Evaluation of essential oils from 22 Guatemalan medicinal plants for in vitro activity against cancer and established cell lines

1 College of Life Sciences, Brigham Young University (BYU), Provo, UT USA. 2 Secretaria General del Consejo, Superior Universitario de Centroamerica (CSUCA), Ave. Las Americas 1-03, Zona 14, Interior Club Los Arcos, Guatemala City, Guatermala. 3 Benson Agriculture and Food Institute, Welfare Services, Salt Lake City, Utah (UT), USA. 4 Facultad de Agronomía, Edificio T-8, Ciudad Universitaria, Zona No.12, Guatemala City, Guatemala. 5 Benson Institute Guatemala, Chiquimula, Guatemala.


INTRODUCTION
The use of medicinal plants is important to the health care of Guatemalans (Hautecoceur et al., 2007;Cates et al., 2013) and there is a need to determine the efficacy of these plants against human diseases (Comerford, 1996;Kufer et al., 2005;Adams and Hawkins, 2007).For example, essential oils from 11 species collected in Guatemala yielded high levels of inhibition and low MIC values against pathogenic microbes (Miller et al., 2015).
In the study reported here, the activity of essential oils from 22 species against tongue, skin, and stomach cancer cell lines and a non-cancerous Vero line is reported.A neutral red assay was used to determine the cytotoxicity of each oil using IC 50 (half the maximal inhibitory concentration) and CC 50 (cytotoxic concentration to cause death of 50% of viable cells) values, a therapeutic index was then calculated for each oil, and the degree of inhibition of cancer cell proliferation using the [ 3 H]-thymidine incorporation assay was determined.
Essential oils are complex mixtures of monoterpenes, sesquiterpenes and phenolics (Carson and Riley, 1995;Radulescu et al., 2004), and are known to have biological activity against cancer cell lines (Edris, 2007;Bhalla et al., 2013;Gautam et al., 2014;Raut and Karauppayil, 2014).Oil activity has been shown to be a sum of the effects of individual components based on the ratio of the different constituents and not necessarily on the quantity of one component (Kalemba and Kunicka, 2003;Houghton et al., 2007).
This variation indicates strong potential for synergistic interactions among oil components (Biavatti, 2009;Rosso et al., 2015) and specific mechanisms of action toward a particular cancer cell line or disease organism (Wittstock and Gershenzon, 2002;Rajesh and Howard, 2003;Savelev et al., 2003;Salminen et al., 2008).These characteristics suggest that the development of essential oils might be useful in anticancer treatments and cancer therapies (Patel and Gogna, 2015).

Plant selection and tissue collection
Plants species were collected in Guatemala from 2007 to 2009 in the villages of Tuticopote, Salitrón, Roblarcito, Olopa, and San Juan Ermita in the Chiquimula Department.Additional collections were made at the Museo Odontológico de Guatemala and the Jardín Botánico Maya, Guatemala City and the Coleccion y Huerto Productivo de Plantas Medicinales, Centro Experimental y Docente de Agronomia, Guatemala City.Vouchers with detailed collection information were deposited in the Herbaria at the CUNORI Campus, University of San Carlos, Chiquimula, Guatemala and at Brigham Young University (BRY) herbarium, Provo, UT USA.
For each sample about 300 g of plant tissue was bagged, labelled, placed on dry ice, and stored in a -80°C ultralow Miller et al. 43 (Isotemp Basic, Thermo Electron Corporation, Asheville, NC USA) at BYU.

Essential oil extraction and preparation
Essential oils were extracted using a steam distillation apparatus (Scientific-Glass, Rancho Santa Fe, CA, USA) following Luque de Castro (1999) and Charles and Simon (1990).In order to obtain sufficient oil for the assays, multiple samples of each species were extracted for approximately 4 h followed by oil removal from the receiver of the apparatus by pipette.
To aid in the separation of oils from the water and glass surfaces, 125 μL of diethyl-ether (Mallinckrodt-Baker, Phillipsburg, NJ, USA) was added to the receiver.The oil/diethyl-ether mixture was removed, placed in vials and dehydrated using anhydrous sodium sulfate (EMD Chemicals, Darmstadt, Germany).Oils were separated from sodium sulfate by adding an additional 200 μL of diethyl-ether, and then the diethyl-ether was evaporated from the oil/diethyl-ether mixture using pressurized nitrogen (approximately 35 s).The resulting purified essential oil was then placed in an amber vial, weighed, and stored at -80°C until analyzed (Miller et al., 2015).Oil yields from multiple extractions of tissue from each plant species were averaged and expressed as % yield (w/w) (Table 1).

Method adaptation
Assessment of the bioactivity of essential oils can be problematic due to the highly volatile nature of the oils and their lack of solubility (Donaldson et al., 2005).Volatile components were found to crosscontaminate adjacent wells of 96-well plates even at low concentrations, thereby leading to inaccurate estimations of minimum inhibitory concentration (MIC) and IC50 values.Donaldson et al. (2005) proposed the addition of 2% biological grade agar (w/v) to the culture media to remedy this problem in microbial tube dilution assays.In order to adapt this method to allow the use of 96-well plates, 15% biological grade agar (v/v) was added to the cell culture media.The addition of 15% agar (v/v) mixed with cell culture media consistently showed no inhibitory *Corresponding author.E-mail: andrewmiller35@gmail.com.Tel: (801) 422-2582.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License effects on the growth of untreated cells in preliminary trials.The resulting mixture of inert agar also maintained a stable emulsion over a 24 h period and minimized oil volatility.

Cell culture techniques
DMEM agar-media was prepared by adding melted molecular biology grade agar (Fisher, Fair Lawn, NJ, USA) to media at 15% v/v ratio at room temperature and then allowing the mixture to cool.FBS (10%) was then added followed by 5 mL of 1M HEPES, 2.5 mL of 100 mM sodium pyruvate, and 5 mL of 10 mg/mL gentamycin.Ham's F-12 Kaighn's Modification agar-media was prepared in the same manner with the omission of sodium pyruvate.All cell lines were grown to 90% confluency in 175cm 2 flasks (Sarstedt) at 37°C and 5% CO2 and then seeded in 96-well plates.Stomach cells were seeded at a density of 7.0 x 10 4 , skin cells at 6.0 x 10 4 , tongue cells at 5.0 x 10 4 , and Vero C 1008 cells at 2.0 x 10 4 .Each well was filled with 150 μL of complete media and then placed in an incubator at 37°C and 5% CO2.
Seeded plates were removed from the incubator after 24 h and the media was also removed.Two essential oils and their controls were analyzed on each plate and each concentration was replicated three times.Essential oils were serially diluted in agar media resulting in final concentrations of 7.0, 3.5, 1.75, 0.88, 0.44, 0.22, 0.11 and 0.05 μL/mL.200 μL of the appropriate essential oil concentration was then added to each well.Controls consisted of 200 μL of agar media in wells with no additives.All edge wells were filled with 200 μL of sterilized double distilled water (DDH2O) to control edge effects.Each plate was returned to the incubator for an additional 24 h

Neutral red assay to determine IC50 and CC50 values
The neutral red (NR) assay was chosen for determining IC50 and CC50 because of its accuracy in the quantitative assessment of in vitro cytotoxicity (Borenfreund and Puerner, 1985;Babich and Borenfreund, 1991;Schröterová et al., 2009).Plates were removed from the incubator after 24 h and phosphate buffered saline was used to gently wash and remove all traces of the essential oil and the agar-media from the wells.NR dye solution was made using 0.33 mg/mL NR solution (3-aminom-dimethylamino-2-methylphenazine hydrochloride in DBPS; Sigma-Aldrich) and then added to make a 10% NR media mixture.
This solution was added to each well excluding edge wells which were filled with sterilized DDH2O.Plates were then incubated for 3 h after which the NR media mixture was removed and discarded.A fixative solution (1% CaCl2 in 0.5% formaldehyde; Mallinckrodt, Phillipsburg, NJ, USA) was added, removed after 30 s of exposure, and then a solubilization solution was added (1% acetic acid; EM Science, Gibbstown, NJ, USA, in 50% ethanol; Decon Labs, King of Prussia, PA, USA).Cell viability was measured using a Fusion α-HT Universal Microplate Analyzer (Packard Instruments, Meriden, CT, USA) with 540 nm filter and 690 nm reference filter.
Final values were generated by subtracting the 690 value from the 540 value followed by correction of the data by subtracting the average value generated from the blank edge wells.The values of the three replicate trials were first averaged and then used to create a dose-response curve from which final IC50 and CC50 values were determined.A Therapeutic Index was calculated using the ratio CC50/ IC50 (Greer et al., 2010).

Determination of cell proliferation
Oils form this assay were selected based on the lowest IC50 values within a cancer cell line and the consideration of the IC50 values among cancer cell lines within a plant species (Table 4).The [ 3 H]thymidine incorporation assay is a measure of cell proliferation levels based on the synthesis of new DNA (Sugihara et al., 1992;Marimpietri et al., 2007;Zhang et al., 2008), and consequently, a high value which indicates the percent decrease in cell proliferation (Table 4).
Cancer cells were seeded into 96-well plates at previously noted densities, incubated (37°C, 5% CO2) for 4 h, and then oils were added at a concentration that yielded the IC50 value for that oil.This mixture was then incubated for 24 h.The oil/media mixture was removed and the cells were washed once with fresh media followed by the addition of 200 ml of fresh media with thymidine (Amersham, Piscataway, NJ, USA).
0.1 μL thymidine (185 GBq/mmol) was used for skin and tongue lines and 0.15 μL for stomach line.Plates were incubated for 4 h and then harvested using a multi-well harvester (Inotech Biosystems International, Rockville, MD, USA) with collection onto glass fiber filters.Each filter was placed into approximately 2.5 mL of scintillation fluid (MP Biomedicals, Solon, OH, USA) and results of radioactivity were measured in cpm on a scintillation counter (Beckman Coulter, Brea, CA, USA).Those values were used to calculate a percent decrease in cell proliferation relative to controls.

Statistical analysis
For cell proliferation assay, four replicates of each oil along with their controls were assayed on each plate.Statistical significance was measured using the Student's t-test comparing the cpm values of the essential oil treatment for each species relative to the controls (P ≤ 0.05).

IC 50 and CC 50
All oils assayed showed inhibitory activity against one or more cancer cell lines (Table 2).Highly inhibitory IC 50 values of 0.10 μL/mL or less were observed against cancer cell lines in eight instances from four species (12% of total recorded IC 50 values).Additionally, 28 moderately inhibitory IC 50 values (between 0.10 and 0.30 μL/mL) were observed for 15 species (42% of total recorded IC 50 values), resulting in a total of 36 instances of an IC 50 of 0.30 μL/mL or less.Overall, 10 IC 50 values (45%) for skin cell line, 12 (54%) for stomach cell line, and 14 values (64%) for tongue cell line were below 0.30 All essential oils were shown to be cytotoxic to Vero C 1008 cell line at some concentration (Table 2).10 oils (45%) produced highly cytotoxic CC 50 values of 0.10 μL/mL or less, and 9 oils (41%) produced moderately inhibitory CC 50 values (between and 0.30 μL/mL).In total 19 oils (86%) produced a CC 50 value below 0.30 μL/mL against the Vero cells.2).
14 (21%) therapeutic index (TI) values, where TI value was equal to or greater than 1 indicated a significantly higher cytotoxcity to cancer cells compared to the established Vero cell line (Table 3).For R. chalepensis all three TI values were over 1, and two values over 1 which were recorded for C. limetta, C. aurantium, Rosmarinus officinalis L. (Lamiaceae), and O. vulgare, and one value greater than or equal to 1 was recorded for E. globulus, P. oocarpa, and L. graveolens.Ten TI values were not calculated due to IC 50 or CC 50 values being below the smallest measurable value for this assay (Table 3).

Cell proliferation
DNA synthesis as measured by cancer cell proliferation was significantly decreased (P ≤ 0.026) by, exposure to essential oils (Table 4).All oils resulted in a decrease in cell proliferation by at least 50% and 15 of the 22 oils resulted in a decrease of 95% or greater.For skin cell line oils from T. ambrosioides, O. vulgare demonstrated high percentages (>97%) of decreased cell proliferation at low oil concentrations of 0.05 μL/mL, respectively.L. graveolens demonstrated a high percentage (>96%) in decreased proliferation for tongue cell line at a concentration of 0.05 μL/mL.None of the oils tested against the stomach cell line showed similar decreases in cell proliferation at low oil concentrations (<10 μL/mL).Average decrease in cell proliferation was greatest for oils effective against the skin cancer cell line (99%), followed by oils against the tongue cell line (88%) and stomach cell line (80%).

DISCUSSION IC 50 and CC 50
The IC 50 values for 59% of the oils assayed have not been reported previously (Table 2).All oils assayed showed some inhibitory effect on cancer cells lines (Table 2) and many displayed high inhibition at low concentrations.For C. aurantium, C. limetta, E. globulus, L. graveolens, and O. vulgare, (and to some extent for R. officinales and R. chalepensis) the IC 50 values, therapeutic indices, and cell proliferation decreases are consistent in showing significant inhibition of cancer cells (Tables 2, 3, and 4).
Oil from C. aurantiifolia was the most effective oil against all three cancer cell lines with an IC 50 less than 0.05 μL/mL for each line (Table 2).Oil from O. vulgare produced highly inhibitory IC 50 values against skin and tongue cell lines and L. graveolens produced a highly inhibitory IC 50 value against the tongue cell line.The average IC 50 value for each of these oils against the three cancer cell lines was 0.12 μL/mL indicating potential for broad scale cancer cell inhibition.Both oils have been reported to have similar composition (Salgueiro et al., 2003) which may explain their comparable levels of activity and effectiveness (Al-Kalaldeh et al., 2014;Begnini et al., 2014).Oil from T. ambrosioides also produced highly inhibitory IC 50 values against skin and tongue cell lines (  Yuangang et al. (2010) showed that the essential oil from C. zeylanicum was moderately active against prostate and lung cancer cells and Manosroi et al. (2006) found that the essential oil from P. guajava was moderately active against human mouth epidermal carcinoma.Oils from both of these plants are known to contain βcaryophyllene oxide, which has been noted for signal cascade inactivation resulting in down-regulation of proliferation and angiogenesis in some cancer cell lines (Park et al., 2011;Kim et al., 2014).Oils from R. officinalis also have been reported to have high inhibitory values against a variety of cell lines (Hussain et al., 2010;Wang et al., 2012).
Therapeutic indices further indicated that several oils show potential because TI values greater than 1 indicate reduced cytotoxicty to cells from the established cell line (Table 3).TI values for C. limetta, C. aurantium, L. graveolens and O. vulgare indicate the potential of these oils against the tongue cell line.Additionally TI values of C. limetta, C. aurantium and E. globulus indicate potential against the stomach cell line while oils from O. vulgare and R. officinalis showed similar results against the skin cell line.The oil from R. chalepensis was the only oil that generated three TI values greater than 1, although none of the individual IC 50 values were highly inhibitory.
Additional testing and identification of active components of the oil from this species are needed to determine if similar compounds are active against both non-cancerous and cancerous cells.Average TI values of 2.05 and 1.52 across tongue and stomach cell lines, respectively, were calculated for C. limetta and C. aurantium (Table 3) possibly indicating broad spectrum activity.O. vulgare showed a similar pattern against the the tongue and skin cell lines with TIs of 1.25 and 1.11, respectively.This result is significant because only two low IC 50 values among the 22 species tested occurred against skin cancer cells (Table 2).

Conclusion
This study provided an increased understanding about the activity of essential oils against cancer cell lines and cytotoxicity from medicinal plants commonly used in Guatemala.IC 50 values indicated that essential oils can be highly effective against one or more cancer cell lines with oils from C. aurantiifolia, L. graveolens, O. vulgare, R. chalepensis, and T. ambrosioides showing potential for future development.
Results from therapeutic indices and cell proliferation assay consistently indicate that essential oils from C. limetta, C. aurantium, L. graveolens, O. vulgare, E. globulus, R. officinalis, and R. chalepensis were more toxic to cancerous cells than cells from the established cell line which shows broad as well as cell line specific activity.
Future research should include identification of active compounds, determining mechanisms of action of these compounds, possible synergistic interactions, and animal and clinical studies.

Table 2 .
IC50 values (μL/mL) for cancer cell lines, and CC50 (μL/mL) values for the Vero C 1008 line, for essential oils of Guatemalan medicinal plants in vitro.
#Oils not previously reported to have been tested on cancer cell lines in vitro, *IC50 values are below the measurable values of this assay, **IC50 values are above the measurable values of this assay.

Table 3 .
Therapeutic Index values for essential oils from Guatemalan medicinal plants for activity on cancer and established cell lines in vitro.

Table 4 .
The effect of essential oils on per cent decrease in cell proliferation as measured by [ 3 H]-thymidine incorporation (% ± se).