Antimicrobial activity of Rosmarinus officinalis against oral pathogens: Relevance of carnosic acid and carnosol

Article abstract of DOI:10.1002/cbdv.200900301

The in vitro inhibitory activity of crude EtOH/H₂O extracts from the leaves and stems of Rosmarinus officinalis L. was evaluated against the following microorganisms responsible for initiating dental caries: Streptococcus mutans, S. salivarius,

Full text of DOI:10.1002/cbdv.200900301

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1835
Antimicrobial Activity of Rosmarinus officinalis against Oral Pathogens:
Relevance of Carnosic Acid and Carnosol
a
a
a
a
by Wagner A. Bernardes ), Rodrigo Lucarini ), Marcos G. Tozatti ), Maria G. M. Souza ),
a
a
a
M a´ rcio L. Andrade Silva ), Ademar A. da Silva Filho ), Carlos H. Gomes Martins ),
Antonio E. Miller Crotti ), Patricia M. Pauletti ), Milton Groppo ), and Wilson R. Cunha* )
a
a
b
a
a) N u´ cleo de Pesquisas em Ci eˆ ncias Exatas e Tecnol o´ gicas, Universidade de Franca, Av. Dr. Armando
Sales de Oliveira, 201 14404-600, Franca, SP, Brazil (fax: þ55-16-3711-8878;
e-mail: wrcunha@unifran.br)
b) Departamento de Biologia da Faculdade de Filosofia Ci eˆ ncias e Letras de Ribeir a˜ o Preto,
Universidade de S a˜ o Paulo, Ribeir a˜ o Preto, SP, Brazil
The in vitro inhibitory activity of crude EtOH/H O extracts from the leaves and stems of Rosmarinus
2
officinalis L. was evaluated against the following microorganisms responsible for initiating dental caries:
Streptococcus mutans, S. salivarius, S. sobrinus, S. mitis, S. sanguinis, and Enterococcus faecalis. Minimum
inhibitory concentrations (MIC) were determined with the broth microdilution method. The bioassay-
guided fractionation of the leaf extract, which displayed the higher antibacterial activity than the stem
extract, led to the identification of carnosic acid (2) and carnosol (3) as the major compounds in the
fraction displaying the highest activity, as identified by HPLC analysis. Rosmarinic acid (1), detected in
another fraction, did not display any activity against the selected microorganisms. HPLC Analysis
revealed the presence of low amounts of ursolic acid (4) and oleanolic acid (5) in the obtained fractions.
The results suggest that the antimicrobial activity of the extract from the leaves of R. officinalis may be
ascribed mainly to the action of 2 and 3.
Introduction. – Dental caries is a common oral bacterial pathology caused by a
biofilm consisting of microorganisms present on the tooth surface [1][2]. This disease
has been associated with Streptococcus spp., mainly Streptococcus mutans and S.
sobrinus [3][4]. Several antimicrobial substances, such as ampicillin, chlorhexidine,
sanguinarine, metronidazole, and phenolic and quaternary ammonium antiseptics, have
been very effective in preventing dental caries [3][5]. However, various adverse effects
such as tooth and restoration staining, increase of calculus formation, diarrhea, and
disarrangements of the oral and intestinal flora have been associated with the use of
such compounds [3][6]. Thus, these drawbacks justify the search for new effective
anticariogenic agents that could be employed in caries prevention.
The use of plant extracts and their constituents with known antimicrobial properties
can be of great significance in therapeutic treatments. Within this context, the
antimicrobial properties of plant extracts and isolated compounds have been
investigated by a number of researchers worldwide [6][7]. Recent studies undertaken
in our laboratory have demonstrated the great importance of natural products, both
plant extracts and isolated compounds, as natural antimicrobial agents in oral care
products [8][9].
ꢀ
2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Rosmarinus officinalis L. (Lamiaceae) is an edible evergreen shrub native to the
Mediterranean area, and it is widely used around the world for culinary and medicinal
purposes [10–12]. Its main constituents are rosmarinic acid (1), carnosic acid (2),
carnosol (3), ursolic acid (4), oleanolic acid (5), genkwanin, apigenin, and luteolin
(
Fig. 1) [13–15]. Despite several pharmacological applications of R. officinalis, studies
on its antimicrobial properties against oral bacteria have been scarce [16].
Fig. 1. Chemical structures of rosmarinic acid (1) and its derivatives (1a and 1b), carnosic acid (2),
carnosol (3), ursolic acid (4), and oleanolic acid (5)
As part of our ongoing research on medicinal plants [17–20], we report herein the
in vitro antimicrobial activity of crude EtOH/H O extracts obtained from stems
2
(
HEROS) and leaves (HEROL) of R. officinalis against some important oral
pathogens. Additionally, we have carried out a bioassay-guided fractionation and
HPLC analysis of the HEROL, the more active extract, in order to identify the main
compounds responsible of this biological activity.
Results and Discussion. – The effects of the extracts of R. officinalis (HEROS and
HEROL) on the growth of the selected cariogenic bacteria are shown in the Table. The
lowest MIC values were obtained for the EtOH/H O extract obtained from leaves
2
(
HEROL).
Among the seven fractions (Frs. 1–7) achieved by the fractionation of the HEROL,
Fr. 3 displayed the highest antibacterial activity against the selected bacteria (Table).
HPLC analysis of this fraction (Fig. 2) showed that its major compounds are carnosic
acid (2, tR 32.16 min) and carnosol (3, tR 27.41 min). Regarding their antimicrobial
activity, both 2 and 3 displayed significant inhibitory effects against the selected oral
pathogens (Table). They displayed better antibacterial activity than thymol, a
commercially available antibacterial agent, which was used as positive control (Table).

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1837
Table. Minimum Inhibitory Concentrations (MIC) of Extracts and Fractions of R. officinalis, Rosmarinic
Acid (1) and Its Derivatives (1a and 1b), Carnosic Acid (2), and Carnosol (3) against Oral Pathogens
Sample
MIC [mg/ml]
E. faecalis
S. salivarius
S. sanguinis
S. mitis
S. mutans
S. sobrinus
a
HEROS )
>400
350
>400
200
70
400
>400
>400
>400
>400
>400
>400
70
>400
160
>400
300
12
200
400
300
300
>400
400
300
30
350
50
>400
50
50
50
200
90
100
>400
>400
300
50
400
170
>400
60
350
90
>400
90
50
200
400
300
>400
>400
>400
200
>400
80
>400
70
a
HEROL )
Fr. 1
Fr. 2
Fr. 3
Fr. 4
Fr. 5
Fr. 6
Fr. 7
1
12
10
200
400
300
>400
>400
300
200
15
200
400
300
>400
>400
>400
300
40
1
1
2
3
a
b
30
75
100
35
35
35
50
Chlorhexidine
Thymol
0.37
>400
0.09
400
0.74
>400
0.37
300
0.09
>400
0.09
300
a) HEROS and HEROL are crude EtOH/H O extracts from stems and leaves of R. officinalis,
2
respectively.
Fig. 2. HPLC Profile obtained with Method 1 for Fr. 3 of the EtOH/H O extract of the leaves of R.
2
officinalis showing the major compounds: carnosol (3, tR 27.41 min) and carnosic acid (2, tR 32.16 min)
The literature reports sparse information about the antimicrobial activities of R.
officinalis, and most of this work refers to its essential oil [21][22]. A recent in vitro
antimicrobial evaluation of commercial rosemary extract formulations against some
bacteria showed that Gram-positive bacteria were more sensitive to the formulations
than Gram-negative ones, especially in the case of oil-soluble extracts containing
carnosic acid as the major phenolic compound [23]. Moreover, a recent study also
demonstrated the antimicrobial activities of the leaf extract of R. officinalis as well as of
carnosic acid against bacteria and yeasts with dermatological relevance [24].
On the other hand, rosmarinic acid (1, t 7.47 min), which was detected as major
R
compound in Fr. 7, did not display antimicrobial activity against the tested oral

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
pathogens. In addition, the acetyl and methyl ester derivatives, 1a and 1b, respectively,
prepared from 1 were not able to improve its inhibitory activity.
The HPLC analysis also showed that both ursolic acid (4) and oleanolic acid (5) are
present in small concentrations in Frs. 2 and 3 of the HEROL. Concerning the
antibacterial activity of these triterpene acids, previous studies undertaken in our
laboratory revealed that both 4 and 5 possess moderate activity against oral pathogens
[
9].
The antibacterial activities of the two extracts and the isolated compounds against
the selected oral pathogens were also compared to chlorhexidine, an antiseptic that
possesses several detrimental side effects [3][6]. The high bactericidal activity of
chlorhexidine is due to its ability to inhibit glycosydic and proteolytic activities.
Moreover, it is able to reduce matrix metalloproteinase activities in a huge variety of
oral bacteria [25][26].
Several phenolic compounds, such as thymol, have been reported for their
inhibition of oral bacteria [27]. It was shown that the OH moiety attached to the
aromatic ring is required for the high antibacterial activity of these type of compounds
[
27][28]. However, it was observed that rosmarinic acid, which is a polyphenolic
compound, did not show antibacterial activity against the selected oral pathogens. In
addition, it has been reported that some polyphenolic compounds present in tea
extracts possess low anticariogenic activity against oral pathogens [29][30]. Thus, the
occurrence of phenol groups may not be the only essential chemical characteristics for
the antibacterial activity of the phenolic compounds.
In summary, we suggest that the antimicrobial activity against oral pathogens of R.
officinalis leaf extract may be mainly attributed to the effects of carnosic acid (2) and
carnosol (3). Therefore, with respect to an antimicrobial application, 2 and 3 should be
considered as appropriate compounds for the quality control of R. officinalis leaf
extract and its formulations.
The authors are grateful to FAPESP and CNPq for financial support.
Experimental Part
Plant Material. Rosmarinus officinalis L. (Lamiaceae) was collected in the urban perimeter of
Patrocꢂnio city (188 56’ 35’’ S, 468 59’ 31’’ W, Minas Gerais, Brazil, in May 2007). The plant material was
identified by M. G. A voucher specimen (collector M. G., number 1871, SPFR 11912) was deposited with
the Herbarium of the Departmento de Biologia, Faculdade de Filosofia, Ci eˆ ncias e Letras da
Universidade de S a˜ o Paulo, (Herbarium SPFR).
Extraction and Fractionation. The aerial parts of R. officinalis were divided into stems and leaves,
dried in a stove with circulating air (408), and powdered by means of a blender. The obtained powders
(
130 g each) were exhaustively extracted with EtOH/H O 8 :2 (v/v) by maceration at r.t., followed by
2
filtration. The filtered extracts were concentrated under reduced pressure, affording the stem (HEROS,
.6 g) and leaf (HEROL, 18.6 g) crude extracts. Because of its higher antimicrobial activity, the leaf
EtOH/H O extract of R. officinalis (12.0 g) was chromatographed over 300 g silica gel 60 (SiO , 0.063–
2
2
2
0
.200 mm; Merck) by vacuum liquid chromatography [31] to afford seven fractions of 1000 ml each: Fr. 1
(
(
hexane), Fr. 2 (hexane/AcOEt 75 :25 (v/v)), Fr. 3 (hexane/AcOEt 50 :50 (v/v)), Fr. 4 (AcOEt), Fr. 5
AcOEt/EtOH 75 :25 (v/v)), Fr. 6 (AcOEt/EtOH 50 :50 (v/v)), and Fr. 7 (EtOH). All the fractions
obtained were analyzed by HPLC. A portion of the most active fraction (Fr. 3) was dissolved in MeOH/
H O 1:1 (v/v) and chromatographed on a prep. RP-HPLC Shimadzu Shim-pack ODS column (250ꢀ
2
2
0 mm; 5 mm), equipped with a pre-column of the same material using H O with 0.1% AcOH (A) and
2

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1839
MeOH (B) as eluents (isocratic step of 50% B during 5 min, followed by a linear gradient to 100% B in
5 min, and maintaining this composition for 10 min; the system was then re-equilibrated to the initial
2
composition in 10 min) at a flow rate of 10 ml/min, affording compounds 2 and 3. Fr. 7 led to the isolation
of 1, under the same conditions as used for the analysis of Fr. 3. Moreover, Fr. 2 provided compounds 4
and 5.
HPLC Analysis. Two HPLC methods were used for the identification of the compounds in the
rosemary fractions. The HPLC equipment employed in this work was a Shimadzu LC-6AD system
equipped with a degasser DGU-20A5, a UV-DAD detector SPD-M20A series with a CBM-20A module,
and a Reodyne manual injector. Separations of the micromolecules were carried out on a Shimadzu
Shim-pack ODS column (250ꢀ4.60 mm; 5 mm) equipped with a pre-column of the same material. The
MeOH used was HPLC grade (J. T. Baker), and ultrapure H O was obtained by passing redist. H O
2
2
through a Direct-Q UV3 system. The eluents utilized for the chromatographic analysis of 1, 2, and 3
Method 1) were H O with 0.1% AcOH (A) and MeOH (B). An isocratic step of 50% B during 5 min
(
2
was run, followed by a linear gradient to 100% B in 25 min, and maintaining this composition for 10 min;
the system was then re-equilibrated to the initial composition in 15 min. The flow rate was 1.7 ml/min.
The mobile phase for the chromatographic analysis of 4 and 5 (Method 2) was MeOH/H O with 0.1%
2
AcOH 85 :15. Additionally, the compounds were identified by comparison of their tR and UV spectra
with those of the corresponding standards. All standards were acquired from Sigma-Aldrich (St. Louis,
MO, USA).
Preparation of Rosmarinic Acid Derivatives. Rosmarinic acid (1.0 g) was treated with excess Ac O in
2
pyridine, to give the AcO derivative 1a (0.73 g). In another preparation, rosmarinic acid (0.53 g) was
treated with CH N in Et O, to yield the Me ester derivative 1b (0.54 g). The derivatives were purified by
2
2
2
column chromatography (CC) on Sephadex LH-20 (Acros Organics, New Jersey, USA).
Structure Identification. Structures of all the compounds were determined by spectroscopic methods.
1H- and C-NMR spectra (at 400 and 100 MHz, resp.) were recorded on a Bruker DPX-400
spectrometer in (D )DMSO or CDCl using Me Si as internal standard. HR-ESI-MS were recorded on a
13
6
3
4
Bruker Ultra-TOF mass spectrometer.
Microorganisms. All the bacterial strains were acquired from the American Type Culture Collection.
The following microorganisms were used in this study: Enterococcus faecalis (ATCC 4082),
Streptococcus salivarius (ATCC 25975), S. mitis (ATCC 49456), S. mutans (ATCC 25275), S. sobrinus
(
ATCC 33478), and S. sanguinis (ATCC 10556).
Antimicrobial Assay. The MIC values (the lowest concentration of the extracts or pure compounds
from R. officinalis capable of inhibiting microorganism growth) were determined in triplicate using the
broth microdilution method in 96-well microplates [32]. The samples were dissolved in DMSO at 0.5 mg/
ml, followed by dilution in tryptic soy broth; concentrations ranging from 400 to 1 mg/ml were achieved.
The final DMSO content was 5% (v/v), and this soln. was used as negative control. The inoculum was
5
ꢁ1
adjusted for each organism to yield a cell concentration of 5ꢀ10 colony forming units (CFU)·ml . One
inoculated well was included, to control the adequacy of the broth. To ensure medium sterility, one non-
inoculated well containing no antimicrobial agent was also included. Chlorhexidine and thymol were
used as positive controls. To determine the MIC values for chlorhexidine, concentrations ranging from
5
.90 to 0.01 mg/ml were used. The microplates were sealed with plastic film and incubated at 378 for 24 h.
After incubation, resazurin (30 ml) in aq. soln. (0.02%) was added to the microplates. This procedure was
based on the methodology described by Palomino et al. [33].
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