http://informahealthcare.com/phbISSN 1388-0209 print/ISSN 1744-5116 online

Editor-in-Chief: John M. PezzutoPharm Biol, Early Online: 1–7

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.932391

ORIGINAL ARTICLE

The antibacterial and antifungal activity of essential oils extracted fromGuatemalan medicinal plants

Andrew B. Miller1, Rex G. Cates1, Michael Lawrence1, J. Alfonso Fuentes Soria2, Luis V. Espinoza3,Jose Vicente Martinez4, and Dany A. Arbizu5

1Department of Biology, College of Life Sciences, Brigham Young University (BYU), Provo, UT, USA, 2Secretarıa General del Consejo Superior

Universitario Centroamericano, Guatemala City, Guatemala, 3Benson Agriculture and Food Institute, Welfare Services, Salt Lake City, UT, USA,4Facultad de Agronomıa, Edificio T-8, Ciudad Universitaria, Guatemala City, Guatemala, and 5Benson Institute Guatemala, Chiquimula, Guatemala

Abstract

Context: Essential oils are prevalent in many medicinal plants used for oral hygiene andtreatment of diseases.Objective: Medicinal plant species were extracted to determine the essential oil content.Those producing sufficient oil were screened for activity against Staphylococcus aureus,Escherichia coli, Streptococcus mutans, Lactobacillus acidophilus, and Candida albicans.Materials and methods: Plant samples were collected, frozen, and essential oils were extractedby steam distillation. Minimum inhibitory concentrations (MIC) were determined using a tubedilution assay for those species yielding sufficient oil.Results: Fifty-nine of the 141 plant species produced sufficient oil for collection and 12 speciesnot previously reported to produce essential oils were identified. Essential oil extracts from32 species exhibited activity against one or more microbes. Oils from eight species were highlyinhibitory to S. mutans, four species were highly inhibitory to C. albicans, and 19 species yieldedMIC values less than the reference drugs.Discussion: Results suggest that 11 species were highly inhibitory to the microbes testedand merit further investigation. Oils from Cinnamomum zeylanicum Blume (Lauraceae),Citrus aurantiifolia (Christm.) Swingle (Rutaceae), Lippia graveolens Kunth (Verbenaceae), andOriganum vulgare L. (Lamiaceae) yielded highly significant or moderate activity against allmicrobes and have potential as antimicrobial agents.Conclusion: Teas prepared by decoction or infusion are known methods for extracting essentialoils. Oils from 11 species were highly active against the microbes tested and merit investigationas to their potential for addressing health-related issues and in oral hygiene.

Keywords

Antimicrobial activity, aromatic plants,Guatemala, MIC

History

Received 7 February 2014Revised 24 April 2014Accepted 4 June 2014Published online 21 October 2014

Introduction

Over 65% of the world population relies on traditional

medical approaches for treatment of diseases and oral hygiene

(Fabricant & Farnsworth, 2001). But in rural communities

such as those found in Guatemala, the estimate is that 75–90%

of the population rely on medicinal plants as their main source

of health care (Chivian & Bernstein, 2008; Fowler, 2006;

Goldman et al., 2002; Hautecoeur et al., 2007). Investigations

regarding the use of traditional medicines and the role of

natural products found in these plants to human health

continue to yield significant information and treatments

(Kingston, 2010) but one area that lags is the role of

medicinal plants in oral health (Colvard et al., 2006). This

appears to be the case even though traditional preparations are

known to extract natural products that confer significant

activities against microbes that are related to oral hygiene

(Cates et al., 2013; Jebashree et al., 2011).

With regard to the focus of this paper on essential oils,

traditional preparations like teas created by decoction or

infusion are common methods for extracting oils (Bilia

et al., 2000; Carnat et al., 1999; Radulescu et al., 2004).

Adams and Hawkins (2007) and Kufer et al. (2005) noted that

Guatemalan villagers use teas, bath with plant material boiled

in water, inhale steam, and use poultices as ways to prepare

medicinal plants for external and internal use. For example,

leaf and bark tissues from 64 of 81 medicinal plants (79%)

used in the community of San Andres are boiled or steeped

(Comerford, 1996). Furthermore, fragrant and aromatic

plants such as members of the Asteraceae, Lamiaceae,

Rutaceae, and Verbenaceae produce essential oils which

historically have been important in traditional medicines

(Edris, 2007). Plants containing essential oils have bioactiv-

ity against tick larvae, a host of bacteria, fungi, parasitic proto-

zoans, viruses, and cancer cell lines (Anthony et al., 2005;

Boyom et al., 2003; Burt, 2004; Edris, 2007; Kalemba &

Correspondence: Rex G. Cates, Department of Biology, College of LifeSciences, Brigham Young University, 401 WIDB, Provo, UT 84602,USA. Tel: +1 801 422 2582. Fax: +1 801 422 0090. E-mail:rex_cates@byu.edu

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Kunicka, 2003; Kim et al., 2008; Lahlou, 2004; Martinez-

Velazquez et al., 2011). In many cases, the mode of action is

known for various components of essential oils (Bakkali et al.,

2008). Assessing the effects of essential oil extracts from

Guatemalan medicinal plants on disease-causing microbes

would add information regarding their use and potential

as therapeutics (Adams & Hawkins, 2007; Booth et al., 1993;

Goldman et al., 2002; Hartecoeur et al., 2007; Kufer

et al., 2005).

Consequently, a study was undertaken to determine which

plant species produce essential oils from a total of 141 species

used by villagers. Oils from species that yielded a sufficient

quantity of oil were tested against Staphylococcus aureus,

Streptococcus mutans, Escherichia coli, Lactobacillus acid-

ophilus, and Candida albicans to determine minimum

inhibitory concentrations (MIC). Streptococcus mutans,

L. acidophilus, and C. albicans were included because

they also are associated with dental plaque, caries, and

other oral cavity issues (Kleinberg, 2002).

Materials and methods

Plant tissue collection

Medicinal plants were collected from 2006 to 2009 in the

villages of Tuticopote Abajo, Salitron, and Roblarcito of

the Torja River basin, in Olopa and San Juan Ermita San

Francisco Chanco of the Chanco River basin, and from the

Pinalito Association, Chiquimula Department (Ardon, 2008;

Galvez, 2008). Additional collections were made at the

Museo Odontologico de Guatemala y Jardın BotanicoMaya

and the Coleccion y Huerto Productivo de Plantas

Medicinales, Facultad de Agronomıa, Guatemala City,

Guatemala. Vouchers were deposited in the herbaria at the

CUNORI Campus, University of San Carlos, Chiquimula,

Guatemala, and at Brigham Young University (BYU), Provo,

UT, USA. About 300 g of plant tissue was bagged, labeled,

placed on dry ice, and stored in a �80�C ultralow at BYU.

Essential oil extraction and preparation

Essential oils were extracted by steam distillation (Scientific-

Glass, Rancho Santa Fe, CA) from 50 g fresh plant tissue

following Luque de Castro et al. (1999) and Charles and

Simon (1990). Oils were removed from the distillation

receiver by pipette after adding 125 ml of diethyl-ether

(Mallinckrodt-Baker, Phillipsburg, NJ). This mixture was

dehydrated using anhydrous sodium sulfate (EMD Chemicals,

Darmstadt, Germany). Oils were separated from the sodium

sulfate by adding 200 ml of diethyl-ether and then evaporating

the diethyl-ether under pressurized nitrogen (�35 s). Purified

essential oil was placed in an amber vial, weighed, and stored

at �80 �C until bioassayed.

Microbial strains

Essential oil extracts were bioassayed for activity against

E. coli (ATCC 11229; ATCC, Manassas, VA), S. aureus

(ATCC 6538P; Becton, Dickinson and Co. Laboratories,

Cockeysville, MD), S. mutans (ATCC 33402; ATCC),

L. acidophilus (ATCC 11975; ATCC), and C. albicans

(ATCC 90028; ATCC). Escherichia coli, S. aureus, and

S. mutans were cultured in tryptic soy broth (Becton,

Dickinson and Co., Cockeysville, MD), L. acidophilus in

MRS broth (Becton, Dickinson and Co., Cockeysville, MD),

and C. albicans in Sabouraud dextrose broth (Sigma-Aldrich,

St. Louis, MO). Streptococcus mutans and L. acidophilus

were incubated at 5% CO2 at 37 �C while E. coli, S. aureus,

and C. albicans were incubated at 37 �C.

Determination of MIC

MIC values were obtained using the tube dilution bio-

assay following Donaldson et al. (2005) and Eloff (1998).

To reduce essential oil volatility and increase solubility,

2% agar (w/v) was added to each broth. Essential oil

(20 ml) was serially diluted across five borosilicate test

tubes (13� 100 mm) resulting in final oil concentrations

of 5.00, 2.50, 1.25, 0.63, and 0.31ml/ml. Each test tube

was inoculated with 20 ml of microbial broth and controls

consisted of test tubes containing 20 ml of the microbial

broth without oil. All tubes were incubated as noted above

and control and experimental groups were replicated three

times.

After 24 h, 800ml of p-iodonitrotetrazolium chloride dye

solution (INT) (Sigma-Aldrich, Atlanta, GA) was added to

each tube. INT is a colorimetric indicator that changes

from clear to purple after exposure to CO2 indicating

bacterial respiration, metabolic activity, and growth (Mann

& Markham, 1998). Color changes were observed after

30 min and samples in tubes without color change were plated

to confirm growth inhibition. Samples of controls also were

plated to confirm growth. INT was not used for S. mutans and

L. acidophilus due to unreliable and indistinct color changes.

MICs for these microbes were determined by plating samples

from each tube.

MIC was defined as the lowest concentration of essential

oil that inhibited greater than 95% growth of the microorgan-

ism, and the MIC of 0.31 ml/ml with no variation among

replicates was considered as highly inhibitory. Two positive

control drugs were used to verify assay repeatability and

provide a comparison to the MIC values derived from the

essential oils (Hoffman et al., 1993; McCutcheon et al., 1994;

Ritch-Krc et al., 1996). Gentamycin (10 mg/ml; Sigma-

Aldrich, Atlanta, GA) was used against E. coli, S. aureus,

S. mutans, and L. acidophilus and nystatin (1 mg/ml in

DMSO; Sigma-Aldrich, Atlanta, GA) against C. albicans.

These drugs (20 ml) were administered and diluted following

the same procedure used for essential oils.

Results

Species yielding essential oils with activity

Of the 141 plant species screened 59 (42%) produced

sufficient essential oil for collection (Table 1). Forty-five

(76%) species yielded an average of 50.2 % (w/w).

Seven species yielded 0.2–0.4%, four yielded 40.4–0.6%,

two yielded 40.6–1.0%, and one species yielded over 1.0%

(Table 1). Twelve species not previously reported to produce

essential oils were identified (noted in Table 1). However,

Stigmaphyllon ellipticum A. Juss. (Malpighiaceae) and

Clematis dioica L. (Ranunculaceae) yielded small amounts

2 A. B. Miller et al. Pharm Biol, Early Online: 1–7

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suggesting that confirmation of essential oil production is

needed.

Forty-five (76%) of the 59 species produced sufficient

amounts of essential oil for testing against at least one

microbe (Table 2). Extracts from 32 (71%) of the 45 species

produced a MIC against one or more microbes and 13 species

were not active against any microbe. Thus, 22.7% of the 141

species collected showed activity against one or more

microbes. Of the 12 species identified for the first time as

producing essential oils, the oil from Cissus verticillata (L.)

Nicolson & C.E. Jarvis (Vitaceae) yielded a highly

significant MIC of 0.31 ml/ml against S. mutans (Table 2).

Table 1. Species, family, common name, tissue type, and mean oil yield per species for Guatemalan medicinal plants extracted by steam distillation.

Species Family Common name Tissue type Oil yield (�X % w/w)

Achillea millefolium L. Asteraceae Milenrama Aerial portion 0.12Anacardium occidentale L. Anacardiaceae Maranon Leaf 0.01Anethum graveolens L. Apiaceae Hinojo Aerial portion 0.14Anthemis oppositifolia Lam.a Asteraceae Ixmaramac Aerial portion 0.09Arnica montana L.a Asteraceae Arnica Aerial portion 0.07Baccharis latifolia (Ruiz & Pav.) Pers. Asteraceae Conrrobo negro Aerial portion 0.07Baccharis trinervis Pers. Asteraceae Corrimiento Aerial portion 0.11Bixa orellana L. Bixaceae Achiote Seed and pod 0.03Buddleja americana L.a Scrophulariaceae Salvia santa Leaf 0.20Buddleja davidii Franch. Scrophulariaceae Hoja de lanza Aerial portion 0.07Bursera simaruba (L.) Sarg. Burseraceae Palo de jiote Leaf 0.02Casimiroa edulis La Llave Rutaceae Matasano Leaf 0.02Cinnamomum zeylanicum Blume Lauraceae Canela Leaf 0.92Cissus verticillata (L.) Nicolson & C.E. Jarvisa Vitaceae Tabardillo Aerial portion 0.03Citrus aurantiifolia (Christm.) Swingle Rutaceae Limon criollo Leaf 0.40Citrus aurantium L. Rutaceae Naranja Leaf 0.06Citrus limetta Risso Rutaceae Lima Leaf 0.10Clematis dioica L.a Ranunculaceae Bejuco de cancer Aerial portion 50.01Cupressus lusitanica Mill. Cupressaceae Cipres Leaf 0.26Cymbopogon citratus (DC.) Stapf. Poaceae Te limon Leaf 0.03Elephantopus spicatus (Juss. ex Aubl.a Asteraceae Oreja de coche Aerial portion 0.03Eucalyptus globulus Labill. Myrtaceae Eucalipto Leaf 0.46Eupatorium semialatum Benth. Asteraceae Venadillo Aerial portion 0.04Fleischmannia pycnocephala (Less.) R.M. King & H. Robb Asteraceae Violeta Aerial portion 0.04Ilex aquifolium L. Aquifoliaceae Trueno Leaf 0.02Ixora coccineaa Rubiaceae Coralillo Leaf 0.03Lantana camara L. Verbenaceae Cinco negritos Aerial portion 0.05Lippia dulcis Trev. Verbenaceae Hierba dulce Aerial portion 0.09Lippia graveolens Kunth Verbenaceae Oregano Leaf 0.47Liquidambar styraciflua L. Hamamelidaceae Liquidambar Leaf 0.02Litsea guatemalensis Mez Lauraceae Laurel Leaf 0.20Mangifera indica L. Anacardiaceae Mango Leaf 0.02Mentha piperita L. Lamiaceae Menthol piperita Aerial portion 0.93Murraya paniculata (L.) Jack Rutaceae Limonaria Leaf 0.05Neurolaena lobata (L.) Cass. Asteraceae Tres puntas Leaf 0.05Ocimum basilicum L. Lamiaceae Albahaca Aerial portion 0.45Ocimum micranthum Willd. Lamiaceae Albahaca Aerial portion 0.15Origanum vulgare L. Lamiaceae Oregano de castillo Leaf 1.05Persea americana Mill. Lauraceae Aguacate Leaf 0.03Pimenta dioica (L.) Merr. Myrtaceae Pimienta Leaf 0.26Pinus oocarpa Schiede ex Schltdl.a Pinaceae Pino Leaf 0.09Piper auritum Kunth Piperaceae Santa Maria Leaf 0.33Pluchea odorata (L.) Cass. Asteraceae Siguapate Leaf 0.03Psidium guajava L. Myrtaceae Guayabo Leaf 0.13Rhus terebinthifolia Schltdl. & Cham.a Anacardiaceae Sal de vanado Leaf 0.03Rosmarinus officinalis L. Lamiaceae Romero Leaf 0.14Ruta chalepensis L. Rutaceae Ruda Aerial portion 0.05Spondias purpurea L. Anacardiaceae Jocote Leaf 0.01Stevia connata Lag. Asteraceae Guapillo Root 0.08Stigmaphyllon ellipticum A. Juss.a Malpighiaceae Contra hierba Leaf 50.01Tagetes erecta L. Asteraceae Flor de muerto Aerial portion 0.02Tagetes filifolia Lag. Asteraceae Anıs de monte Aerial portion 0.51Tagetes lucida Cav. Asteraceae Pericon Aerial portion 0.24Teloxys ambrosioides (L.) W.A. Weber Chenopodiaceae Apasote Aerial portion 0.04Thymus vulgaris L. Lamiaceae Tomillo Aerial portion 0.02Verbena litoralis Kuntha Verbenaceae Verbena Aerial portion 0.02Vernonia leiocarpa DC.a Asteraceae Suquenay Leaf 0.11Vetiveria zizanioides Nashb Poaceae Vetiver grass Root 0.03Zingiber officinale Roscoe Zingiberaceae Jengibre Rhizome 0.03

aSpecies not previously reported to have essential oil.bVillagers referred to F. pycnocephala as Violeta and V. zizanioides as Valeriana.

DOI: 10.3109/13880209.2014.932391 Antibacterial and antifungal activity of essential oils 3

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Also, Buddleja americana L. (Scrophulariaceae) and Pinus

oocarpa Schiede ex Schltdl. (Pinaceae) were moderately

inhibitory (0.42–0.83 ml/ml) to S. mutans. Fourteen species

were not tested because of insufficient oil to make serial

dilutions.

Bioassay

Seventeen bioassays (22% of all assays) from the extracts of

11 species produced a highly inhibitory MIC of 0.31 ml/ml

(Table 2). Twenty bioassays (26%) from 17 species displayed

moderately inhibitory MIC values (0.42–0.83ml/ml). Oils

from an additional 18 bioassays (22%) from 14 species

produced MIC values that were neither highly nor moderately

inhibitory but produced MIC values that were more inhibitory

than the reference drug (Table 2). Overall, 55 (71%) of the

recorded MIC values were equal to or lower than those of the

reference drugs.

Oils from 29 (91%) species tested against S. mutans

yielded MIC values and eight were highly inhibitory

(Table 2). Nineteen species yielded MIC values that were

less than the reference drug. Oils from 27 (60%) of the 45

species tested inhibited L. acidophilus but none of the MIC

values was highly inhibitory. Fourteen of the MICs were

less than the reference drug. Candida albicans was inhibited

by 56% of the oils tested and oils from four species were

Table 2. MIC (ml/ml)a and MIC range data (parentheses) for essential oil extracts from Guatemalan medicinal plants tested for activity againstmicrobial taxa.

MIC (range)

Species E. coli S. aureus S. mutans L. acidophilus C. albicans

Achillea millefolium –b – 1.46 (0.63–2.50) 3.75 (1.25–5.00) 2.50 (2.50)Anacardium occidentale –Anethum graveolens – – 2.50 (2.50) 5.00 (5.00) 0.63 (0.63)Arnica montana – – 2.71 (0.63–5.00) – –Baccharis latifolia – – 0.94 (0.31–1.25) –Baccharis trinervis – AOc

Bixa orellana – – 0.31 (0.31) – –Buddleja americana – – 0.63 (0.63) – –Cinnamomum zeylanicum 0.83 (0.6–1.25) 1.04 (0.63–1.25) 0.31 (0.31) 1.46 (0.63–2.50) 0.63 (0.31–1.25)Cissus verticillata – – 0.31 (0.31) – –Citrus aurantiifolia 2.50 (1.25–5.00) 0.42 (0.31–0.63) 0.63 (0.63) 1.25 (1.25) 0.31 (0.31)Citrus aurantium – 2.92 (1.25–5.00) 2.92 (1.25 –5.00) 0.42 (0.31 – 0.63)Citrus limetta – 3.33 (2.50 – 5.00) 1.67 (1.25 – 2.50) 4.16 (2.50 – 5.00) 0.42 (0.31–0.63)Cupressus lusitanica – – 0.42 (0.31–0.63) 1.25 (1.25) –Cymbopogon citratus AO 0.63 (0.31–1.25) AO AO AOEucalyptus globulus – 5.00 (5.00) 2.50 (2.50) 1.25 (1.25) 5.00 (5.00)Eupatorium semialatum – – AOFleischmannia pycnocephala – 0.31 (0.31) –Ilex aquifolium 0.42 (0.31–0.63)Lippia dulcis AO –Lippia graveolens 0.31 (0.31) 0.31 (0.31) 0.31 (0.31) 0.83 (0.63–1.25) 0.31 (0.31)Litsea guatemalensis – – 0.31 (0.31) 2.08 (1.25–2.50) –Mangifera indica –Mentha piperita 1.25 (1.25) 1.04 (0.63–1.25) 0.42 (0.31–0.63) 1.67 (1.25–2.50) 1.04 (0.63–1.25)Murraya paniculata – AONeurolaena lobata –Ocimum basilicum – – – – –Ocimum micranthum – – 0.42 (0.31–0.63) 2.92 (1.25–5.00)Origanum vulgare 0.31 (0.31) 0.31 (0.31) 0.31 (0.31) 1.04 (0.63–1.25) 0.31 (0.31)Persea americana – –Pimenta dioica 0.42 (0.31–0.63) 0.83 (0.63–1.25)Pinus oocarpa – – 0.52 (0.31–0.63) 1.04 (0.63–1.25) –Piper auritum – – 2.08 (1.25–2.50) 2.08 (1.25–2.50) 0.83 (0.63–1.25)Pluchea odorata – – 0.31 (0.31)Psidium guajava – – 0.42 (0.31–0.63) – –Rosmarinus officinalis – – 0.52 (0.31–0.63) 2.08 (1.25–2.50) –Ruta chalepensis – – 2.50 (2.50) 2.08 (1.25–2.50) 0.31 (0.31)Stevia connata –Tagetes erecta – AOTagetes filifolia 0.52 (0.31–0.63) – 1.04 (0.63–1.25) – 0.52 (0.31–0.63)Tagetes lucida 0.31 (0.31) 4.17 (2.50–5.00)Teloxys ambrosioides – – – – 0.63 (0.63)Verbena litoralis – –Vernonia leiocarpa –Vetiveria zizanioides 0.42 (0.31–0.63)Gentamycin 2.50 (2.50) 0.83 (0.63–1.25) 0.83 (1.25–2.50) 3.33 (2.50–5.00)Nyastatin 2.50 (2.50)

aBlank spaces indicate insufficient oil to carry out the bioassay.bIndicates oil not active at any concentration.cAO is activity observed but insufficient oil for three replicates. AO designation not used in the Results section or in calculations in Table 3.

4 A. B. Miller et al. Pharm Biol, Early Online: 1–7

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highly inhibitory. Oils from 12 species produced MIC values

that were less than the reference drug. Staphylococcus aureus

was inhibited by 28% of the oils tested, oils from two species

were highly inhibitory, and four MIC values were less

than the reference drug (Table 2). Escherichia coli was

inhibited by 21% of the species producing oils, three

were highly inhibitory, and six were less than the

reference drug.

Potential specificity was demonstrated by Bixa orellana L.

(Bixaceae), C. verticillata, Fleishmannia pycnocephala

(Less.) R. M. King & H. Rob (Asteraceae), and Pluchera

odorata (L.) Cass. (Asteraceae), all of which produced oils

highly active against S. mutans (Table 2). It was noteworthy

that Lippia graveolens Kunth (Verbenaceae) and Origanum

vulgare L. (Lamiaceae) were highly active against E. coli,

S. aureus, S. mutans, and C. albicans and showed moderate

activity against L. acidophilus (Table 2). Ocimum basilicum L.

(Lamiaceae) was not active against any of the five microbes.

Family distribution of species containing essential oils

Oils were collected from species known to 23 families and

nine families were represented by more than one species

(Table 1). Twenty-five percent of these species were from

the Asteraceae, 10% from the Lamiaceae, 10% from the

Rutaceae, and 7% from the Verbenaceae. Of the nine families

represented by more than one species six families had

multiple species producing MIC values, but only three

families had more than one species with a highly inhibitory

MIC value of 0.31 ml/ml (Table 3).

Discussion

Essential oil production

To our knowledge, 12 species have not been reported

previously as producing essential oils (Table 1). Of these,

the essential oil of C. verticillata was highly inhibitory to

S. mutans, and B. americana and P. oocarpa oils showed

moderate inhibition against S. mutans. Essential oils have

been reported from the fruits of Spondias purpurea L.

(Anacardiaceae) but have not been reported from leaf tissue

(Koziol & Macia, 1998). The activity of oils from

Anthemis oppositifolia Lam. (Asteraceae), Arnica montana

L. (Asteraceae), B. americana, C. verticillata, Cupressus

lusitanica Mill. (Cupressaceae), Ilex aquifolium L.

(Aquifoliaceae), Litsea guatemalensis Mez. (Lauraceae),

Piper auritum Kunth (Piperaceae), P. odorata, Tagetes

lucida Cav. (Asteraceae), Tagetes filifolia Lag., the seeds of

B. orellana, the leaves of C. aurantium L., Citrus aurantiifolia

(Christm.) Swingle (Rutaceae), C. limetta Risso (Rutaceae),

and the aerial portions of Anethum graveolens L. (Apiaceae)

is being reported for the first time.

Essential oil yields especially for Thymus vulgaris L.

(Lamiaceae) and to some extent for Rosmarinus officinalis L.

(Lamiaceae) were lower than expected (Table 1).

Alternatively, for plants for which there are comparable

data, most species in this study are in the range of published

essential oil yields (e.g., O. basillicum, O. vulgare) (Hussain

et al., 2008; Kokkini et al., 1997). Quantitative and qualitative

production of essential oils varies due to genetically based

chemotypes (Thompson et al., 2003), phenological stages

(Jordan et al., 2006), seasonal changes (Kokkini et al., 1997),

temperature (Hussain et al., 2008), and other environmental

factors (Burt, 2004). Thymus vulgaris and R. officinalis were

collected in the summer in pre-flowering condition which is a

season and phenological stage known for reduced oil

production (Hussain et al., 2008), but other factors could be

involved. This suggests that an investigation addressing the

essential oil yield with regard to genetic-based chemotypes,

phenology, and environmental factors of the 11 most active

species may be beneficial in locating plants high in oil

production.

Essential oil activity as determined by MIC

Essential oils from several species examined in this study and

also noted in other studies (Burt, 2004; Edris, 2007) merit

further investigation as to their antibacterial and antifungal

properties. Lippia graveolens and O. vulgare exhibited

remarkable activity against E. coli, S. mutans, S. aureus,

and C. albicans, and moderate activity against L. acidophilus.

Pozzatti et al. (2008) also reported inhibitory activity for oils

extracted from these two species against C. albicans. Similar

MIC values found in this study for the oil from L. graveolens

against E. coli, S. aureus, and C. albicans were reported by

Salgueiro et al. (2003), and these authors noted that the most

active compounds were thymol, carvacrol, and p-cymene.

Oils from B. orellana, Cinnamomum zeylanicum Blume

(Lauraceae), C. verticillata, C. aurantiifolia, F. pycnocephala,

L. graveolens, L. guatemalensis, O. vulgare, P. odorata,

Ruta chalepensis L. (Rutaceae), and T. lucida registered a

Table 3. Family distribution of Guatemalan medicinal plant speciescontaining essential oils, number and percent of species producinga MIC (ml/ml) against a microbe, and number of species with a MICof 0.31ml/ml.

FamilyNo. spp.

producing oil

No. (%) spp.yielding

MICa

No. (%) spp.with

MIC of 0.31a

Anacardiaceae 4Apiaceae 1 1 (100)Aquifoliaceae 1 1 (100)Asteraceae 15 7 (47) 3 (20)Bixaceae 1 1 (100) 1 (100)Burseraceae 1Chenopodiaceae 1 1 (100)Cupressaceae 1 1 (100)Hamamelidaceae 1Lamiaceae 6 4 (67) 1 (14)Lauraceae 3 2 (67) 2 (67)Malpighiaceae 1Myrtaceae 3 3 (100)Pinaceae 1 1 (100)Piperaceae 1 1 (100)Poaceae 2 2 (100)Ranunculaceae 1Rubiaceae 1Rutaceae 6 4 (67) 2 (33)Scrophulariaceae 2 1 (50)Verbenaceae 4 1 (20) 1 (20)Vitaceae 1 1 (100) 1 (100)Zingiberaceae 1

aBlank spaces indicate either no activity and/or insufficient oil to carryout a bioassay.

bData from Table 2 indicates that an MIC of 0.31 was recorded in allthree replicates.

DOI: 10.3109/13880209.2014.932391 Antibacterial and antifungal activity of essential oils 5

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highly inhibitory MIC of 0.31 ml/ml against one or more

microbes and merit further examination. Additionally, MIC

values in the range of 0.42 ml/ml and 0.52 ml/ml included

replicates that were highly inhibitory (defined as 0.31 ml/ml).

Oils from 16 species were active at this level against at least

one microbe except for L. acidophilus (Table 2) suggesting

that these species may merit further study. Lippia graveolens

and Pimenta dioica (L.) Merr. (Myrtaceae) were moderately

active (MIC¼ 0.83 ml/ml) against L. acidophilus. On occa-

sion, an essential oil extract with a MIC value of 0.42 ml/ml

was microbe specific such as I. aquifolium, Psidium guajava

L. (Myrtaceae), and Vetiveria zizanioides Nash (Poaceae)

against S. mutans. Jardim et al. (2008) found that an essential

oil extract from Teloxys ambrosioides (L.) W. A. Weber

(Chenopodiaceae) demonstrated a high level of inhibition

against a number of fungi; and in our study, this species

was moderately active against C. albicans. Also supporting

our results were the findings that the essential oil extracts

from the leaves of T. ambrosioides and Eucalyptus globulus

Labill. (Myrtaceae) were not active against E. coli or

S. aureus (Mulyaningsih et al., 2011; Owolabi et al., 2009).

Finally, several species in our study also were noted by

Burt (2004) as producing essential oils that were highly active

against various pathogens. To that list of species, we add

the significant MIC values of essential oils from R. officinalis,

O. vulgare, C. citratus, and L. graveolens against S. mutans,

and R. officinalis and L. graveolens against C. albicans.

With regard to essential oils with potential for treating

oral diseases, eight species were found to be highly active

against S. mutans and four were highly active against

C. albicans (Table 2). Essential oils from an additional

six species were moderately active against C. albicans.

Lippia graveolens and P. dioica yielded some activity against

L. acidophilus (MIC of 0.83 ml/ml). MIC values from the

oils of C. zeylanicum, C. aurantiifolia, L. graveolens, and

O. vulgare showed either highly significant or moderate

activity against all three microbes and merit further investi-

gation as potential oils for treating microbial diseases of the

oral cavity.

Family distribution of active species based onMIC values

Essential oils have been reported from various species of each

family tested in this study (Bakkali et al., 2008; Lahlou,

2004). In our study, essential oils from 11 species in seven

families were highly active against one or more microbes

(Tables 2 and 3). In addition, for two of these species

(C. zeylanicum and L. guatemalensis), methanol and acetone

extracts were active against S. mutans and breast cancer cells,

respectively (Cates et al., 2013). The high activity of oils

from some species in the Asteraceae and Rutaceae is also

notable. Oils from species of the same family are known

to produce some of the same compounds which may

increase their likelihood of inhibiting particular microbes

(Edris, 2007).

Conclusion

Species in the Asteraceae, Lamiaceae, Rutaceae, and

Verbenaceae and other families are used as traditional

medicines in Guatemala and this study identified 12 species

not previously reported to produce essential oils. Additionally,

oils from B. orellana, C. zeylanicum, C. verticillata,

C. aurantiifolia, F. pycnocephala, L. graveolens, L. guatema-

lensis, O. vulgare, P. odorata, R. chalepensis, and T. lucida

yielded highly inhibitory MICs of 0.31 ml/ml against the

microbes tested. While in vitro studies indicate the potential

of these oils in treating diseases, in vivo investigations

are needed to determine the potential of these oils or their

components to treat oral, gastric, dermal, and fungal infec-

tions and to determine their level of cytotoxicity.

Acknowledgements

Dr. Allen C. Christensen of the Benson Agriculture and

Food Institute and Wade J. Sperry and Ferren Squires from

LDS Church Welfare Services provided support for this

project. We are indebted to Cleria A. Espinoza for her

translation of documents and tireless devotion to this project.

We thank Dr. Ivan G. Rodriguez, Director and Administrator

of the Museo Odontologico de Guatemala y Jardın Botanico

Maya, for his collaboration in this project and devotion

to improving the oral hygiene of Guatemalans. David E.

Mendieta, Juan Castillo, Jorge Vargas, Dr. Armando Caceres,

Mario Veliz, Mervin E. Perez (all from USAC), and

Marco Estrada Muy (CSUCA) were instrumental in plant

identification. We thank villagers who patiently helped us

understand their needs.

Declaration of interest

The authors report no declaration of interest. The authors

thank M.Sc. Arg. Sergio Enrique Veliz Rizzo, Secretario

Ejecutivo, Consejo Nacional De Areas Protegidas for granting

us permit number SEVR/JCCC/spml Exp. 6647. Financial

and logistical supports were provided by the Benson

Agriculture and Food Institute, SANT Foundation, and

the Professional Development Fund, Department of

Biology, BYU.

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