ZEBRAFISH

Volume 00, Number 00, 2018 Original Article

ª Mary Ann Liebert, Inc.
DOI: 10.1089/zeb.2017.1519

Effects of Essential Oil from Thymus vulgaris

on Viability and Inflammation in Zebrafish Embryos

Katherine M. Polednik, Abby C. Koch, and Lisa K. Felzien

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Abstract

Innate immunity provides the initial response against pathogens and includes the inflammatory response.
Regulation of the initiation and duration of neutrophil and mononuclear cell influx during inflammation de-
termines both the successfulness of pathogen elimination and the level of resulting tissue damage. Zebrafish
embryos provide excellent opportunities to visualize the inflammatory response. Neutrophil granules may be
stained with Sudan black, and variation in neutrophil counts may be used to monitor the level of the response.
Inflammation may be triggered by injuring the caudal fin, providing an opportunity for testing possible anti-
inflammatory compounds in a whole-animal system. The use of homeopathic compounds as anti-inflammatory
treatments is common in alternative medicine. Effects of unfractionated essential oil from Thymus vulgaris and
its specific component, carvacrol, have been examined in cells in culture and in rodents. Our work extends this
research to zebrafish, and includes toxicity and morphological studies as well as examination of anti-
inflammatory effects following tail fin injury. Our results show that zebrafish are more sensitive to thyme oil
compared to cells in culture, that cardiac defects arise due to thyme oil treatment, and that thyme oil reduces
neutrophil infiltration during an inflammatory response.

Keywords: inflammation, thyme oil, toxicity

Introduction IL-1, have been reported during zebrafish tail amputation.8 Tail
injury methods are typically performed in a mostly sterile en-

I nflammation is a complex, biological, protective re-

sponse of body tissues to harmful stimuli. Prolonged in-
flammation can cause tissue damage and also plays a role in the
clinical implications of diseases such as atherosclerosis, car-
diovascular disease, type II diabetes, arthritis, osteoporosis,
Alzheimer’s disease, and periodontal disease.1–3 Thus, identi-
fying mechanisms for controlling the inflammatory response is
crucial. Zebrafish embryos provide an excellent opportunity for
observing the inflammatory response in vivo.4 Examining the
inflammatory response of innate immunity independently from
adaptive immunity is possible in zebrafish embryos, as innate
immunity arises early during embryonic development, while
adaptive immunity does not develop until four to six weeks
after hatching.4 Due to a high degree of sequence and func-
tional homology with humans, including similar signaling
pathways,5 zebrafish can provide detailed insights into human
immune system function. Both tail amputation and needle-
induced injuries in zebrafish embryos have been used to de-
termine neutrophil and mononuclear cell migration to an injury
site, as well as the resolution of the inflammatory response, in
part, through apoptosis of leukocytes.6,7 In addition, increases
in the expression of some proinflammatory cytokines, such as
vironment so that the response to injury rather than infection is
examined. These conditions mimic situations that occur in
many of the human conditions associated with an increased
inflammatory response.
Homeopathic remedies have become a more prevalent and
sometimes preferred method for treating common ailments.
Essential oils, a large contributor to naturopathy, have played
a pivotal role in cosmetic, pharmaceutical, and medicinal
practices.3,9 Distillation procedures allow components from
aromatic plants to be extracted, concentrated, and readily used
by consumers.9 Applications of these compounds include the
following: lavender (Lavandula) as a sedative, muscle relax-
ant, antidepressant, and burn and insect bite relief10; tea tree
(Melaleuca alternifolia) as an antiseptic, antimicrobial, and
preservative within cosmetics11; nutmeg (Myristica fragrans
Houtt.) as an astringent, antithrombotic, antifungal, and anti-
inflammatory agent12; and basil (Ocimum basilicum L.) as an
aromatic and flavor additive in cooking, and treatment for
various medical conditions, including headaches, kidney mal-
function, diarrhea, and constipation.3 Extracts from Thymus
vulgaris have been shown to have various medicinal properties,
including antibacterial and antifungal actions,5,13 antioxidant

Department of Biology, Rockhurst University, Kansas City, Missouri.

1
 2 POLEDNIK, KOCH, AND FELZIEN
functions,3,14 and anti-inflammatory activity.15 Thyme oil re-
duces chemically induced edema in the peritoneum and ear in
rats and reduces leukocyte migration, in vitro.16 In mice, a
combination of thyme and oregano oils reduces overt symp-
toms, damage to the colon, and expression of proinflammatory
cytokines associated with a model of colitis.17 While progress
has been made in understanding the role of thyme oil in re-
ducing inflammation in whole-animal models, studies in which
leukocyte migration may be observed in vivo are not possible in
rodent models. Establishing the appropriate dosage and effects
of thyme oil on inflammation in zebrafish, where leukocyte
migration may be observed in vivo,4 will provide the basis for
future determination of the mechanisms of action for the anti-
inflammatory effects of thyme oil.
Extracts of T. vulgaris contain two major bioactive, mono-
terpene components, carvacrol (4-isopropyl-2-methylphenol)
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and thymol (2-isopropyl-5-methylphenol).3,12 Other compo-
nents include the following: linalool, a-terpineol, and 1,8-
cineole.3 Thymol and carvacrol have been tested separately for
anti-inflammatory effects on cells in culture, and mostly simi-
lar functions have been observed. Both compounds repress
proinflammatory cytokine expression in human macrophage
cell cultures treated with oxidized low-density lipoprotein
(LDL) and lipopolysaccharide (LPS).18,19 However, some
studies have shown differences or even opposing effects by
the two compounds, specifically in the areas of regulating
leukocyte migration16 and in regulation of transcription factors
that control proinflammatory cytokines.18 Due to the fact that
the common usage of thyme oil is in the nonfractionated form
and the identification of some variation in the actions of thyme
oil compounds, this study focuses on the anti-inflammatory
effects of the complete thyme oil preparation and will add to
cell culture studies by examining the effects of thyme oil on
leukocyte migration in vivo.
In addition to determining the medically relevant activities
of herbal remedies, the safety of such methods must also be
addressed. Zebrafish embryos are prime indicators of toxicity
and are cost-effective alternatives compared to rodent
models.4,20 Therefore, the zebrafish model was used to ex-
amine whole animal toxicity at various thyme oil dosages
given at the beginning of development, as well as to deter-
mine morphological effects associated with detrimental
dosages delivered at times relevant for studying inflamma-
tion. Following identification of the maximum nonlethal
dosage of thyme oil, small cuts were made in the caudal fins
of 72 h embryos and neutrophil migration to the site of injury
was determined by staining with Sudan black. Determining
the number of first responder cells (neutrophils) is an effec-
tive method for understanding the effects of thyme oil on the
magnitude of the inflammatory response.21 Elucidating both
the overall effects of thyme oil and the specific actions re-
garding the inflammatory response provides a better under-
standing of the complexities associated with the multitude of
effects caused by the compounds present in thyme oil.
Methods
Analysis of thyme oil components
The doTERRA thyme oil used in this study underwent che-
mical analysis using gas chromatography-mass spectroscopy
(GC-MS). A Hewlett Packard GC system, HP800 Series, con-
nected to a mass spectrometer was used for this experiment. The
column temperature was programmed at 50C for 1 min, and
then 10C/min to 250C, and finally left at 250C for 5 min. The
injection port temperature was 260C, while that of the detector
was 250C (Split ratio: 1/60). The carrier gas was helium with a
flow rate of 1.0 mL/min. The analyzed essential oil volume was
1 lL. Percentages of the constituents were calculated by elec-
tronic integration. Retention indices were calculated for separate
compounds relative to NIST Mass Spectral Library 5, 6. The MS
conditions were as follows: ionization voltage, 70 eV; ion source
temperature, 150C; and electron ionization mass spectra were
acquired over the mass range 50–550 m/z. The thyme oil (lot no.
30220001), serving as the experimental reagent, was obtained
from the doTERRA company in Pleasant Grove, Utah. These
specifications were similar to the methods used in similar re-
search intended to determine the composition of essential oils.22
Zebrafish embryo cultures and toxicity studies
Wild-type AB zebrafish were obtained from the Zebrafish
International Resource Center and maintained using described
care and breeding procedures.10 Embryos were cultured in
sterile E3 medium12 at 28C.
For initial toxicity studies (Fig. 1), embryos were treated
immediately after collection, within 2 hours postfertiliza-
tion (hpf ), with dimethylsulfoxide (DMSO), various levels
of thyme oil diluted in DMSO, or 0.005% coconut oil
(doTERRA) diluted in DMSO. Each solution was mixed in E3
medium in a 1:1000 dilution before addition of the embryos.
Final concentrations of thyme oil were 0.005% (v/v), 0.001%
(v/v), and 0.0005% (v/v). Embryos were grown for 72 h in a
sterile, covered dish and analyzed using a Zeiss Discovery.V8
stereoscope. Live embryos were counted every 2–24 h, while
dead embryos were removed from the culture. The experiment
shown (Fig. 1) is representative of three experiments performed
with three separate clutches. For each experiment, 20–30 em-
bryos were subjected to each treatment.
For toxicity studies of later stage embryos (Figs. 2–4), em-
bryos were treated 48 or 72 h after collection with DMSO or
various levels of thyme oil or coconut oil diluted in DMSO.
Each solution was mixed in E3 medium in a 1:1000 dilution
before addition of the embryos. Final concentrations of thyme
oil were 0.005%, 0.001%, and 0.0005%; the final concentration
of coconut oil was 0.005%. Embryos at 48 hpf were allowed to
continue growth for another 24 h in a sterile, covered dish.
Embryos treated at 72 hpf were monitored for 3 h following
treatment. Heart rates, in beats per minute (BPM), were docu-
mented following anesthesia with 0.016% Tricaine (ACROS).
All experiments were carried out in accordance with the
accepted standards of humane animal care and approved by
the Rockhurst Animal Care and Use Committee.
Zebrafish tail fin injury
Embryos used for fin injury were grown in sterile E3
medium. 0.003% phenylthiourea (PTU; Wards Science) was
added to the culture after 8 h to block melanization. Larvae at
72 hpf were pretreated for 2 h with DMSO, final concentra-
tions of 0.001% (v/v), 0.0005% (v/v), and 0.00005% (v/v)
thyme oil, or 300 lM hydrocortisone (Calbiochem). Stock
solutions of thyme oil were diluted in DMSO at 1:1000 to
reach the final concentrations, and hydrocortisone was pre-
pared in DMSO.
 THYME OIL EFFECTS ON VIABILITY AND INFLAMMATION
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Before injury, embryos were anesthetized with 0.016%
Tricaine to allow proper handling and ability to induce an
adequate injury. Individual fish were placed on a sterile 4%
agar dissecting pad. With the use of a dissecting microscope,
tails were clipped with sterile 31 gauge needles and fish were
returned to preinjury solutions in the 28C incubator for 6 h.
Sudan black staining and imaging
Six hours postinjury, the fish were fixed overnight with a
4% paraformaldehyde solution and then stained using a Su-
dan black neutrophil staining method.23 Embryo images were
generated by using a Nikon LABOPHOT-2 compound mi-
croscope and a Jenoptik ProgResC3 imaging system.
Neutrophil counts and statistical analysis
Neutrophil counts were made by three separate individuals
through single-blind analysis, in which two of the three in-
dividuals were unaware of the treatments given before each
injury. Numbers generated from the three separate counts
were averaged to achieve the final neutrophil count on each
picture.
Statistical analysis of neutrophil migration was performed
on average counts generated by single-blind analysis by
Student’s t-test using Minitab software (Minitab, Inc., State
College, PA). A 95% confidence interval was used for
ANOVA analysis.
Results
Chemical analysis of thyme oil components
The composition of the thyme oil used in this study was
examined by GC-MS and compared to compound types and
concentrations provided by the manufacturer’s (doTERRA)
handbook.24 Relative concentrations of 15 compounds from
NIST Mass Spectral Library were compared to the stated
3
FIG. 1. Effects of thyme oil treatment
on embryo survival rate. Embryos were
treated at 1 hpf with the controls, DMSO
(carrier control), and 0.005% coconut
oil (oil control), and 0.005%, 0.001%,
and 0.0005% thyme oil. Embryos
were allowed to grow in the presence of
treatments and were examined three
times during the first day of develop-
ment (2, 4, and 9 hpf) and then every
24 h, up to 72 hpf. Toxicity of 0.005%
thyme oil was observed beginning at 2 h
and continued until complete lethality
was reached after 24 h. This experiment
is representative of three experiments
with three separate embryo clutches and
20–30 embryos per treatment in each
experiment. DMSO, dimethylsulfoxide;
hpf, hours postfertilization.
compound intervals from doTERRA, as seen in Table 1. All
of the stated compounds were identified in the same relative
concentrations as was reported. Discrepancies can be related
to GC-MS primarily serving as a qualitative technique and
not being modified for quantitative analysis in this study. The
compounds present in the highest concentrations were thy-
mol and p-cymene. Of all 15 compounds present in the thyme
oil preparation, thymol and p-cymene have been shown to be
the most toxic.25–27
Effects of thyme oil on zebrafish embryo survival
The survival of embryos treated with 0.005% (v/v),
0.001% (v/v), and 0.0005% (v/v) thyme oil is shown in
Figure 1. Treatment with 0.005% thyme oil at 1 hpf resulted
in a rapid decline in embryonic survival, with only 60% of
embryos alive at 4 hpf, 20% alive at 9 hpf, and complete
lethality by 24 hpf. Reducing the dosage by fivefold, to
0.001%, resulted in embryonic death that was delayed and
reduced, with 77% survival at 72 hpf. This level of lethality
was similar to levels observed with the diluent, DMSO,
which had a survival rate of 81% at 72 hpf. The 0.0005%
thyme oil concentration displayed a slight decline beginning
at 9 hpf, but a final survival rate of 88% at 72 hpf. A solution
of 0.005% (v/v) coconut oil was used as an oil control and
minimal lethality was observed, with a survival rate of 93% at
72 hpf.
Malformation effects of thyme oil on 72 hpf zebrafish
When embryos were treated immediately postfertilization
with 0.005% thyme oil, embryonic death occurred rapidly, with
a 40% death rate within 4 hpf, compared to 0%, 9%, and 3%
with DMSO and 0.005% coconut oil, 0.001% thyme oil, and
0.0005% thyme oil, respectively. Before 4 hpf, no structural
abnormalities were present; however, a developmental delay
was observed in the 0.005% group, in which development
 4 POLEDNIK, KOCH, AND FELZIEN
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FIG. 2. Morphological changes in 72 hpf embryos after 24h

treatment with thyme oil. Embryos were treated at 48 hpf with
DMSO (A), 0.005% coconut oil (B), 0.0005% thyme oil
(C), 0.001% thyme oil (D), or 0.005% thyme oil (not shown).
Embryos were monitored at 72 hpf, and physical changes were
documented. All embryos treated with 0.005% thyme oil were
dead by 72 hpf. Embryos treated with 0.005% coconut oil and
0.0005% thyme oil showed normal physical features, similar
to those seen with DMSO. Some embryos treated with
0.001% thyme oil demonstrated pericardial edema (arrow in
D) and developmental delays seen in the yolk sac and pigment
production. The frequency of pericardial edema was variable,
at 20%, 37%, and 78% in three separate experiments. Average
heart rates for each of the treatments were as follows: 215
BPM for DMSO, 211 BPM for 0.005% coconut oil, 164 BPM
for 0.0005% thyme oil, and 118 BPM for 0.001% thyme oil.
This figure is representative of three experiments with three
separate clutches of embryos and 25–40 embryos per treat-
ment in each experiment. BPM, beats per minute.
nearly halted at the point of treatment. No developmental delays
were seen in the DMSO, 0.005% coconut oil, 0.001% thyme oil,
or 0.0005% thyme oil groups.
In addition to early toxicity effects on embryos, we also
wished to explore the effects of thyme oil at stages later in
FIG. 3. Effects of thyme oil on the heart in 72 hpf em-
bryos. Embryos were subjected to short-term treatment at
72 hpf with DMSO (A), 0.005% coconut oil (B), 0.0005%
thyme oil (C), 0.001% thyme oil (D), and 0.005% thyme oil
(E). The 0.005% thyme oil dosage resulted in death by
50–120 min following treatment, depending on the experi-
ment. A visible factor corresponding with embryo death was
blood pooling near the heart (arrow in E), which was seen in
60%–80% of dying or dead embryos in each experiment. This
figure is representative of three separate experiments with
three separate clutches of embryos. In each experiment, 15
embryos were subjected to each treatment and examined
for possible lethality. Physical features of three to nine
embryos per treatment, per experiment were documented
through microscopy, for a total of 28 total embryos per
treatment.
 THYME OIL EFFECTS ON VIABILITY AND INFLAMMATION 5
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FIG. 4. Effects of thyme oil on heart rate in 72 hpf embryos. Embryos were subjected to short-term treatment at 72 hpf
with DMSO, 0.005% coconut oil, 0.0005% thyme oil, 0.001% thyme oil, and 0.005% thyme oil. Heart rates (BPM) in
embryos from each treatment were counted every 30 min over a course of 180 min. 0.005% thyme oil caused 100% death
after 90 min, and a sharp decrease in heart rate beginning at 30 min following treatment. The 0.001% and 0.0005% thyme oil
dosages caused a less marked decrease in heart rate compared to the DMSO and coconut oil controls. This graph is
representative of three separate experiments with three separate clutches of embryos. The amount of time to reach full
lethality ranged from 50 to 120 min, depending on the experiment. In each experiment, heart rates in three to nine embryos
per treatment, were determined, for a total of 28 embryos per treatment. The representative experiment shown was
performed with eight to nine embryos per treatment. Error bars represent standard errors.

development, including teratogenicity end points, such as
malformation of the head, tail, or heart, scoliosis, deformity
of yolk, and growth retardation.28 Figure 2 shows images of
72 hpf embryos treated at 48 hpf with DMSO, 0.005% co-
conut oil, 0.001% thyme oil, and 0.0005% thyme oil. Treat-
ment at 48 hpf with 0.005% thyme oil resulted in 100%
lethality by 72 hpf. In some embryos, the 0.001% thyme oil
dosage caused pericardial edema and a developmental delay
observed by decreased pigmentation and premature yolk size
and shape. The frequency of these observations varied in
individual experiments, with rates at 20%, 37%, and 78% in
three separate experiments. These abnormalities were not
observed in embryos treated with DMSO, 0.005% coconut
oil, or 0.0005% thyme oil. Because of the edema present in

TERRA Thyme Oil Using Gas Chromatography–Mass Spectroscopy

Table 1. Chemical Analysis of do
Retention time Compound Experimental Stated interval
5.896 Alpha pinene 1.89% NR
6.676 Beta myrcene 2.75% 1%–2.8%
7.65 p-cymene 11.45% 14%–28%
7.941 Gamma terpinene 7.94% 4%–11%
8.607 Linalool 9.70% 3%–6.5%
10.524 Thymol methyl ether 1.67% NR
11.37–11.705 Thymol 23.97% 37%–55%
11.791 Carvacrol 4.54% 0.5%–5.5%
12.700 Copaene 3.50% NR
13.333 Caryophyllene 5.75% NR
13.756 4,7,10-cycloundecatriene 3.43% NR
14.502 Naphthalene 1.256% NR
15.331 Caryophyllene oxide 1.235% NR
Total percentage composition 80.16%
Thyme oil was run through a GC-MS instrument, and retention times were recorded. Resulting peaks were analyzed to determine the
identity of compounds in the solution. The intensity of peaks generated from analysis provides an estimate of the relative concentrations of
compounds. The stated interval shows manufacturer-provided ranges of compounds expected in the thyme oil preparation.
GC-MS, gas chromatography–mass spectroscopy; NR, not reported.
 6 POLEDNIK, KOCH, AND FELZIEN
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FIG. 5. Needle injury of the tail fin induces inflammation.
Embryos at 72 hpf were either left uninjured (A) or sub-
jected to tail fin injury under anesthesia with 31 gauge
needles (B). Uninjured embryos were maintained in E3
medium and injured embryos were returned to E3 medium
for 6 h. Embryos were fixed and stained with Sudan black, a
lipophilic stain that identifies neutrophils. Injuries resulted
in a clear infiltration of neutrophils to the site of inflam-
mation, and the uninjured control demonstrates that normal
incubation conditions do not induce neutrophil infiltration.
the pericardium of some embryos, heart rate was examined.
Average rates were 215 BPM for DMSO, 211 BPM for
0.005% coconut oil, 164 BPM for 0.0005% thyme oil, and
118 BPM for 0.001% thyme oil.
Additional microscopic observations were made with larvae
treated at 72 hpf (Fig. 3). No physical changes were observed
with DMSO, 0.005% coconut oil, 0.0005% thyme oil, or
0.001% thyme oil. Interestingly, 100% larval death occurred
within 50–120 min (range based on observations in separate
experiments) following 0.005% thyme oil treatment, compared
to 100% lethality at 24 h following treatments on fish treated
just after fertilization and at 48 hpf. This increased rate of le-
thality within a short period of time may have been due to the
absence of the chorion, and direct and immediate effects by
thyme oil on the young fish or a greater sensitivity at later
developmental stages to the compounds in thyme oil. As em-
bryos died, blood pooling near the heart was seen. To further
examine possible effects on the heart, average heart rates were
calculated for embryos in each treatment group over a time
course of 180 min. Figure 4 shows a decreasing heart rate in
embryos treated with 0.005% thyme oil, until the point of full
lethality at 90 min. In the experiment shown, some embryos
began to die at 60 min, which contributed to a reduced heart rate
average. Embryos still living at the 60 min time point had an
average heart rate of 39 BPM, compared to an average heart
rate of 34 BPM at 30 min, when all embryos were still alive.
Smaller declines in heart rate were observed in the presence of
0.0005% thyme oil (145 BPM at 180 min) and 0.001% thyme
oil (100 BPM at 180 min). Heart rates at 180 min for embryos
treated with DMSO and 0.005% coconut oil were 177 and 165
BPM, respectively.
Effects of thyme oil on neutrophil infiltration during injury
While thyme oil toxicity has not been well studied, several
groups have shown an inhibitory effect of thyme oil or car-
vacrol on white blood cell migration in vitro.16 To determine
whether this inhibitory activity could be observed in vivo, a
tailfin injury procedure using small needles was developed to
mimic the more common method of tail fin amputation. Si-
milar methods of needle injury6 have been utilized to study
the effects of heat shock on neutrophil infiltration. Experi-
mental procedures for examining the effect of compounds on
neutrophil migration require multiple embryo transfers and
incubations, which could possibly trigger small lesions to the
tail. Thus, we compared the presence of neutrophils identified
by Sudan black staining in the tail fin due to normal handling
versus the impact of the needle injury at 72 hpf. Figure 5
shows a representative comparison of neutrophils in the tail
fins of injured versus noninjured embryos and demonstrates
that the needle injury is a reliable method for triggering an
influx of neutrophils far beyond those neutrophils present due
to small levels of damage caused by handling alone.
To examine the effects of thyme oil on neutrophil infil-
tration at sites of injury, 72 hpf embryos were pretreated with
nonlethal doses of thyme oil, DMSO as a negative control,
and cortisol, a known anti-inflammatory agent. Figure 6
shows neutrophil counts in tail regions from four separate
experiments. Figure 6A represents an initial experiment us-
ing the two lowest dosages of thyme oil from the toxicity
study (0.00005% and 0.0005%). A reduction in neutrophils
was observed with 0.0005%, but not 0.00005% thyme oil,
leading to the exclusion of the lowest dosage in subsequent
experiments. Figure 6B represents an initial experiment,
which included cortisol as a control, 0.0005% thyme oil, and
0.001% thyme oil. In this experiment, cortisol and 0.001%
thyme oil caused reductions in neutrophil infiltration, but
0.0005% thyme oil did not. Figure 6C and D represent two
experiments in which 0.0005% and 0.001% thyme oil con-
centrations were used. In these experiments, both 0.0005%
and 0.001% caused reductions in the number of infiltrating
neutrophils. Taken together, three of the four individual ex-
periments showed modest reductions in neutrophils present at
the site of injury in the presence of 0.0005% thyme oil. The
experiment that did not show a reduction at 0.0005% thyme
oil had a lower level of neutrophils observed in the DMSO
control, which may have affected the outcome. The 0.001%
thyme oil dosage showed modest neutrophil reductions in
three of three experiments containing that treatment. Figure 7
contains sample tail images showing neutrophils stained with
Sudan black for thyme oil treatment at dosages of 0.0005%
and 0.001%, compared to the DMSO control.
Inherent variation exists in tail injury experiments, due to
possible differences in cut size and location. To address this
variation, a single researcher (Polednik) performed all in-
juries, and as many injuries as was reasonable within an
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FIG. 6. Effects of thyme oil on neutrophil infiltration during tail fin injury. At 72 hpf, embryos were pretreated with the
indicated solutions, injured with 31 gauge needles, returned to treatment for 6 h, and fixed and stained with Sudan black.
Neutrophils in the distal tail region were counted, averaged, and graphed, with error bars representing standard errors. Each
panel shows an individual experiment performed with a separate clutch of embryos, with numbers of embryos stated for
each treatment. (A) Represents an experiment examining 0.0005% thyme oil, which reduced infiltrating neutrophils, and
0.00005% thyme oil, which did not show a reduction in neutrophils. (B) Represents an experiment using cortisol as an anti-
inflammatory control, which reduced infiltrating neutrophils, 0.0005% thyme oil, which did not show a decrease in
neutrophils, and 0.001% thyme oil, which reduced infiltrating neutrophils. (C, D) Show decreased neutrophil infiltration
following treatment with both 0.0005% and 0.001% thyme oil.

experiment were completed. To avoid researcher bias, all
three authors counted neutrophils through a single-blind ap-
proach, in which only one author was aware of the treatments.
Data from the four experiments shown in Figure 6 were
pooled to provide a large enough sample size for statistical
analysis. Figure 8 summarizes the interval number of neu-
trophils seen in each treatment with pooled data. Significant
differences in the number of neutrophils present at the wound
were seen for both thyme oil treatments. There was a sig-
nificant reduction in neutrophil migration in the fish treated at
72 hpf with 0.001% thyme oil (mean: 13.31, p < 0.05) and
0.0005% thyme oil (mean: 11.67, p < 0.05).
Discussion
The composition of essential oils has been shown to differ
due to the type of plant, harvest time, and extraction tech-
niques. This study utilized a complete thyme oil preparation
rather than individual components to mimic standard usage
of thyme oil as an anti-inflammatory homeopathic treatment.
 8 POLEDNIK, KOCH, AND FELZIEN
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FIG. 7. Thyme oil treatment reduces neutrophil infiltration
caused by needle injury. At 72 hpf, embryos were pretreated
with DMSO (A), 0.0005% thyme oil (B), and 0.001% thyme
oil (C), and tails were injured with 31 gauge needles. Em-
bryos were returned to treatment after injury for 6 h, fixed,
and stained with Sudan black. Images are representative of at
least 25 embryos subjected to each treatment.
To determine the composition of the thyme oil preparation
used, a chemical analysis was performed (Table 1), with re-
sults compared to the stated intervals provided by the
doTERRA company. Levels of thymol and carvacrol are of
greatest interest, as these compounds have been shown to
affect inflammation: thymol both blocking and inducing in-
flammation, and carvacrol consistently acting as an anti-
inflammatory compound.29 While the exact levels of thymol
and carvacrol cannot be fully determined through GC-MS,
relative levels indicate a thymol level approximately fivefold
higher than the carvacrol level of the sample used.
The concentration of p-cymene and thymol was consider-
ably lower than the stated intervals. However, both were still in
the highest concentration within the oil. Both chemicals are
also the most toxic toward humans. When in contact with the
skin, both act as irritants, and when ingested, both are asso-
ciated with toxicity to the central nervous system.25–27 The
concentration of linalool was significantly higher than the
stated intervals. This compound was found to be associated
with skin irritation and respiratory tract irritation, as well as a
specific target organ toxin. These toxicities are characteristic
of many of the other compounds identified in the GC-MS
analysis, and are likely involved in the cytotoxicity observed
with zebrafish. Others have examined uptake of drugs in
zebrafish by performing mass spectrometry on yolk samples.30
Follow-up experiments such as this, which can determine the
specific components of thyme oil that pass through both the
chorion and embryonic epithelial layer, will add important
insights to this work.
Determination of appropriate nontoxic thyme oil concentra-
tions that also had enough critical anti-inflammatory compounds
to have an effect was challenging. Reported concentrations of
similar experiments.17,19,28,31 were too high to maintain viability
in zebrafish embryos. At these concentrations, 100% of the
zebrafish embryos died within 24 h of treatment, with nearly
40% having perished by 4 h posttreatment. The toxicity portion
of this study was designed to address these concerns and to
determine nonlethal concentrations of thyme oil on 72 hpf em-
bryos. The high level of dilution for thyme oil needed to avoid
toxic effects, 0.001% for treatment immediately following fer-
tilization and 0.0005% to avoid later heart edema in 48 hpf
embryos, was surprising and may provide insights about dosages
of naturopathic compounds, which are not typically evaluated or
refined.
Testing the effects of a pure compound from thyme oil,
such as carvacrol, was considered. However, since carvacrol
has a significant skin irritation warning associated with it and
most consumers use thyme oil as a homeopathic remedy, it
was more clinically relevant to conduct the study around
thyme oil as a whole. Testing with coconut oil was relevant to
determine if the fish could survive in an environment with an
oil introduction. Other sources have cited using DMSO as a
negative control compound, and testing with coconut oil
showed little deviation from the DMSO control, indicating
that the presence of oil was not contributing greatly to the fish
death, morphological changes, or decreases in heart rate. This
ruled out the possibility that the fish were dying due to de-
creased gas exchange due to the interactions of the oils with
the chorion. Concentrations of 0.0005% and 0.001% thyme
oil showed high viability (above 75%) for a total of 72 hpf. At
the 8-h benchmark needed to complete the inflammation
study, the viability of fish treated with these concentrations
was above 80%. The near immediate death of our embryos in
0.005% thyme oil was consistent with research that suggests
that 0.016% thyme oil is associated with high levels of cy-
totoxicity in Propionibacterium acnes in humans.32 The
toxicity for human cells, however, should also be explored at
these dosages, as this is well above the minimum nontoxic
dosage for zebrafish.
Our further toxicity studies were focused on damage to
organs and structures present in embryos at the pharyngula and
hatching stages. Treatment at 48 hpf with 0.001% thyme oil
and monitoring at 72 hpf showed pericardial edema and de-
velopmental delay, as shown through reduced pigmentation
and the size and shape of the yolk structure. These features
were observed at varying degrees in separate experiments,
which may be due to small variations in the rate of develop-
ment in different clutches. For example, variation in the
number of embryos emerging from their chorions at the time of
treatment seemed to coincide with the percentage of abnormal
development, with a higher percentage of abnormality present
 THYME OIL EFFECTS ON VIABILITY AND INFLAMMATION
Downloaded by North Carolina Agr & Tech State Univ from www.liebertpub.com at 05/30/18. For personal use only.
in clutches when fewer embryos were hatching at the time of
treatment. Pericardial edema was frequently seen in embryos
that remained unhatched at the 72 hpf time point, suggesting
that the effect may be caused when embryos are less mature at
the time of treatment, as indicated by the absence of hatching
at 48 hpf. The enlargement of the pericardium may be due to
tissue swelling and/or structural weakening. Importantly, these
malformations were often occurring while the embryos were in
chorion, suggesting that the oil components were capable of
crossing the embryonic barrier. This is in agreement with the
hatching literature and may have contributed to the delayed
growth of the embryos.
In 72 hpf embryos undergoing short-term treatment with
0.005% thyme oil, all embryos died as early as 50 min. In
embryos that were either recently dead or dying, blood
pooling near the anterior portion of the yolk sac was observed
in 60%–80% of the embryos treated with 0.005% thyme oil.
Others have shown similar toxic effects by a variety of drugs
on zebrafish development, including heart edema, belly
edema, and vessel weakening.28 It is unclear as to the specific
effects on the heart structure, and further studies, involving
necropsy and blood vessel staining, would be necessary to
determine whether the vessels were weakened by the oil,
pressure was too high due to swelling, or tissue necrotic ef-
fects disrupted the heart structure.
Using the dosages determined by viability experimentation,
the effects of thyme oil on inflammation were examined. While
an anti-inflammatory effect was observed in the presence of
0.001% and 0.0005% thyme oil, the effect was incomplete.
Treatment with 0.0005% and 0.001% thyme oil resulted in a
31% and 21% reduction in neutrophil infiltration, respectively.
To our knowledge, this is the first in vivo evidence that thyme
oil reduced wound-induced inflammation. Our evidence is
consistent with previous experiments. At much higher con-
centrations in mouse and human macrophages, thyme oil
significantly decreased the production of proinflammatory cy-
tokines, resulting in anti-inflammatory effects.17,19 This effect
on gene expression regulation resembles the mechanism of
action of cortisol, which regulates cytokine expression by
9
FIG. 8. Interval plot showing
ranges and means of neutro-
phils present at injury sites
during thyme oil treatment. At
72 hpf, embryos were pre-
treated with DMSO, 0.001%
thyme oil, and 0.0005% thyme
oil, tails were injured, and em-
bryos were stained with Sudan
black. Neutrophils in close
proximity to the injury site were
counted and graphed in an in-
terval plot. Means (dots) and
error bars, representing the 95%
confidence interval, are shown.
These data represent four sepa-
rate experiments, with n = 27
for DMSO, n = 27 for 0.0005%
thyme oil, and n = 26 for
0.001% thyme oil.
controlling glucocorticoid receptor function. In fact, the car-
vacrol component of thyme oil has been shown to act as an
agonist to the nuclear receptor family, peroxisome proliferator-
activated receptors.33 Others have also shown that injection
of cortisol in zebrafish embryos caused pericardial edema
at 48 hpf and lower resting and stress-induced heart rates at
72 hpf.34 Genes involved in heart function and development
were repressed in the presence of cortisol, suggesting a possible
specific effect on heart development and function. Determining
whether carvacrol and other related compounds in thyme oil
may work in a similar manner to cortisol, through regulation of
a variety of genes involved in reducing inflammation and
regulating the development and/or function of the circulatory
system, will be important.
Considering possible nonspecific connections between the
effects on the heart and the inflammatory response is also
important. Pericardial edema and reduced heart rate likely
decrease blood flow and could contribute to a reduction in
neutrophil infiltration. It is unlikely, however, that these issues
are the only reason for the reduction, as we did not observe a
positive correlation between reduced heart rate and decreased
neutrophil infiltration. While 0.0005% thyme oil caused an
overall 31% decrease in neutrophil migration to the injury site
and a 20% decrease in heart rate, 0.001% thyme oil caused a
21% decrease in neutrophil migration to the injury site and
a 51% decrease in the heart rate. In addition, we did not see a
connection between the timing and appearance of pericardial
edema, and the timing and reduction of neutrophil infiltration.
Only when embryos were treated at 48 hpf with 0.001% thyme
oil did we see evidence of pericardial edema, and effects were
not observed until 72 hpf. Our inflammation study was per-
formed beginning at 72 hpf, and embryos were exposed for a
total of 8 h to thyme oil treatment before fixation and staining
at 80 hpf. Pericardial edema seemed specific to treatment at
48 hpf, possibly due to developmental events occurring at that
time. Taken together, our results support a dosage of 0.0005%
thyme oil as the dosage with minimal negative effects on the
heart and development, and maximal effects on neutrophil
infiltration, a critical finding for future studies.
 10
While it is likely that the thyme oil compounds that act as
irritants are cytotoxic for zebrafish cells, the mechanism of
thyme oil toxicity is still unknown. Thyme oil has been
shown to induce apoptosis in squamous cell oral cancers.29
Thus, examining whether zebrafish cells undergo apoptotic
versus necrotic death in the presence of thyme oil will be
important to address. Studies on phagocytic cell infiltration
during zebrafish tail injury has demonstrated that the de-
crease in these cells over time is due to a combination of
reentry into the bloodstream and apoptosis.35 Use of the
transgenic fish line with fluorescently labeled neutrophils,
Tg(mpx:GFP), to monitor neutrophil migration into tissues
as well as reentry into the blood stream over time will be an
important next step in this work. Examining the mechanisms
for cell death causing cytotoxicity as well as possible cell
death resulting in a reduction in neutrophils recruited during
Downloaded by North Carolina Agr & Tech State Univ from www.liebertpub.com at 05/30/18. For personal use only.
inflammation will provide additional insights into the
mechanisms of action of thyme oil.
Acknowledgments
We would like to thank Elizabeth Evans and Aaron Bossert
for their assistance with zebrafish care and maintenance,
IACUC approval and oversight, and development of the
needle injury model; Paula Morehouse and Dale Harak for
their assistance with GC-MS analysis of thyme oil; Samantha
Zavertnik and Kanaporn Temrutrinit for their contributions to
the development of the needle injury model; and Joanna
Cielocha and Aaron Bossert for their assistance with imaging
and assembly of figures.
Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Lisa K. Felzien, PhD
Department of Biology
Rockhurst University
1100 Rockhurst Road
Kansas City, MO 64110
E-mail: lisa.felzien@rockhurst.edu