Evidence-Based Complementary and Alternative Medicine

Volume 2012 (2012), Article ID 751435, 12 pages

doi:10.1155/2012/751435

Research Article

Antimicrobial Activity of Essential Oils against Streptococcus mutans and their Antiproliferative Effects

Lívia Câmara de Carvalho Galvão,¹ Vivian Fernandes Furletti,² Salete Meyre Fernandes Bersan,¹ Marcos Guilherme da Cunha,¹ Ana Lúcia Tasca Góis Ruiz,² João Ernesto de Carvalho,² Adilson Sartoratto,² Vera Lúcia Garcia Rehder,² Glyn Mara Figueira,² Marta Cristina Teixeira Duarte,² Masarahu Ikegaki,³ Severino Matias de Alencar,⁴ and Pedro Luiz Rosalen¹

¹Department of Pharmacology, Anesthesiology and Therapeutics, Piracicaba Dental School, University of Campinas (UNICAMP), 13414-903 Piracicaba, SP, Brazil

²Research Center for Chemistry, Biology and Agriculture, University of Campinas (UNICAMP), P.O. Box 6171, 13083-970 Campinas, SP, Brazil

³School of Pharmacy and Dentistry, Federal University of Alfenas, 37130-000 Alfenas, MG, Brazil

⁴Department of Agri-food Industry, Food and Nutrition, Escola Superior de Agricultura “Luiz de Queiroz” (ESALQ), University of São Paulo, P.O. Box 9, 13418-900 Piracicaba, SP, Brazil

Received 26 January 2012; Accepted 26 February 2012

Academic Editor: William C. S. Cho

Copyright © 2012 Lívia Câmara de Carvalho Galvão et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

This study aimed to evaluate the activity of essential oils (EOs) against Streptococcus mutans biofilm by chemically characterizing their fractions responsible for biological and antiproliferative activity. Twenty EO were obtained by hydrodistillation and submitted to the antimicrobial assay (minimum inhibitory (MIC) and bactericidal (MBC) concentrations) against S. mutans UA159. Thin-layer chromatography and gas chromatography/mass spectrometry were used for phytochemical analyses. EOs were selected according to predetermined criteria and fractionated using dry column; the resulting fractions were assessed by MIC and MBC, selected as active fractions, and evaluated against S. mutans biofilm. Biofilms formed were examined using scanning electron microscopy. Selected EOs and their selected active fractions were evaluated for their antiproliferative activity against keratinocytes and seven human tumor cell lines. MIC and MBC values obtained for EO and their active fractions showed strong antimicrobial activity. Chemical analyses mainly showed the presence of terpenes. The selected active fractions inhibited S. mutans biofilm formation () did not affect glycolytic pH drop and were inactive against keratinocytes, normal cell line. In conclusion, EO showed activity at low concentrations, and their selected active fractions were also effective against biofilm formed by S. mutans and human tumor cell lines.

1. Introduction

Despite the implementation of measures to control and treat dental caries with fluoride, they remain the most prevalent dental disease in many countries [1]. Caries are a multifactorial infectious disease caused by accumulation of biofilm on tooth surface [2]. Manifestations of the disease occur when there is an imbalance between the biofilm and the host due to changes in biofilm matrix pH caused by diet, microorganisms, or salivary flow and their components [3, 4].

Streptococcus mutans is considered the most cariogenic of all oral streptococci [5]. S. mutans is able to colonize the tooth surface and to produce large amounts of extra and intra-cellular polysaccharides. This microorganism is also highly acidogenic and aciduric, and it metabolizes several salivary glycoproteins, thus being responsible for the initial stage of oral biofilm formation and caries lesions [6].

Several products have been used to control dental caries, such as fluoride, chlorhexidine, and their associations [7]. However, natural products have contributed significantly to the discovery of chemical structures to create new medicaments to be used as innovative therapeutic agents against this prevalent disease [8, 9].

Essential oils (EOs) are important for their detected antimicrobial activity [10–12] including that against S. mutans [13]. They are complex, volatile, natural compounds formed by aromatic plants as secondary metabolites [14]. They are known for their bactericidal, virucidal, fungicidal, sedative, anti-inflammatory, analgesic, spasmolytic, and locally anesthetic properties [14]. The presence of complex chemical structures constituted of several groups, such as terpenes and terpenoids, aromatic and aliphatic constituents, all characterized by low molecular weight, may explain their successful bacteriostatic and bactericidal action [14].

Additionally, it was attested that the antimicrobial activity of a natural product, such as EO, is important to evaluate its effects on human normal cell lines and also against human tumor cell lines in order to evidence potential toxicity on human healthy and tumor cell lines [15]. For this reason, it is important that extensive studies involving EO as well as other sources of natural medicines are carried out.

The aim of this study was to evaluate the activity of EO and fractions against planktonic cells of S. mutans and also the selected active fractions of EO were chemically characterized and evaluated against mutans biofilm and antiproliferative activity on human cells.

2. Materials and Methods

2.1. Medicinal Plants

We studied 20 medicinal and aromatic plants (Table 1), which were obtained from the germoplasm bank of the Collection of Medicinal and Aromatic Plants (CPMA) of the Research Center for Chemistry, Biology and Agriculture (CPQBA), University of Campinas (UNICAMP), São Paulo, Brazil (http://www.cpqba.unicamp.br/), and identified by Glyn M. Figueira, curator of CPMA.

Table 1: Medicinal and aromatic plants from the germplasm bank of the CPMA/CPQBA/UNICAMP selected for this study with their yield, MIC and MBC values, and MBC : MIC ratio.

Medicinal species
Family
Popular name
Source
CPMA number
Voucher number¹
Yield (%)
MIC (μg/mL)
MBC (μg/mL)
MBC : MIC ratio²
Popular use

Aloysia gratissima (Gillies & Hook)
Verbenaceae
Brazilian lavender
Leaf
714
UEC 121.393
1.1
125–250
250–500
2 : 1
Digestive; antispasmodic

Aloysia triphylla (L’Hér.) Britton
Verbenaceae
Aloisia
Leaf
274/700
UEC 121.412
0.3
125–250
125–250
1 : 1
Sedative; antispasmodic

Alpinia speciosa (Pers.) Burtt & Smith
Zingiberaceae
Colony
Root
447
UEC 145.185
0.2
125–250
250–500
2 : 1
Antimicrobial

Baccharis dracunculifolia DC
Asteraceae
Broom weed
Leaf
1841
—
0.8
62.5–125
250–500
4 : 1
Tonic; eupeptic, antipyretic

Cinnamomum zeylanicum Blume
Lauraceae
Cinnamon
Leaf
455
IAC 19624
0.2
250–500
500–1000
2 : 1
Carminative; antispasmodic

Coriandrum sativum L.
Apiaceae
Coriander
Leaf
664
—
0.3
31.2–62.5
62.5–125
2 : 1
Antimicrobial; antifungal

Cymbopogon citratus (DC) Stapf.
Poaceae
Lemon grass
Leaf
503
UEC 85.210
1.1
125–250
250–500
2 : 1
Sedative; analgesic; anticough

Cymbopogon martini (Roxb.) J. F. Watson
Poaceae
Palmarosa
Leaf
354
UEC 127.115
0.6
125–250
250–500
2 : 1
Antiseptic; antifungal

Cymbopogon winterianus Jowitt
Poaceae
Lemon verbena
Leaf
712
UEC 121.414
1.5
125–250
250–500
2 : 1
Repellent, insecticide

Cyperus articulatus Vahl
Cyperaceae
Priprioca
Bulbs
222
UEC 121.396
0.5
125–250
250–500
2 : 1
Anti-inflammatory

Elyonurus muticus Spreng
Poaceae
Agripalma
Leaf
1701
UEC 20.580
0.6
125–250
125–250
1 : 1
Antibacterial

Eugenia florida DC.
Myrtaceae
Guamirim-cereja
Leaf
1685
IAC 49207
0.3
125–250
500–1000
4 : 1
Anti-inflammatory

Eugenia uniflora L.
Myrtaceae
Pitanga
Leaf
1816
—
0.7
125–250
250–500
2 : 1
Antihypertensive; diuretic

Lippia alba (Mill.) N.E. Brown
Verbenaceae
False lemon balm
Leaf
467/509
UEC 121.413
0.3
125–250
250–500
2 : 1
Treatment of migraines

Lippia sidoides Cham.
Verbenaceae
Rosemary
Leaf
398/399
—
4.7
62.5–125
125–250
2 : 1
Bactericide; fungicide

Mentha piperita L.
Lamiaceae
Mint
Leaf
560
UEC 127.110
2.2
250–500
250–500
1 : 1
Antifungal; antibacterial

Mikania glomerata Spreng.
Asteraceae
Guaco
Leaf
766
UEC 102.047
0.4
62.5–125
125–250
2 : 1
Anti-inflammatory; bronchodilator

Siparuna guianenses Aubl.
Monimiaceae
Wild lemon
Leaf
2025
—
0.3
62.5–125
125–250
2 : 1
Tranquilizer; diuretic

Syzygium aromaticum (L.) Merr. & L. M. Perry
Myrtaceae
Cloves
Leaf
455
IAC 19624
0.5
62.5–125
250–500
4 : 1
Seasoning; antibacterial

Ziziphus joazeiro Mart.
Rhamnaceae
Joazeiro fruit
Leaf
2119
—
0.5
250–500
500–1000
2 : 1
Astringent; anti-inflammatory

¹A voucher herbarium specimen is a pressed plant sample deposited for future reference. Vouchers deposited at UEC herbarium (http://www.ib.unicamp.br/herbario/) at Biology Institute (IB) of UNICAMP, SP, Brazil. (—) Species with no voucher number registered. 2 The EOs were considered bactericidal when the MBC : MIC ratio was between 1 : 1 to 2 : 1, and bacteriostatic if this ratio was higher than 2 : 1.

The plants were collected from November 2009 to January 2011, during the morning, after the dew point has been reached. The vouchers of each species were deposited in the herbarium of the Institute of Biology, at UNICAMP-UEC, and also registered in the herbarium of CPQBA, receiving identification numbers (CPMA number).

2.2. Essential Oil Extraction

EOs were obtained from 100 g of aerial fresh plant parts by hydrodistillation using a Clevenger-type system, for 3 hours. The aqueous phase was extracted with 50 mL of dichloromethane. Then, the organic layer was separated, dried over anhydrous sodium sulphate (Na₂SO₄), and filtered; the solvent was removed by vacuum evaporation at room temperature, resulting in EO. Oil samples were stored at −25°C in sealed glass vials [11].

2.3. Fractionation of Essential Oils

In order to select the EO that should be fractionated, we predetermined some criteria: best antimicrobial activity (MIC < 250 μg/mL), extract yield (>0.5%, except for Coriandrum sativum EO), commercial availability, presence of the EO in aerial parts of plants, and easy cultivation. The resulting fractions were also submitted to the antimicrobial assay.

Fractionation was performed using dry column chromatography (cellulose 2 cm × 20 cm) with Si gel 60 (Merck, Darmstadt, Germany) as the stationary phase and dichloromethane as the mobile phase, previously chosen by thin-layer chromatography (TLC), visualized under UV 254 nm, followed by anisaldehyde solution application and drying at 105°C for 5 min. After elution, columns were cut into different parts for each EO, according to polarity and extraction, using dichloromethane. The fractions so obtained were analyzed using TLC and gas chromatography coupled to mass spectrometry (GC-MS) and then bioguided using the antimicrobial assays [16]. All chemical wastes generated during this study were treated according to the Environmental Ethics Committee of UNICAMP (324/2009).

2.4. Analyses of the Selected Active Fractions using GC-MS

The chemical composition of each selected active fraction was evaluated using a Hewlett-Packard 6890 gas chromatograph equipped with an HP-5975 mass selective detector and HP-5 capillary column (30 m × 0.25 mm × 0.25 μm). GC-MS was performed using split injection with the injector set at 220°C, the column set at 60°C with a heating ramp of 3°C/min and a final temperature of 240°C, and the MS detector set at 250°C. Helium was used as a carrier gas at 1 mL/min. The GC-MS electron ionization system was set at 70 eV. The quantitative analyses were performed using a Hewlett-Packard 5890 gas chromatograph equipped with a flame ionization detector under the same conditions previously described. A sample of each EO or its selected active fraction was solubilized in ethyl acetate (15 mg/mL) for the analysis. Retention indices (RIs) were determined using injection of hydrocarbon standards and EO samples under the same conditions described above. The oil components were identified by comparison with data described in the literature and the profiles in the NIST 05 mass spectral library [11, 17].

2.5. Microorganisms

For the development of this study, Streptococcus mutans UA159 was used.

2.6. Antimicrobial Assay

We tested 20 EOs using the antimicrobial assay and selected them according to pre-determined criteria (item 2.3) before being fractionated and continuing the bioguided study.

MIC test was carried out using tissue culture microplates (96 wells) containing 100 μL/well BHI (Brain Heart Infusion, Difco, Franklin Lakes, NJ, USA) medium [18]. The stock solutions of EO and fractions from selected EO (item 2.3) were diluted with propylene glycol (4 mg/mL), transferred to the first well, and serial dilutions were performed to obtain concentrations ranging from 7.81 to 1000 μg/mL. We used 0.12% chlorhexidine (Sigma-Aldrich, St. Louis, MO, USA) as positive control and propylene glycol 6.25% as negative control. The bacterial inoculum (1 × 10⁶ UFC/mL) was added to all wells, and the plates were incubated at 37°C and 5% CO₂ for 24 hours. MIC was defined as the lowest concentration of EO or fraction from selected EO that inhibited microorganism visible growth indicated by resazurin 0.01% (Sigma-Aldrich, St. Louis, MO, USA) [19].

To determine MBC, an aliquot of each incubated well with concentrations higher than MIC was subcultured on BHI medium supplemented with 5% defibrinated sheep blood using a Whitley Automatic Spiral Plater (Don Whitley Scientific Limited, Shipley, West Yorkshire, UK). MBC was defined as the lowest concentration of EO or fraction that allowed no visible growth on the test medium.

To determine the nature of antibacterial effect of EO and fractions, the MBC : MIC ratio for bacteria was used [20]. When MBC : MIC ratio for S. mutans was between 1 : 1 and 2 : 1, the EO or fraction from selected EO was considered bactericidal against this microorganism [20], and when the ratio was higher than 2 : 1, it was considered bacteriostatic.

2.7. Action of Selected Active Fractions from Selected EO against S. mutans Biofilm

We tested 20 EOs, and those that fulfilled the pre-determined criteria (item 2.3) were selected to be chemically fractionated. The resulting fractions were also tested using the antimicrobial assay and selected according to MIC and MBC results and yields. The selected active fractions were then assessed regarding their action against S. mutans biofilm.

2.7.1. Inhibition of S. mutans Biofilm Growth

In order to evaluate the antimicrobial activity of EO selected active fractions against the formation of S. mutans biofilm, the samples were placed, at different concentrations (7.81–1000 μg/mL), in the wells of sterile polystyrene U-bottom microtiter plates, previously treated with saliva (the use of human saliva in this study was approved by the Research Ethics Committee of the Piracicaba Dental School, State University of Campinas (UNICAMP) (Approval 087/2011)) [21]. S. mutans cells (1.0 × 10⁷ cells/mL in BHI medium) were added to wells containing BHI medium with 2% sucrose and the samples were incubated at 37°C for 18 hours. Biofilm growth was revealed and quantified using the crystal violet staining method and measuring absorbance at 575 nm [11, 22].

After 18 hours of incubation, the spent medium was aspirated, nonadhered cells were removed, the wells were washed three times with sterile distilled water, and the plates were dried for 45 min before carrying out biofilm quantification [22].

2.7.2. Glycolytic pH-Drop Assay

The effect of EO selected active fractions against S. mutans biofilm was measured using the standard glycolytic pH-drop assay [23]. Biofilm growth was carried out as previously described (item 2.7.1), in sterile polystyrene U-bottom microtiter plates without fractions. The biofilms so obtained were washed twice with 0.9% NaCl solution and salt solution (50 mM KCl + 1.0 mM MgCl₂), containing EO selected active fractions at different concentrations (1000, 500, and 250 μg/mL), and vehicle (25% propylene glycol, v/v) was added. The pH was adjusted to 7.2 with 0.1 M KOH solution, and glucose was added to a final concentration of 1%, and pH-drop was assessed using Orion pH glass electrode attached to Orion 290 A⁺ pHmeter (Orion Scientific, Houston, TX, USA) for 90 min.

2.8. Scanning Electron Microscopy (SEM)

In order to evaluate S. mutans integrity using SEM, biofilms were first developed in Lab-Tek chambered coverglass (Nunc, Naperville, IL, USA), as described previously (item 2.7.1), were treated with vehicle (6.12% propylene glycol) or had their active fractions selected at concentrations able to inhibit more than 90% of S. mutans biofilm formation. Samples were fixed in 4% glutaraldehyde (v/v) in phosphate-buffered saline (PBS) at room temperature for 12–24 hours. After this procedure, the biofilms were dehydrated through a graded series of ethanol (50% to 100%), dried to a critical point, coated with gold, and observed using a scanning electron microscope JEOL JSM5600LV (JEOL Ltd., Tokyo, Japan) [11, 24].

2.9. Antiproliferative Assay

The in vitro antiproliferative assay [25] was performed in the present study using a human keratinocyte (HaCat) cell line, kindly donated by Dr. Ricardo Della Coletta (FOP, UNICAMP, Brazil), and seven human tumor cell lines (U251 (glioma), MCF-7 (breast), NCI-ADR/RES (ovarian expressing phenotype multiple drugs resistance), 786-0 (renal), NCI-H460 (lung, nonsmall cells), PC-3 (prostate), and OVCAR-03 (ovarian), kindly provided by M. A. Frederick (National Cancer Institute, USA). Stock and experimental cultures were grown in medium containing 5 mL RPMI-1640 (Gibco-BRL, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (Gibco-BRL, Grand Island, NY, USA). A penicilline-streptomicine mixture (1000 U/mL : 1000 mg/mL, 1 mL/L RPMI) was added to experimental cultures. Cells in 96-well plates (100 μL cells/well) were exposed to each EO and selected active fractions in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) (0.25, 2.5, 25, and 250 μg/mL) at 37°C and 5% CO₂ for 48 hours. Final DMSO concentration did not affect cell viability. Before (T₀ plate) and after sample addition (T₁ plates), cells were fixed with 50% trichloroacetic acid and cell proliferation was determined by spectrophotometric quantification (540 nm) of cellular protein content using sulforhodamine B assay. Using the concentration-response curve for each cell line, the total growth inhibition (TGI) was determined by nonlinear regression analysis using the software Origin 8.0 (OriginLab Corporation, Northampton, MA, USA) [26, 27].

2.10. Statistical Analysis

An exploratory data analysis was performed to determine the most appropriate statistical test. Inhibition of biofilm growth, and glycolytic pH-drop data were compared using the nonparametric Kruskal-Wallis test. P value < 0.05 was considered statistically significant. Triplicates from at least three separated experiments were conducted in each assay.

3. Results

3.1. Essential Oils and Fraction Yields

The EO yields, expressed in relation to dry weight of plant material (%, w/w), are shown in Table 1.

According to pre-determined criteria (item 2.3), four EOs were selected to be fractionated using dry column as follows: A. gratissima, B. dracunculifolia, C. sativum, and L. sidoides.

The yields of the fractions from selected EO were expressed as a function of the respective EO yield (%, w/w) and are shown in Table 2. The yields of A. gratissima fractions ranged from 14.4% to 29%, B. dracunculifolia from 20.1% to 30.6%, C. sativum from 4.9% to 30.9%, and L. sidoides from 1.7% to 33.3%.

Table 2: Selected EO and their fractions with yield results, MIC and MBC values, and MBC : MIC ratio.

Essential oil
Fraction

Identification
MIC (μg/mL)
MBC (μg/mL)
Identification
Yield (%)
MIC (μg/mL)
MBC (μg/mL)
MBC : MIC ratio¹

Aloysia gratissima (Ag)
125–250
250–500
Ag₁
28.9
250–500
500–1000
2 : 1

Ag₂
17.9
250–500
500–1000
2 : 1

Ag₃
20.1
62.5–125
500–1000
8 : 1

Ag₄ ²
14.4
31.2–62.5
62.5–125
2 : 1

Baccharis dracunculifolia (Bd)
62.5–125
250–500
Bd₁
30.5
250–500
500–1000
2 : 1

Bd₂
22.1
15.6–31.2
125–250
8 : 1

Coriandrum sativum (Cs)
31.2–62.5
62.5–125
Cs₁
6.6
125–250
500–1000
4 : 1

Cs₂
4.9
125–250
250–500
2 : 1

Cs₃
12.7
15.6–31.2
31.2–62.5
2 : 1

Cs₄
30.9
15.6–31.2
31.2–62.5
1 : 1

Lippia sidoides (Ls)
62.5–125
125–250
Ls₁
13.6
250–500
500–1000
2 : 1

Ls₂
33.3
62.5–125
250–500
4 : 1

Ls₃
26
62.5–125
125–250
2 : 1

Ls₄
6.1
62.5–125
125–250
2 : 1

Ls₅
1.7
62.5–125
125–250
2 : 1

1 The fractions from selected EO were considered bactericidal when the MBC : MIC ratio was between 1 : 1 to 2 : 1, and bacteriostatic if this ratio was higher than 2 : 1. 2 The fractions in bold font were selected as active fractions and evaluated against S. mutans biofilm and for their antiproliferative action. The subscript numbers of the fractions represent the numbers of parts obtained using the dry column fractionation.

3.2. Antimicrobial Activity

MIC and MBC values for all tested EO are shown in Table 1. MIC values ranged from 31.2 to 500 μg/mL, and MBC values ranged from 62.5 to 1000 μg/mL. The highest activities were observed for A. gratissima and A. triphylla (125–250 μg/mL), B. dracunculifolia, L. sidoides, M. glomerata, S. guianenses, S. aromaticum (62.5–125 μg/mL), and C. sativum (31.2–62.5 μg/mL).

Based on pre-determined criteria (item 2.3), four EOs (A. gratissima, B. dracunculifolia, C. sativum, and L. sidoides) were selected to be fractionated. MIC and MBC values of fractions from selected EO are shown in Table 2. MIC values obtained for all fractions ranged from 15.6 to 500 μg/mL, and MBC values ranged from 31.2 to 1000 μg/mL. The highest activities were observed for the fractions Ag₄ (31.2–62.5 μg/mL), Bd₂ (15.6–31.2 μg/mL), Cs4 (15.6–31.2 μg/mL), and Ls₃ (62.5–125 μg/mL).

The MBC : MIC ratio (Table 1) showed that most EOs are bactericidal, except for B. dracunculifolia, E. florida, and S. aromaticum, which are considered bacteriostatic against S. mutans. Among the selected EO chosen to be fractionated, only that obtained from B. dracunculifolia was bacteriostatic. Most fractions from selected EO were bactericidal, except for Ag₄, Cs₁, Ls₂, and Bd₂, considered bacteriostatic against S. mutans (Table 2). Based on yield and antimicrobial activity, Ag₄, Bd₂, Cs₄, and Ls₃ fractions were selected for further evaluations.

3.3. Selected Active Fractions Activity against S mutans Biofilm

Figure 1 shows the development of S. mutans biofilm inhibiton after treatment with selected active fractions. Their growth was measured by optic density at 575 nm. The result showed that the selected active fractions tested at different concentrations were significantly different () from the vehicle. Moreover, Cs₄ and Bd₂ fractions presented a better performance since they inhibited more than 90% of biofilm formation at lower concentrations (31.2 μg/mL).

Figure 1: Influence of selected active fractions Ag₄, Bd₂, Cs₄, and Ls₃ from selected essential oils at different concentrations against Streptococcus mutans biofilm formation. All fractions tested were significantly different from the vehicle at all concentrations tested. Kruskal-Wallis test ().

3.4. pH-Drop Assay

The influence of selected active fractions from EO on glycolytic pH-drop of S. mutans biofilm formation in the presence of excess glucose was not significant () for all fractions tested (Ag₄, Bd₂, Cs₄, and Ls₃).

3.5. Chemical Characterization of Fractions Constituents

The chemical composition of the selected EO and the selected active fractions is shown in Table 3.

Table 3: Major compounds of the selected active fractions from essential oils with their retention time (Rt), retention index (RI), and relative percentage.

Rt (min)
RI
Compound

Relative percentage¹

Ag EO
Ag₄
Bd EO
Bd₂
Cs EO
Cs₄
Ls EO
Ls₃

4.02
899
Cyclohexanone
—
—
—
—
—
—
6.5
—

4.22
850
3-hexen-1-ol
—
—
—
0.8
3.6
5.1
—
—

5.87
977
Beta-pinene
12.0
—
—
—
—
—
—
—

7.2
1024
p-cymene
—

—
—
—
—
17.3
—

13.08
1140
Trans-pinocarveol
—
4.9
—
—
—
—
—
—

14.09
1165
Trans-pinocamphone
16.0
36.7
—
—
—
—
—
—

14.61
1177
Cis-pinocamphone
6.0
17.0
—
—

—
—
—

16.7
1274
2-decen-1-ol <E>
—
—
—
—
23.6
26.9
—
—

16.86
1277
1-decanol
—
—
—
—
33.9
35.4
—
—

17.76
1299
Trans-pinocarvyl acetate
8.2
—
—
—
—
—
—
—

19.74
1300
Thymol
—
—
—
—
—

65.8
97.8

19.95
1303
Carvacrol
—
—
—
—
—
—
—
0.6

21.84
1349
Ethyl ester benzenepropanoic
—
—
—
11.7
—
—
—
—

22.57
1416
Trans-caryophyllene
7.2
—
10.7
—
—
—
10.5
—

24.86
1473
2-dodecen-1-ol
—
—
—
—
13.1
14.5
—
—

25.04
14.78
Germacrene D
—
—
4.9
—
—
—
—
—

25.66
1493
Bicyclogermacrene
4.2
—
6.8
—
—
—
—
—

27.97
1553
M² = 204
6.4
—
—
—
—

—
—

30.59
1566
Trans-nerolidol
—
—
31.7
52.2
—
—
—
—

31.05
1578
Spathulenol
—
—
13.6
11.5
—
—
—
—

31.23
1582
Caryophyllene oxide
6.4
7.0
—
6.3
—
—
—
0.7

31.9
1600
Guaiol
8.5
12.7
—
—
—
—
—
—

33.44
1641
Epi alpha cadinol
—
—
—
3.1
—
—
—
—

32.47
1674
2-tetradecen-1-ol <E>
—
—
—
—
5.5
5.2
—
—

34.40
1668
Bulnesol
—
3.5
—
—
—
—
—
—

1 The selected active fractions Ag₄, Bd₂, Cs₄, and Ls₃ had their actions against S. mutans biofilm and their antiproliferative activity evaluated. Ag EO, Bd EO, Cs EO, and Ls EO correspond to the following essential oils: Aloysia gratissima, Baccharis dracunculifolia, Coriandrum sativum, and Lippia sidoides, respectively. Only the compounds with relative percentage above 3% are listed. 2 M: molecular weight of a nonidentified compound.

The analyses of EO and fractions indicated the presence of volatile compounds, mainly mono- and sesquiterpenes.

We identified 28 compounds in the EO of A. gratissima, representing 92.73% of the EO, 25 compounds in the EO of B. dracunculifolia, representing 93.45% of the EO, 15 compounds in the EO of C. sativum, representing 91.93% of the EO, and four compounds in the EO of L. sidoides, representing 100% of the EO. We also identified 19 compounds in fraction Ag₄, representing 94.6% of the fraction, 10 compounds in fraction Bd₂, representing 83.06% of the fraction, nine compounds in fraction Cs₄, representing 89.71% of the fraction, and five compounds in fraction Ls₃, representing 99.7% of the fraction.

The major compounds identified in each selected EO were: trans- and cis-pinocamphone, beta-pinene, and guaiol in A. gratissima; trans-nerolidol and spathulenol in B. dracunculifolia; 2-decen-1-ol and 1-decanol in C. sativum; and thymol in L. sidoides. The major compounds identified in each selected fractions were trans- and cis-pinocamphone and guaiol in Ag₃; trans-nerolidol, spathulenol, and ethyl ester benzenepropanoic in Bd₂; 2-decen-1-ol and 1-decanol in Cs₄; thymol in Ls₃.

3.6. Scanning Electron Microscopy (SEM)

The effect of selected active fractions against S. mutans biofilm formation was evaluated by SEM. Figure 2 shows a reduction in biofilm formation. Biofilms were first developed as described previously (Section 2.7.1), were treated with vehicle, or had their active fractions selected at concentrations able to inhibit more than 90% of S. mutans biofilm formation (Ag₄ at 62.5 μg/mL, Bd₂ and Cs₄ at 31.2 μg/mL, and Ls₃ at 125 μg/mL).

(a)

(b)

(c)

(d)

(e)

Figure 2: Scanning electron microscopy of Streptococcus mutans biofilms treated with the selected active fractions from selected essential oils and the vehicle. Images a, b, c, and d show the reduction of biofilm formation after treatment with Ag₄, Bd₂, Cs₄, and Ls₃ fractions, respectively, compared with the treatment with the vehicle (image (e)) (magnification of 7000x).

3.7. Antiproliferative Assay

Most EOs and their selected active fractions did not present activity against the human normal cell line evaluated in this study or presented high concentrations to totally inhibit its growth. TGI values are shown in Table 4.

Table 4: Total growth inhibition (TGI) of selected essential oils and their selected active fractions tested against normal human cell and tumor cell lines.

Cell line

TGI (μg/mL)¹

Ag EO
Ag₄
Bd EO
Bd₂
Cs EO
Cs₄
Ls EO
Ls₃
Dox

Glioma (U251)
>250
55.6
38.2
51.4
8.3
61.5
>250
94.9
0.92

Breast (MCF-7)
>250
45.2
46.0
67.7
13.6
111.6
>250
56.6
3.3

Ovarian (NCI-ADR/RES)
>250
50.6
59.2
10.5
90.0
13.1
>250
112.3
1.6

Kidney (786-0)
>250
5.9
49.5
47.1
29.8
72.1
>250
26.7
>250

Lung (NCI-H460)
>250
42.7
87.6
76.8
105.0
110.3
>250
79.8
4.9

Prostate (PC-3)
99.9
>250
>250
>250
118.1
141.9
26.7
>250
11.7

Ovarian (OVCAR-3)
<0.25
47.6
<0.25
58.0
<0.25
73.7
<0.25
60.4
7.6

Keratinocytes (HaCaT)
>250
>250
92.3
95.7
129.4
145.6
>250
>250
2.3

1 Data result from three replicates per treatment in two independent tests at 25°C for 48 hours. Ag EO, Bd EO, Cs EO, and Ls EO correspond to the following essential oils: Aloysia gratissima, Baccharis dracunculifolia, Coriandrum sativum, and Lippia sidoides, respectively. Ag₄, Bd₂, Cs₄, and Ls₃ are the selected active fractions evaluated. Dox: doxorubicin (positive control).

Among the EO evaluated, B. dracunculifolia and C. sativum were the most active inhibitors of human tumor cell lines growth, presenting selectivity for U251 (TGI = 38.2 μg/mL and TGI = 8.3 μg/mL, resp.) and OVCAR-3 (TGI < 0.25 μg/mL for both). On the other hand, A. gratissima and L. sidoides displayed the lowest activity, both presenting selectivity for OVCAR-3 (TGI < 0.25 μg/mL for both) and L. sidoides for PC-3 (TGI = 26.7 μg/mL). The reference compound, doxorubicin, presented antiproliferative activity against all cell lines, except for kidney (Table 4).

Table 4 also shows the activity of selected active fractions. Ag₄ and Ls₃ fractions presented better results than A. gratissima and L. sidoides EO, respectively, since these fractions were not active against human normal cell lines (TGI > 250 μg/mL) and showed lower TGI values, being selective for 786-0 (TGI = 5.9 μg/mL and TGI = 26.7 μg/mL, resp.). Cs₄ fraction had better results than C. sativum EO only against NCI-ADR/RES (TGI = 13.1 μg/mL and TGI = 90 μg/mL, resp.). Bd₂ displayed a better performance than B. dracunculifolia EO against NCI-ADR/RES (TGI = 10.5 μg/mL and TGI = 59.2 μg/mL, resp.), 786-0 (TGI = 47.1 μg/mL and TGI = 49.5 μg/mL, resp.), and NCI-H460 (TGI = 76.8 μg/mL and TGI = 87.6 μg/mL, resp.).

4. Discussion

The activity of natural products, especially EO, against microorganisms has been recently confirmed by several studies focusing on antimicrobial activity of EO against planktonic cells. However, bacteria growing in biofilms exhibit a specific phenotype and are often, but not always, more resistant to antimicrobial agents than their planktonic counterparts [10, 11]. Thus, it is important to search for natural products that have antibiofilm properties and antimicrobial activity against oral pathogens [28].

This study aimed to evaluate the activity of EO and their fractions against planktonic cells of S. mutans, and the active fractions were evaluated against biofilm formed by S. mutans. Also, EO and their active fractions were chemically characterized and their activity against human normal and tumor cell lines proliferation were determined.

The antimicrobial assay revealed low MIC values for almost all 20 EOs and 15 fractions from the selected EO tested. EO and the selected active fractions presented strong activity against S. mutans, since natural products are considered strong inhibitors of microbial activity, when MIC values are lower than 500 μg/mL [29].

These results demonstrate that the EO studied and especially those selected (A. gratissima, B. dracunculifolia, C. sativum, and L. sidoides) have potential for bioprospection of new active biomolecules. The fractionation process adopted showed good results, since the fractions obtained were more active than the original EO (Table 2). This bioguided study is a model for bioprospecting new drugs [30], and it can be considered successful since we found active fractions presenting higher activity than their respective EO.

Most EO and fractions studied showed MBC : MIC ratio that enables them to be classified as bactericidal compounds. This could be explained by their hydrophobicity, an important characteristic that exists in EO and their fractions [31] and may allow them to partition the lipids of the bacterial cell membrane, turning them more permeable and leading to leakage of ions and other cell constituents [32, 33]. On the other hand, B. dracunculifolia EO and its selected active fraction (Bd₂) present compounds that could be capable of infiltrating the cell and interact with cellular metabolic mechanisms [34], demonstrating their bacteriostatic effect. Nevertheless, despite presenting bactericidal or bacteriostatic effect, the selected EO proved to be active against both S. mutans planktonic cells and biofilm, demonstrating the effectiveness of the substances present in these EO, since it is difficult to disrupt S. mutans biofilm [35].

The selected active fractions were also tested against S. mutans biofilm, and they were able to disrupt its formation at all tested concentrations. This disruption was observed using SEM, which showed the change the selected active fractions caused in the structure of S. mutans biofilm.

At the concentrations tested, it was possible to observe huge failures in S. mutans biofilm surface treated with the active fractions when compared with the treatment with the vehicle, which presented a more homogeneous biofilm surface. These changes were also observed in another study that tested the action of C. sativum and its bioactive fraction against Candida albicans [11]. Moreover, the simple conformational change in biofilm, caused by the action of the selected active fractions, could make it more susceptible and less virulent [4].

However, when the selected active fractions were tested in order to evaluate their ability to reduce S. mutans acid production, no significant results were observed (). Therefore, the selected active fractions could not act on this important virulence factor of S. mutans, different from the findings of another work with B. dracunculifolia extracts, which showed significant reduction in production of acid by this microorganism [36]. The difference between B. dracunculifolia EO and the active extracts from this plant may be attributed to the extraction method, which results in different compound mixtures with different mechanisms of action [37].

It is known that EOs are composed of numerous different chemical compounds, and their antimicrobial activity might be attributed to several different mechanisms, which could explain the variations in their mode of action [38].

The present data suggest the occurrence of a separation during the fractionation process of the selected EO in such a way that the selected active fractions presented higher amounts of bioactive compounds than their respective EO. The main biologically active compounds found in the selected active fractions were thymol, carvacrol, 2-decen-1-ol, trans-nerolidol, spathulenol, ethyl ester benzenepropanoic, trans-pinocamphone, cis-pinocamphone, and guaiol. These compounds have been extensively described in the literature for their effect on microorganisms [39, 40].

Both forms of trans- and cis-pinocamphone are major constituents of Ag₄ fraction and were also found in Hyssopus officinalis L. EO [41]. These compounds are responsible for the antibacterial, antifungal, and antioxidant activities of H. officinalis EO, demonstrating that they pass through the cell wall and the plasma membrane, disrupting their structure [41]. The bactericidal activity of Ag₄ fraction observed in the present study may be a consequence of this mode of action.

Trans-nerolidol and spathulenol, two compounds present in Bd₂ fraction, have been considered active against unknown Gram-positive and Gram-negative bacteria [13]. Although spathulenol shows activity against S. mutans, its mechanism of action still remains unknown [13].

Other studies showed that certain alcohols, such as 2-decen-1-ol, have higher antimicrobial activity than aldehydes against Candida ssp. [11, 16]. These alcohols were found in Cs₄ fraction and may be responsible for the action against S. mutans biofilm. Furthermore, considering the mode of action of C. sativum EO, it seems to result in bacterial cell permeabilization, leading to the impairment of other cell functions, such as membrane potential, respiratory activity, or efflux pump activity [42].

Thymol is an optic isomer of carvacrol, and both substances seem to make bacterial membrane more permeable [43]. In our study, both were found in Ls₃ fraction as its major components. Previous studies have shown that these compounds present antimicrobial activity against fungi and bacteria [44], including species of the genus Streptococcus [12].

After determining the antimicrobial activity of a natural product, it is important to verify if it also exhibits antiproliferative activity, mainly after its fractionation, a procedure that may concentrate toxic compounds in the fractions that present biological activity.

Based on TGI values, the selected EO and selected active fractions could be classified as inactive (TGI > 50 μg/mL), weakly active (15 μg/mL < TGI < 50 μg/mL), moderately active (6.25 μg/mL < TGI < 15 μg/mL), and strongly active (TGI < 6.25 μg/mL) [45]. The absence of activity was clearly observed in this study since all selected EO and selected active fractions were inactive against the human normal cell line tested.

All EOs tested were selective against the ovarian tumor cell line, showing potent activity. Ag₄ showed potent activity against the kidney tumor cell line, and Bd₂ and Cs₄ fractions showed only moderate activity against the ovarian tumor cell line. These results show the specificity of these EO and their fractions against some tumor cell lines, an important and desired characteristic for potential new chemotherapic drugs [15].

It is known that EO compounds, such as monoterpenes, have shown effects on mevalonate metabolism, linked to the maintenance of cell membrane, which could contribute to terpene tumor suppressive action [46]. Thereby, the presence of monoterpenes in the selected active fractions of our study may explain their antiproliferative actions against some tumor cell lines [47]; however, more studies are required to find the compounds of EO responsible for their anticancer activity, since little is known about essential oils and their antiproliferative activity.

5. Conclusion

The results of the present study indicate that all EO and fractions tested showed good antimicrobial activity, but only those showing activity at low concentrations were taken into consideration and fractionated for bioprospection of new agents against S. mutans. Among these fractions, the selected active fractions were able to disrupt S. mutans biofilm formation, did not inhibit normal cell line growth, and were more specific against human tumor cell lines. These features enable them to be tested in further studies and help the discovery of new bioactive molecules.

Acknowledgment

The authors thank São Paulo Research Foundation (FAPESP, no. 2009/12353-0 and no. 2011/14757-0) and the National Council for Scientific and Technological Development (CNPq, no. 308644/2011-5) for the financial support.

References

Brasil, Ministério da Saúde, Departamento de Atenção Básica, Coordenação Nacional de Saúde Bucal, Projeto SB Brasil 2010—Pesquisa Nacional de Saúde Bucal, Primeiros resultados, Brasília, Brazil, 2011

P. D. Marsh, “Are dental diseases examples of ecological catastrophes?” Microbiology, vol. 149, no. 2, pp. 279–294, 2003.

W. H. Bowen, S. M. Amsbaugh, S. Monell-Torrens, J. Brunelle, H. Kuzmiak-Jones, and M. F. Cole, “A method to assess cariogenic potential of foodstuffs,” The Journal of the American Dental Association, vol. 100, no. 5, pp. 677–681, 1980.

J. K. Kajfasz, I. Rivera-Ramos, J. Abranches et al., “Two Spx proteins modulate stress tolerance, survival, and virulence in Streptococcus mutans,” Journal of Bacteriology, vol. 192, no. 10, pp. 2546–2556, 2010.

D. Ajdić, W. M. McShan, R. E. McLaughlin et al., “Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 22, pp. 14434–14439, 2002.

T. M. S. Alves, C. A. Silva, N. B. Silva, E. B. Medeiros, and A. M. G. Valença, “Atividade antimicrobiana de produtos fluoretados sobre bactérias formadoras do biofilme dentario: estudo in vitro,” Pesquisa Brasileira em Odontopediatria e Clínica Integrada, vol. 10, no. 2, pp. 209–216, 2010.

J. D. Bader, D. A. Shugars, and A. J. Bonito, “Systematic reviews of selected dental caries diagnostic and management methods.,” Journal of Dental Education, vol. 65, no. 10, pp. 960–968, 2001.

J. Clardy and C. Walsh, “Lessons from natural molecules,” Nature, vol. 432, no. 7019, pp. 829–837, 2004.

J. G. Jeon, P. L. Rosalen, M. L. Falsetta, and H. Koo, “Natural products in caries research: current (limited) knowledge, challenges and future perspective,” Caries Research, vol. 45, no. 3, pp. 243–263, 2011.

M. Simões, “Antimicrobial strategies effective against infectious bacterial biofilms,” Current Medicinal Chemistry, vol. 18, no. 14, pp. 2129–2145, 2011.

V. F. Furletti, I. P. Teixeira, G. Obando-Pereda, et al., “Action of Coriandrum sativum L. essential oil upon oral Candida albicans biofilm formation,” Evidence-Based Complementary and Alternative Medicine, vol. 2011, Article ID 985832, 9 pages, 2011.

M. A. Botelho, N. A. P. Nogueira, G. M. Bastos et al., “Antimicrobial activity of the essential oil from Lippia sidoides, carvacrol and thymol against oral pathogens,” Brazilian Journal of Medical and Biological Research, vol. 40, no. 3, pp. 349–356, 2007.

F. Silva, “Efeito antimicrobiano in vitro dos compostos isolados da Mikania glomerada sobre os patógenos orais,” [Senior Research Project], Faculdade de Odontologia de Piracicaba, UNICAMP, Piracicaba, Brazil, 2005

F. Bakkali, S. Averbeck, D. Averbeck, and M. Idaomar, “Biological effects of essential oils—a review,” Food and Chemical Toxicology, vol. 46, no. 2, pp. 446–475, 2008.

J. E. Carvalho, “Atividade antiulcerogênica e anticâncer de produtos naturais e de síntese,” Construindo a História dos Produtos Naturais, vol. 7, pp. 1–18, 2006.

A. F. Begnami, M. C. T. Duarte, V. Furletti, and V. L. G. Rehder, “Antimicrobial potential of Coriandrum sativum L. against different Candida species in vitro,” Food Chemistry, vol. 118, no. 1, pp. 74–77, 2010.

R. P. Adams, Identification of Essential Oils Components by Gas Chromatography/Mass Spectrometry, Allured, Carol Stream, Ill, USA, 2007.

Clinical and Laboratory Standards Institute, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard, vol. 26, no. 2, CLSI document M07-A7, Fort Wayne, Ind, USA, 7th edition, 2006.

S. P. Soares, A. H. C. Vinholis, L. A. Casemiro, M. L. A. Silva, W. R. Cunha, and C. H. G. Martins, “Atividade antibacteriana do extrato hidroalcoólico bruto de Stryphnodendron adstringens sobre microorganismos da cárie dental,” Revista Odonto Ciência, vol. 23, no. 2, pp. 141–144, 2008.

Clinical and Laboratory Standards Institute, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, vol. 29, no. 2, CLSI document M07-A8, Wayne, Pa, USA, 8th edition, 2009.

H. Koo, M. F. Hayacibara, B. D. Schobel et al., “Inhibition of Streptococcus mutans biofilm accumulation and polysaccharide production by apigenin and tt-farnesol,” Journal of Antimicrobial Chemotherapy, vol. 52, no. 5, pp. 782–789, 2003.

R. O. Mattos-Graner, S. Jin, W. F. King, T. Chen, D. J. Smith, and M. J. Duncan, “Cloning of the Streptococcus mutans gene encoding glucan binding protein B and analysis of genetic diversity and protein production in clinical isolates,” Infection and Immunity, vol. 69, no. 11, pp. 6931–6941, 2001.

S. Duarte, P. L. Rosalen, M. F. Hayacibara et al., “The influence of a novel propolis on mutans streptococci biofilms and caries development in rats,” Archives of Oral Biology, vol. 51, no. 1, pp. 15–22, 2006.

S. P. Hawser and L. J. Douglas, “Biofilm formation by Candida species on the surface of catheter materials in vitro,” Infection and Immunity, vol. 62, no. 3, pp. 915–921, 1994.

A. Monks, D. Scudiero, P. Skehan et al., “Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines,” Journal of the National Cancer Institute, vol. 83, no. 11, pp. 757–766, 1991.

R. H. Shoemaker, “The NCI60 human tumour cell line anticancer drug screen,” Nature Reviews Cancer, vol. 6, no. 10, pp. 813–823, 2006.

C. Denny, M. E. Zacharias, A. L. T. G. Ruiz et al., “Antiproliferative properties of polyketides isolated from Virola sebifera leaves,” Phytotherapy Research, vol. 22, no. 1, pp. 127–130, 2008.

H. Koo, B. P. F. A. Gomes, P. L. Rosalen, G. M. B. Ambrosano, Y. K. Park, and J. A. Cury, “In vitro antimicrobial activity of propolis and Arnica montana against oral pathogens,” Archives of Oral Biology, vol. 45, no. 2, pp. 141–148, 2000.

M. C. T. Duarte, E. E. Leme, C. Delarmelina, A. A. Soares, G. M. Figueira, and A. Sartoratto, “Activity of essential oils from Brazilian medicinal plants on Escherichia coli,” Journal of Ethnopharmacology, vol. 111, no. 2, pp. 197–201, 2007.

L. M. C. Simões, L. E. Gregório, A. A. Silva Filho et al., “Effect of Brazilian green propolis on the production of reactive oxygen species by stimulated neutrophils,” Journal of Ethnopharmacology, vol. 94, no. 1, pp. 59–65, 2004.

S. Burt, “Essential oils: their antibacterial properties and potential applications in foods—a review,” International Journal of Food Microbiology, vol. 94, no. 3, pp. 223–253, 2004.

K. Knobloch, H. Weigand, N. Weis, H. M. Schwarm, and H. Vigenschow, “Action of terpenoids on energy metabolism,” in Proceedings of the Progress in essential oil research: 16th International Symposium on Essential, E. J. B. Oils, Ed., pp. 429–445, De Gruyter, 1986.

A. Ultee, M. H. J. Bennik, and R. Moezelaar, “The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus,” Applied and Environmental Microbiology, vol. 68, no. 4, pp. 1561–1568, 2002.

M. Marino, C. Bersani, and G. Comi, “Impedance measurements to study the antimicrobial activity of essential oils from Lamiaceae and Compositae,” International Journal of Food Microbiology, vol. 67, no. 3, pp. 187–195, 2001.

P. D. Marsh, “Dental plaque: biological significance of a biofilm and community life-style,” Journal of Clinical Periodontology, vol. 32, no. 6, pp. 7–15, 2005.

D. P. S. Leitão, A. A. Silva Filho, A. C. M. Polizello, J. K. Bastos, and A. C. C. Spadaro, “Comparative evaluation of in-vitro effects of Brazilian green propolis and Baccharis dracunculifolia extracts on cariogenic factors of Streptococcus mutans,” Biological and Pharmaceutical Bulletin, vol. 27, no. 11, pp. 1834–1839, 2004.

J. N. Eloff, “Which extractant should be used for the screening and isolation of antimicrobial components from plants?” Journal of Ethnopharmacology, vol. 60, no. 1, pp. 1–8, 1998.

S. Calsamiglia, M. Busquet, P. W. Cardozo, L. Castillejos, and A. Ferret, “Invited review: essential oils as modifiers of rumen microbial fermentation,” Journal of Dairy Science, vol. 90, no. 6, pp. 2580–2595, 2007.

I. H. N. Bassole, R. Nebie, A. Savadogo, C. T. Ouattara, N. Barro, and S. A. Traore, “Composition and antimicrobial activities of the leaf and flower essential oils of Lippia chevalieri and Ocimum canum from Burkina Faso,” African Journal of Biotechnology, vol. 4, no. 10, pp. 1156–1160, 2005.

N. A. Parreira, L. G. Magalhães, D. R. Morais et al., “Antiprotozoal, schistosomicidal, and antimicrobial activities of the essential oil from the leaves of baccharis dracunculifolia,” Chemistry and Biodiversity, vol. 7, no. 4, pp. 993–1001, 2010.

S. Kizil, N. Haşimi, V. Tolan, E. Kilinç, and H. Karataş, “Chemical composition, antimicrobial and antioxidant activities of hyssop (Hyssopus officinalis L.) essential oil,” Notulae Botanicae Horti Agrobotanici Cluj-Napoca, vol. 38, no. 3, pp. 99–103, 2010.

F. Silva, S. Ferreira, J. A. Queiroz, and F. C. Domingues, “Coriander (Coriandrum sativum L.) essential oil: its antibacterial activity and mode of action evaluated by flow cytometry,” Journal of Medical Microbiology, vol. 60, no. 10, pp. 1479–1486, 2011.

R. J. W. Lambert, P. N. Skandamis, P. J. Coote, and G. J. E. Nychas, “A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol,” Journal of Applied Microbiology, vol. 91, no. 3, pp. 453–462, 2001.

E. Lacoste, J. P. Chaumont, D. Mandin, M. M. Plumel, and F. J. Matos, “Antiseptic properties of the essential oil of Lippia sidoides Cham: application to the cutaneous microflora,” Annales Pharmaceutiques Francaises, vol. 54, no. 5, pp. 228–230, 1996.

G. Fouche, G. M. Cragg, P. Pillay, N. Kolesnikova, V. J. Maharaj, and J. Senabe, “In vitro anticancer screening of South African plants,” Journal of Ethnopharmacology, vol. 119, no. 3, pp. 455–461, 2008.

K. M. Swanson and R. J. Hohl, “Anti-cancer therapy: targeting the mevalonate pathway,” Current Cancer Drug Targets, vol. 6, no. 1, pp. 15–37, 2006.

A. E. Edris, “Pharmaceutical and therapeutic potentials of essential oils and their individual volatile constituents: a review,” Phytotherapy Research, vol. 21, no. 4, pp. 308–323, 2007.