The antibacterial and antifungal activity of essential oils extracted from Guatemalan medicinal plants

Abstract

Context: Essential oils are prevalent in many medicinal plants used for oral hygiene and treatment 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 extracted by steam distillation. Minimum inhibitory concentrations (MIC) were determined using a tube dilution assay for those species yielding sufficient oil.

Results: Fifty-nine of the 141 plant species produced sufficient oil for collection and 12 species not previously reported to produce essential oils were identified. Essential oil extracts from 32 species exhibited activity against one or more microbes. Oils from eight species were highly inhibitory to S. mutans, four species were highly inhibitory to C. albicans, and 19 species yielded MIC values less than the reference drugs.

Discussion: Results suggest that 11 species were highly inhibitory to the microbes tested and merit further investigation. Oils from Cinnamomum zeylanicum Blume (Lauraceae), Citrus aurantiifolia (Christm.) Swingle (Rutaceae), Lippia graveolens Kunth (Verbenaceae), and Origanum vulgare L. (Lamiaceae) yielded highly significant or moderate activity against all microbes and have potential as antimicrobial agents.

Conclusion: Teas prepared by decoction or infusion are known methods for extracting essential oils. Oils from 11 species were highly active against the microbes tested and merit investigation as to their potential for addressing health-related issues and in oral hygiene.

• Antimicrobial activity
• aromatic plants
• Guatemala
• MIC

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 bioactivity against tick larvae, a host of bacteria, fungi, parasitic protozoans, viruses, and cancer cell lines (Anthony et al., 2005; Boyom et al., 2003; Burt, 2004; Edris, 2007; Kalemba & 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 acidophilus, 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, Salitrón, and Roblarcito of the Torjá River basin, in Olopa and San Juan Ermita San Francisco Chancó of the Chancó River basin, and from the Pinalito Association, Chiquimula Department (Ardón, 2008; Gálvez, 2008). Additional collections were made at the Museo Odontológico de Guatemala y Jardín BotánicoMaya and the Colección y Huerto Productivo de Plantas Medicinales, Facultad de Agronomía, 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 μl 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 μl 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 bioassay 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 μl) 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 0.31 μl/ml. Each test tube was inoculated with 20 μl of microbial broth and controls consisted of test tubes containing 20 μl 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, 800 μl 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 microorganism, and the MIC of 0.31 μl/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 μl) 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 <0.2 % (w/w). Seven species yielded 0.2–0.4%, four yielded >0.4–0.6%, two yielded >0.6–1.0%, 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 suggesting that confirmation of essential oil production is needed.

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

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 μl/ml against S. mutans (Table 2). Also, Buddleja americana L. (Scrophulariaceae) and Pinus oocarpa Schiede ex Schltdl. (Pinaceae) were moderately inhibitory (0.42–0.83 μl/ml) to S. mutans. Fourteen species were not tested because of insufficient oil to make serial dilutions.

Table 2. MIC (µl/ml)^a and MIC range data (parentheses) for essential oil extracts from Guatemalan medicinal plants tested for activity against microbial 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		–	AO^c
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	AO
Eucalyptus globulus	–	5.00 (5.00)	2.50 (2.50)	1.25 (1.25)	5.00 (5.00)
Eupatorium semialatum	–	–	AO
Fleischmannia 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		–	AO
Neurolaena 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		–	AO
Tagetes 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.

Bioassay

Seventeen bioassays (22% of all assays) from the extracts of 11 species produced a highly inhibitory MIC of 0.31 μl/ml (Table 2). Twenty bioassays (26%) from 17 species displayed moderately inhibitory MIC values (0.42–0.83 μl/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 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 μl/ml (Table 3).

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

Family	No. spp. producing oil	No. (%) spp. yielding MIC^a	No. (%) spp. with MIC of 0.31^a
Anacardiaceae	4
Apiaceae	1	1 (100)
Aquifoliaceae	1	1 (100)
Asteraceae	15	7 (47)	3 (20)
Bixaceae	1	1 (100)	1 (100)
Burseraceae	1
Chenopodiaceae	1	1 (100)
Cupressaceae	1	1 (100)
Hamamelidaceae	1
Lamiaceae	6	4 (67)	1 (14)
Lauraceae	3	2 (67)	2 (67)
Malpighiaceae	1
Myrtaceae	3	3 (100)
Pinaceae	1	1 (100)
Piperaceae	1	1 (100)
Poaceae	2	2 (100)
Ranunculaceae	1
Rubiaceae	1
Rutaceae	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 carry out a bioassay.

^bData from Table 2 indicates that an MIC of 0.31 was recorded in all three replicates.

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 highly inhibitory MIC of 0.31 μl/ml against one or more microbes and merit further examination. Additionally, MIC values in the range of 0.42 μl/ml and 0.52 μl/ml included replicates that were highly inhibitory (defined as 0.31 μl/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 (MIC = 0.83 μl/ml) against L. acidophilus. On occasion, an essential oil extract with a MIC value of 0.42 μl/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 μl/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 investigation as potential oils for treating microbial diseases of the oral cavity.

Family distribution of active species based on MIC 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. guatemalensis, O. vulgare, P. odorata, R. chalepensis, and T. lucida yielded highly inhibitory MICs of 0.31 μl/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 infections 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. Iván G. Rodriguez, Director and Administrator of the Museo Odontológico de Guatemala y Jardín Botánico 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 Cáceres, Mario Véliz, Mervin E. Pérez (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 Véliz 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.