickness) was
used. Hydrogen at a flow rate of 1.8 mL/min was
employed as the carrier gas, and the GC oven tem-
perature was programmed to rise from 100 to 140°C at
10°C/min, from 140 to 180°C at 2.8°C/min, maintained
at 180°C for 1 min, followed by an increase from 180
to 280°C at 20°C/min, and kept at 280°C for 1 min
more. The temperatures of the injector and detector
ports were kept at 240°C and 290°C, respectively.
The injector was operated in a split mode of 1/50.
Gas chromatography/mass spectrometry (GC/MS) was
performed using a Shimadzu – QP 2010 gas chroma-
tography equipped with an automatic injector AOC –
20Si, a DB-5 column (30 m ¥ 0.25 mm ¥ 0.25 mm),
and a mass spectrometer of the same company, which
was operated in the EI mode (beam energy voltage
70 eV). Hydrogen at a flow rate of 1.8 mL/min was
employed as the carrier gas. Both the injector and
oven temperatures were programmed as described
above. The main constituents were identified by com-
paring their retention indices (RI relative to C9-C25
n-alkanes), which were obtained by GC-FID and
GC/MS analyses, with those reported in the literature,
as well as by comparison of the obtained mass spectra
of the peaks with those either reported in the litera-
ture or available in the Wiley NBS data system library.
In order to perform the antimicrobial evaluations
against oral pathogens, the main sesquiterpenes iden-
tified in C. langsdorffii oleoresin were purchased from
Sigma-Aldrich (trans-caryophyllene, Sigma, technical
grade 98.5%; a-copaene, Aldrich, technical grade Ն
A sample of F6 was analysed by reversed phase
HPLC using an analytical Shimadzu ODS column (4.6 ¥
250 mm, 5 mm, 100 Å; MeCN : H2O 85 : 15; flow rate
1.0 mL/min; UV detection at 210 nm); compounds 5 and
6 were identified as the main constituents of this
fraction.
Semi-synthetic derivatives. About 100.0 mg of com-
pound 2 was treated with ethereal diazomethane and,
after addition of a small amount of acetic acid to destroy
the remaining diazomethane and elimination of the
solvent, the methylated derivative 7 was obtained
(Ambrósio et al., 2008). Compounds 3, 5 and 6 were
submitted to the same treatment, to give compounds 8,
9 and 10, respectively.
Bacterial strains and antimicrobial testing. The
minimal inhibitory concentration values (MIC; lowest
concentration of the compound capable of inhibiting
microorganism growth) and the minimal bactericidal
concentration (MBC; lowest concentration of the com-
pound at which 99.99% or more of the initial inoculum
was killed) were determined in triplicate by using the
microdilution broth method in 96-well microplates
(Porto et al., 2009b). The tested strains were obtained
from the American Type Culture Collection (ATCC).
The following microorganisms were used in the present
work: Streptococcus salivarius (ATCC 25975), Strepto-
90.0%; a-humulene, Aldrich, technical grade
98.0%).
Ն
Isolation of compounds. About 20.0 g of oleoresin was
chromatographed over silica gel 60 H (Merck, art. 7736)
Copyright © 2010 John Wiley & Sons, Ltd.
Phytother. Res. 25: 215–220 (2011)

ANTIMICROBIAL ACTIVITY OF COPAIFERA LANGSDORFFII DESF.
217
coccus sobrinus (ATCC 33478), Streptococcus mutans
(ATCC 25275), Streptococcus mitis (ATCC 49456),
Streptococcus sanguinis (ATCC 10556) and Lactobacil-
lus casei (ATCC 11578).
combination of CHD with CA. The synergy tests were
evaluated in triplicate, and concentrations of each com-
pound (1/32 to 3 times of their MIC values) were com-
bined in standard MIC format against 5 ¥ 105 CFU/mL
of S. mutans. To evaluate the synergism effect between
CHD and CA, the fractional inhibitory concentration
(FIC) index values were calculated on the basis of the
equation previously established in the literature (White
et al., 1996). FIC index values were analysed as follows:
FIC index values Յ 0.5, synergism; FIC index values > 4,
antagonism. Indifference was defined as an FIC index of
greater than 0.5 but of equal to or less than 4 (White
et al., 1996).
Samples were dissolved in dimethyl sulfoxide
(DMSO; Synth) at 1 mg/mL, followed by dilution in
tryptic soy broth (Difco); concentrations ranging from
200.0 to 1.0 mg/mL were achieved. The final DMSO
content was 5% (v/v), and this solution was used as a
negative control. The inoculum was adjusted for each
organism, to yield a cell concentration of 5 ¥ 105
colony forming units (CFU) per mL, according to
guidelines by the Clinical Laboratory Standards
Institute. One inoculated well was included, to allow
control of the adequacy of the broth for organism
growth. One non-inoculated well, free of antimicrobial
agent, was also employed, to ensure medium sterility.
Chlorhexidine dihydrochloride (CHD) was used as
positive control. The microplates (96-wells) were
sealed with plastic film and incubated at 37°C for
24 h. After that, resazurin (30 mL) in aqueous
solution (0.02%) was added to the microplates,
to indicate microorganism viability (Porto et al.,
2009b).
Before the addition of resazurin and in order to deter-
mine MBC, an aliquot of the inoculum was aseptically
removed from each well presenting no apparent growth
and then plated onto tryptic soy agar supplemented
with 5% sheep blood. The plates were incubated as
described previously. Determination of the MBC values
were only performed for the most active compound (2,
CA), against the strains whose MIC values were very
promising (equal to or lower than 10 mg/mL) (Ríos and
Récio, 2005).
RESULTS AND DISCUSSION
Figure 1 shows the chemical structures of the metabo-
lites that were evaluated in the present work. The spec-
tral data of the compounds isolated from the oleoresin
of C. langsdorffii are in agreement with those reported
previously in the literature: 1 (Moreira et al., 2007), 2
(Ohsaki et al., 1994), 3 (Braun and Breitenbach, 1977), 4
(Romero et al., 2009), 5 (Zdero et al., 1991) and 6
(Romero et al., 2009). The structures of the semi-
synthetic methyl derivatives (7–10) were confirmed on
the basis of comparison of their nuclear magnetic reso-
nance (1H and 13C NMR) data with their respective acid
diterpenes. All isolated substances as well as those
obtained by semi-synthetic means were evaluated by
TLC using different solvent systems.According to these
1
procedures and in addition to their H and 13C NMR
data, the purity of such compounds was considered
suitable for the antimicrobial assays. Analysis by GC
and GC/MS of F1 and F2 allowed the identification of
compounds 11 (trans-caryophyllene), 12 (a-copaene)
and 13(a-humulene) as the main metabolites of these
fractions.
Time-kill curves. Time-kill assays were performed in
triplicate based on D’Arrigo et al. (2010) against Strep-
tococcus mutans, which is considered one of the primary
causative agents of dental caries (Chung et al., 2006).
Compound 2 (CA) was chosen for time-kill curve
assays because it displayed the highest antimicrobial
activity.
The MIC values for the investigated sesquiterpenes
and diterpenes are shown in Table 1. Among all
metabolites, compound 2 (CA) displayed the highest
antibacterial activity, furnishing MIC values ranging
from 2.0 to 6.0 mg/mL. On the other hand, compounds 3
and 4 demonstrated a moderate activity (MIC values
between 40 and 80 mg/mL), while the other substances
were not able to efficiently inhibit the growth of most
pathogens (MIC values higher than 100 mg/mL).
Time-kill curve assays (Fig. 2) were constructed
against S. mutans (5 ¥ 105 CFU/mL) only for CA, using
the three different concentrations (7.0, 14.0 and 21.0 mg/
mL). The resulting time-kill curves are presented in
Fig. 2, which reveals that CA is able to kill S. mutans
after 24 h of incubation. Analysis of these data also
shows that CA drastically reduces the number of
microorganisms after 12 h, in the three evaluated
concentrations. No statistical differences were observed
during the evaluated periods (p < 0.05). As for the syn-
ergistic antimicrobial test, the combinations of CHD
with CA did not exhibit any synergic effects when the
checkerboard methodology described by White et al.
(1996) was employed. According to this author, the
obtained FIC index = 2.02 means ‘indifference’.
Tubes containing CA at final concentrations of 7.0,
14.0 and 21.0 mg/mL (respectively one, two, and three-
times the MBC of CA for S. mutans) were inoculated
with the tested microorganism, resulting in a start bac-
terial density of 5 ¥ 105 CFU/mL, and then incubated at
37°C. Samples were removed for the determination of
viable strains at 0, 5, 15 and 30 min and 6, 12, 18 and 24 h
after incubation, followed by dilution, when necessary,
in sterile fresh medium. The diluted samples (50 mL)
were spread onto tryptic soy agar plate supplemented
with 5% sheep blood, incubated at 37°C and counted
after 48 h.Time-kill curves were constructed by plotting
the log10 CFU/mL versus time. The assays were per-
formed in triplicate for each concentration and also
for the positive (chlorhexidine dihydrochloride, CHD,
4 mg/mL) and negative controls (suspension of S. mutans
without added CA). CHD was used at its MBC
(4 mg/mL).
Synergistic antimicrobial activity. Checkerboard assays
were performed according to the protocol described
previously by White et al. (1996). The objective was to
investigate the in vitro antimicrobial efficacy of the
Our research group has concentrated efforts on the
evaluation of the antimicrobial potential of diterpenes,
aiming to discover new natural anticaries agents. It was
Copyright © 2010 John Wiley & Sons, Ltd.
Phytother. Res. 25: 215–220 (2011)

218
A. B. SOUZA ET AL.
12
12
O
15
13
O
11
11
20
13
20
14
14
1
4
1
9
6
9
17
17
8
10
8
7
10
2
3
2
3
R1
5
5
7
H
H
4
6
H
HO
H
R2
18
18
19 R3
19
1
2: R1 = COOH; R2 = H; R3 = H
4
3: R1 = COOH; R2 = OCOCH3; R3 = H
5: R1 = COOH; R2 = H; R3 = COOH
6: R1 = COOH; R2 = OH; R3 = H
7: R1 = COOCH3; R2 = H; R3 = H
8: R1 = COOCH3; R2 = OCOCH3; R3 = H
9: R1 = COOCH3; R2 = H; R3 = COOCH3
10: R1 = COOCH3; R2 = OH; R3 = H
H
H
11
12
13
Figure 1. Chemical structures of the evaluated compounds.
Table 1. In vitro antibacterial activity (MIC) of the metabolites from C. langsdorffii against oral pathogens
Minimum inhibitory concentration – mg/mL (minimum bactericidal concentration – mg/mL)
Microorganism
PC
1
2
3
4
5
6
7
8
9
10
11
12
13
Streptococcus salivarius
Streptococcus mutans
Streptococcus mitis
Streptococcus sobrinus
Streptococcus sanguinis
Lactobacillus casei
0.9
0.9 (4.0)
3.6
0.9
3.6
*
*
2.0 (2.0)
3.0 (7.0)
5.0 (12.0)
3.0 (3.0)
6.0 (15.0)
4.0 (10.0)
12.0
40.0
60.0
40.0
60.0
50.0
80.0
60.0
80.0
40.0
40.0
60.0
*
*
*
*
*
*
*
*
*
*
*
*
120.0
100.0
80.0
150.0
60.0
180.0
150.0
120.0
100.0
80.0
120.0
100.0
80.0
120.0
100.0
80.0
200.0
120.0
150.0
180.0
120.0
150.0
*
*
*
*
*
*
*
*
*
*
*
*
*
200.0
150.0
180.0
200.0
150.0
200.0
*
*
0.9
150.0
80.0
150.0
* Inactive in the evaluated concentration (MIC values higher than 200 mg/mL); Positive control (PC), chlorhexidine dihydrochloride;
Negative control (5% DMSO solution v/v) did not affect the growth of the microorganisms.
10
antiinfection agents against the microorganisms respon-
sible for this pathology. Moreover, it was observed that
minor structure modifications drastically affect the
8
Negative Control
referred antimicrobial activity.
CHD 4 μg/mL
CA 7 μg/mL
CA 14 μg/mL
CA 21 μg/mL
The present work investigated the effect of nine diter-
6
penes and four sesquiterpenes from the oleoresin of C.
langsdorffii against the main microorganisms respon-
4
sible for human caries. From the evaluated metabolites,
it is possible to observe that CA is the most effective
compound (Table 1), since it furnishes very promising
2
MIC values for all the investigated pathogens (MIC
values lower than 10 mg/mL; Ríos and Récio, 2005).
0
0
5
10
15
20
25
These values are much lower than some previously
reported in the literature for compounds belonging to
other classes of natural products, such as triterpenoids
(Rivero-Cruz et al., 2008), monoterpenes (Botelho et al.,
2007), phenolic compounds (Greenberg et al., 2008) and
lignans (Silva et al., 2007).
Time (h)
Figure 2. Time-kill curves for CA against S. mutans. Positive
control: CHD 4 mg/mL.
demonstrated that kaurenoic acid and several
pimarane-type diterpenes, isolated from Brazilian
plants (Ambrósio et al., 2008; Porto et al., 2009b), could
be used as prototypes for the discovery of new effective
Time-kill curve assays (Fig. 2) denote, for all the
evaluated concentrations, that CA is able to completely
kill S. mutans after only 24 h of incubation. Figure 2 also
Copyright © 2010 John Wiley & Sons, Ltd.
Phytother. Res. 25: 215–220 (2011)

ANTIMICROBIAL ACTIVITY OF COPAIFERA LANGSDORFFII DESF.
219
reveals that in the first 12 h, CA only inhibits the growth
of the inoculum.This leads to the interpretation that CA
displays a bacteriostatic effect during this period, but its
bactericidal effect is clearly noted thereafter (between
12 and 24 h). No statistical differences were demon-
strated within the periods of time investigated for each
concentration, allowing us to conclude that no dose-
dependent responses were verified for CA in the assay
conditions. Analysis of Fig. 2 also shows that CA at
23.1 mm (7.0 mg/mL) exhibits a time-kill curve profile
that is very similar to those demonstrated by CHD at
6.9 mm (4.0 mg/mL) (p < 0.05). In addition, the inoculum
concentration is equivalent for both curves. These
results confirm that CHD, the anticariogenic gold stan-
dard, is about three and half times more potent than
CA. Nevertheless, several adverse effects are associated
with the regular use of CHD. This reinforces the great
importance of CA as a prototype for the development
of novel anticaries agents (More et al., 2008).
Antimicrobial compounds have demonstrated differ-
ent action mechanisms (Greenberg et al., 2008). Several
authors have emphasized that diterpenes are an impor-
tant class of plant metabolites for the search of new
antibacterial agents; however, the mechanism(s)
responsible for this property have not yet been very well
elucidated (Urzúa et al., 2008). Urzúa et al. (2008) have
suggested that these metabolites promote bacterial lysis
and disruption of the cell membrane.According to these
authors, the structural features that promote the effi-
cient antibacterial activity include a lipophilic structure,
capable of insertion into the cell membrane, and one
strategically positioned hydrogen-bond-donor group
(HBD; hydrophilic group), which interacts with the
phosphorylated groups on the membrane. In these
studies, it was also emphasized that a second HBD
introduced in the lipophilic region led to reduction in or
suppression of the activity. A careful observation of the
results depicted in Table 1 reveals that compound 2,
which contains only one HBD at C-16, displays much
lower MIC values than those achieved with diterpenes
3–10, which contain two HBD in their structures. Based
on these considerations, our results give support to the
mechanism of action suggested by Urzúa et al. (2008).
However, comparison of the MIC values displayed by
CA (Table 1) and other acid diterpenes previously
reported in the literature, such as ent-kaurenoic acid
(KA) and ent-pimaradienoic acid (Ambrósio et al.,
2008; Porto et al., 2009b) reveals that the antibacterial
activity displayed by this class of natural products is also
ruled by other structural features. These statements
justify the need for information about the anticari-
ogenic potential of a large number of diterpenes, so that
further quantitative structure-active relationship
(QSAR) studies can be accomplished.
Urzúa et al. (2008) have also demonstrated that the
efficiency of hydrogen bond interactions between the
phosphorylated groups on the membrane and the HBD
in the diterpene are very important for the antibacterial
activity. According to these authors, KA is much more
active than its respective methyl ester (KA-Me). The
docking of these compounds into a model using phos-
phatidylcholine bilayer revealed that KA-Me is more
deeply embedded into the hydrophobic region of the
membrane compared with KA, presumably diminishing
the capacity of the molecule to disrupt or damage the
cell membrane. This observation is also in agreement
with our results when the MIC values displayed by the
ent-labdanes diterpenes 2 and 7 are compared (Table 1).
According to several authors (Vuorela et al., 2004;
Newman, 2008), drugs derived from medicinal plants
can serve not only as new drugs themselves but also as
prototypes suitable for optimization through several
approaches, including medicinal and semi-synthetic
strategies. In this sense, modifications of the carboxylic
acid of CA into other hydrophilic moieties, such as an
amino, aldehyde, salt, among others, should be investi-
gated for improvement of its anticariogenic activity.
In conclusion, the results have shown that CA is an
important metabolite in the search for new effective
antibacterial agents against the pathogens responsible
for dental caries. This is an important report, because
very few natural products are known to inhibit the
growth of oral pathogens (Saleem et al., 2010). More-
over, the antimicrobial results displayed by the ent-
labdanes diterpenes described in the present work can
be used to promote docking and QSAR studies, in asso-
ciation with data from other diterpenes reported
previously. This should certainly aid total elucidation of
the structural features involved in the anticariogenic
effect displayed by these natural compounds.
Acknowledgements
This study was supported by Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP) process numbers 2009/09438-3, 2008/
09656-8 and 2007/54762-8.
Conflict of Interest
The authors have declared that there is no conflict of interest.
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