ORIGINAL ARTICLE
identification of eugenol and its effect on Streptococcus mutans.
Osvelia Rodríguez1, 2. Abstract: Dental caries is a disease which affects the human oral cavity. Cu-
Rosa Sánchez1, 2. rrently, the search for active principles of plants with antimicrobial effect seems
María Verde3. promising for dental therapy. In this article the activity of the essential oil of
María Núñez3. Syzygium aromaticum (clove) was evaluated with an emphasis on its antimicro-
Rocío Ríos1. bial properties. The oil was obtained by hydrodistillation, characterized by thin
Abelardo Chávez3. layer chromatography and chemical tests. The main compound was identified in
the oil obtained from the flower buds and its antibacterial activity against plank-
1 Universidad Autónoma de Nuevo León, tonic cells Streptcoccus mutans ATCC700611 was assessed by performing serial
Monterrey, Nuevo León. México. dilutions, from 15 up to 1000 µg/mL, compared with 0.12% chlorhexidine and
2 Universidad Autónoma de Nuevo León, dimethylsulfoxide. MIC was also determined. Subsequently, UFC was analyzed
Escobedo, Nuevo León. México.
and compared with CMR® Test Ivoclar Vivadent. The efficiency in obtaining the
3 Universidad Autónoma de Nuevo León,
oil was 2.20%. By using the CCD technique, a fraction was revealed by UV light,
San Nicolás de los Garza. Nuevo León.
corresponding to eugenol. It had a good response for triterpenoids and flavonoids.
It showed greater antimicrobial activity at concentrations of 1000, 500 and 250µg/
Corresponding author: Osvelia Rodrí- ml. The MIC and MBC of the oil was 125 to 250µg/mL, respectively. Eugenol
guez. Universidad Autónoma de Nuevo
was found as an active principle in the oil obtained. Currently, the impact of using
León, Facultad de Odontología, Departa-
plant extracts has favored the evaluation of alternative, effective and biocompatible
mento de Microbiología. Phone:+52 (81)
83465612. E-mail: osvelia.rodriguezl@ antibacterial agents for the formulations of oral hygiene products applied to the
uanl.mx. Eduardo Aguirre, Mitras Cen- prevention or treatment of oral diseases.
tro, Monterrey Nuevo León CP 64460, Keywords: Eugenol, Streptococcus mutans, Syzygium aromaticum, antimicrobial,
México. E-mail:osveliardzl@yahoo.com. minimum inhibitory concentration MIC.
Receipt: 08/21/2014 Revised: 09/09/2014 Cite as: Rodríguez O, Sánchez R, Verde M, Núñez M, Ríos R & Chávez A. Obtaining the
Acceptance: 10/01/2014 Online: 10/01/2014 essential oil of Syzygium aromaticum, identification of eugenol and its effect on Streptococcus
mutans. J Oral Res 2014; 3(4):218-224.
ISSN Online 0719-2479 - ©2014 - Official publication of the Facultad de Odontología, Universidad de Concepción - www.joralres.com 219
Rodríguez O, Sánchez R, Verde M, Núñez M, Ríos R & Chávez A.
Obtaining the essential oil of Syzygium aromaticum, identification of eugenol and
its effect on Streptococcus mutans.
J Oral Res 2014; 3(4): 218-224.
Abundance 8.50
TIC:2600032.D
8000000
7000000
6000000 9.33
5000000
4000000
3000000
2000000 10.52
9.75
1000000 11.35
7.45 8.95 21.48
Time 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00
Figure 2. Chromatographic profile of the oil from Sysygium aromaticum. Quantification of the principal
component (eugenol), main component in the clove oil by gas chromatography -mass spectrometry.
220 ISSN Online 0719-2479 - ©2014 - Official publication of the Facultad de Odontología, Universidad de Concepción - www.joralres.com
Rodríguez O, Sánchez R, Verde M, Núñez M, Ríos R & Chávez A.
Obtaining the essential oil of Syzygium aromaticum, identification of eugenol and
its effect on Streptococcus mutans.
J Oral Res 2014; 3(4): 218-224.
Table 1. MIC Results, concentrations of oil from Identification of eugenol as the main component in
1000 to 15 µg/ml positive control, chlorhexidine
0.12 %, negative control, DMSO, n=3. the oil.
A sample of the oil obtained was taken to identify and
Extract Concentrations Syzigium aromaticum (clove) quantify the presence of eugenol. A 6890N gas chromatograph
µg/ml 1000 500 250 125 62 31 15 with split/splitless injector and a 5973 mass selective detector
S. mutans - - - - + + + (Agilent Technologies, Germany) with electron-impact
Negative control + + + + + + + ionization source and quadrupole mass analyzer were used.
Positive control - - - - - - -
The separation was carried out with a 5MS HP column
(-) without growth; growth (+).
(30mx0.25mmx0.2μm; Agilent Technologies, Germany).
The carrier gas was helium at a flow rate of 1.0ml min -1. The
Table 2. Results for MIC, CFU, CMR Test Ivoclar C+ program for the furnace temperature consisted of a start-up
(positive control chlorhexidine 0.12%), C- (negative phase at 80°C for 1 min, going up 10°C min-1 to 200°C for
control, DMSO), CC (Growth Control), CRT® Test.
3 min at this temperature. Finally, it was increased to a speed
of 15°C min-1 until reaching 320°C for 8 min. The injector
Oil Concentrations Count in Petri Dishes
µg/ml Repetition 1 Repetition 2 Repetition 3 temperature was 270°C and that of the transfer line 250°C.
1000 < 105 < 105 < 105 Data acquisition was carried out in sweep mode in a range of
500 < 105 < 105 < 105 30-550m/z. The identification of the compounds was carried
250 < 105 < 105 < 105 out using the Wiley 7N.l. database.
125 < 10
5
< 10
5
< 105
62 ≥ 105 ≥ 105 ≥105 RESULTS.
C+ < 105 < 105 < 105 An efficiency of the essential clove oil extraction was
C- ≥ 105 ≥ 105 ≥ 105 2.20%. In the partial phytochemistry identification through
CC ≥ 10
5
≥10
5
≥ 105 chemical reactions, it tested positive in LibermanBuchard for
triterpenes, flavonoids, and ferric chloride test for phenolic
Table 3. Identification of major components of OH groups.
the oil obtained from Sysygium aromaticum,
retention time for each compound identified (rt) In the comparative chromatography using hexane-acetone
and the percentage of the area (abundance). 9:1 as mobile phase, analogy between relationships of fronts
(Rf) was identified between the first fraction and the stan-
Rt (min) Compounds found % Area
dard eugenol, corresponding to 0.65, confirming the presen-
4.89 Alfa-Terpinolene 0.43
6.25 P-Alinalisol ( estragole) 0.58
ce of this compound (Figure 1).
6.91 P-allylphenol 0.19 Clove oil showed antimicrobial activity against
7.45 Thymol 1.41 Streptococcus mutans from concentrations of 1000 up to
8.50 Eugenol 67.5 125µg/ml. MIC was 125µg/ml with bactericidal behavior
8.73 Alfa-copaene 0.28 (Table 1). In 1000µg/ml concentrations, (3 replicates)
8.95 Vanilin 1.15 it was reported <105 CFU growth, as well as for 500,
9.33 Trans-caryophyllene 1626
250 and 125µg/ml concentrations. Regarding lower
9.75 Alfa-humulene 3.14
concentrations, 62µg/mL presented ≥105 CFU, the same
9.99 Germacrene - d 0.14
1052 Eugenol Acetate 4.16 as the negative controls (Table 2).
1136 Caryophyllene Β 2.05 Among the main compounds present in the oil obtained
12.96 cinnamaldehyde 0.52 from Sysygium aromaticum, eugenol was identified as the
ISSN Online 0719-2479 - ©2014 - Official publication of the Facultad de Odontología, Universidad de Concepción - www.joralres.com 221
Rodríguez O, Sánchez R, Verde M, Núñez M, Ríos R & Chávez A.
Obtaining the essential oil of Syzygium aromaticum, identification of eugenol and
its effect on Streptococcus mutans.
J Oral Res 2014; 3(4): 218-224.
major component (its corresponding retention time and per- tions of 512μg/ml, 0.31 and 0.16mg/mL, respectively, for
centage of area are seen in Table 3). Likewise, in the chro- Candida albicans, Staphylococcus aureus and Actinomyces
matographic profile, this compound was clearly identified by viscosus 23. Therefore, based on the described above and
matching the abundance peak. The retention time was 8.50 in accordance with the established criteria, the essential
minutes, representing the main component of clove oil in a oils with MIC between 50 and 500μg/mL are considered
67.5% of the area (Figure 2). to have strong antimicrobial activity. Those with a MIC
between 600 and 1500μg/ml are considered to have mo-
DISCUSSION. derate activity and when MIC is greater than 1500 μg/ml
Several studies have reported eugenol as the main com- they have low activity. In this sense, the essential oil of
pound in clove oil. It is considered as the molecule res- clove is considered a substance with a strong antimicro-
ponsible for the great variety of its activities studied16,17. bial activity as it presents a MIC of 125μg/mL. MBC was
When separating the oil compounds through chromato- 250µg/mL in agreement with the criteria for the establis-
graphy, using hexane/acetone 9:1 as mobile phase allowed hed antimicrobial activity which considers it as a strong
an efficient chromatographic separation, coinciding with activity24.
other reports described, and seeing the presence of euge- On the other hand, in a study on different essential
nol in the study sample. In the same way, in comparative oils of spices, a eugenol level of 82.6% was reported with
chromatography, analogy was identified between the first respect to the influence of heat on the antioxidant acti-
fraction and the standard (eugenol) with R.f 0.65 (Fig.1 vity in the oil of clove. In this study, it was obtained a
A, b), with agreement between the samples analyzed ac- 67.5%25. Other reports mention the presence of eugenol
cording to previously described techniques14. in the clove oil as a compound with values of 89.2% a
In this study, the essential oil of Syzygium aromati- slightly higher percentage than in this study26 . Finally,
cum was obtained, eugenol was identified as a compound 77.4% of eugenol has been reported, due to the fact that
and its antimicrobial activity was assessed, agreeing with the extraction was performed with an equipment using
what has been reported in several studies18,19. Its activity CO2 . Then, the decline in eugenol could be related to the
against Streptococcus mutans was observed, agreeing with system used for the extraction of essential oil 27.
several studies which reported its growth inhibitory acti-
vity in oral pathogens20,21. CONCLUSIONS.
It was noted that the essential oil obtained by hydro- In this study, it was demonstrated the capacity of the
distillation presented strong antimicrobial activity, com- essential oil of clove obtained by hydrodistillation to in-
parable to the concentrations reported in the range of hibit growth of Streptococcus mutans. Eugenol was identi-
micrograms per milliliter, compared to what has been re- fied as the main component of Syzygium aromaticum, in
ported as MIC and MBC for Streptococcus mutans, whe- conjunction with the standard of eugenol analyzed with
re apart from testing clove oil as an antimicrobial and Rf 0.65 and as the compound of greater abundance and
anti-fungal for Candida albicans, a MIC and CMB of with bactericidal activity in a concentration range of 125-
390 and 780µg/mL, respectively, was demonstrated for 1000μg/mL, with MIC 125 μg/mL.
Streptococcus mutans 22 . On the other hand, it has been Phytotherapy provides an important field for research,
reported that MIC of the essential oil of clove from In- where the active metabolites of plants can be tools which
dia on Candida albicans varies from 1000 up to 2500μg/ provide advantages in therapies, being a promising field
ml, for Streptococcus sanguis was 0.31mg/mL. It has also for the search of new alternatives with greater antimicro-
been reported sensitivity to the clove oil for concentra- bial application in the dental practice by using the prin-
222 ISSN Online 0719-2479 - ©2014 - Official publication of the Facultad de Odontología, Universidad de Concepción - www.joralres.com
Rodríguez O, Sánchez R, Verde M, Núñez M, Ríos R & Chávez A.
Obtaining the essential oil of Syzygium aromaticum, identification of eugenol and
its effect on Streptococcus mutans.
J Oral Res 2014; 3(4): 218-224.
ciples derived from plants. CONACyT) through the Grant 256972, the Program of
Support for Scientific and Technological Research (Pai-
ACKNOWLEDGMENTS cyt-U.A.N.L) 2011-2012 CS 1078 and to the Ministry of
Thanks to the National Science and Technology Public Education (Secretaria de Educación Pública, SEP)
Committee (Consejo Nacional de Ciencia y Tecnologia, 11314 for their support.
Obtención del aceite esencial de Syzygium aroma- CMI. Posteriormente, se analizó las UFC, comparándo-
ticum, identificación del eugenol y su efecto sobre se con el Test CMR® Ivoclar Vivadent. La eficiencia en
Streptococus mutans. la obtención del aceite fue de 2.20%. Por la técnica de
Resumen: La caries dental es una enfermedad que CCD se identificó una fracción al revelado UV, corres-
afecta la cavidad oral en los humanos. Actualmente la pondiente al eugenol. Presentó respuesta positiva para
búsqueda de principios activos de plantas con efecto flavonoides y triterpenos. Mostró mayor actividad an-
antimicrobiano representa una promesa en la terapia timicrobiana a las concentraciones de 1000, 500 y 250
Odontológica. El presente trabajo, evaluó la actividad, μg/mL. La CMI y CMB del aceite, resultó a 125 y 250
del aceite esencial de Syzygium aromaticum (clavo) con μg/mL respectivamente. Se comprobó la presencia del
énfasis en su propiedad antimicrobiana. El aceite fue eugenol como principio activo en el aceite obtenido. Ac-
obtenido por hidrodestilación, caracterizado por croma- tualmente la proyección del uso de extractos de plantas
tografía en capa delgada y pruebas químicas. Se iden- ha favorecido la evaluación de agentes antibacterianos
tificó el compuesto principal en el aceite obtenido de alternos, eficaces y biocompatibles para su empleo en las
los botones florales y se evaluó su actividad antibacte- formulaciones de productos de higiene bucal aplicados
riana contra células plantónicas de Streptcoccus mutans a la prevención o tratamiento de enfermedades orales.
ATCC (700611) realizándose diluciones seriadas; desde Palabras clave: Eugenol, Streptococcus mutans, Syzygium
15 hasta 1000μg/mL, comparándose con clorhexidina aromaticum, antimicrobiano, concentración mínima inhi-
al 0.12% y dimetilsulfóxido, además se determinó la bitoria.
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Obtaining the essential oil of Syzygium aromaticum, identification of eugenol and
its effect on Streptococcus mutans.
J Oral Res 2014; 3(4): 218-224.
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224 ISSN Online 0719-2479 - ©2014 - Official publication of the Facultad de Odontología, Universidad de Concepción - www.joralres.com