Académique Documents
Professionnel Documents
Culture Documents
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.
Introduction IL-1, have been reported during zebrafish tail amputation.8 Tail
injury methods are typically performed in a mostly sterile en-
1
2 POLEDNIK, KOCH, AND FELZIEN
functions,3,14 and anti-inflammatory activity.15 Thyme oil re- column temperature was programmed at 50C for 1 min, and
duces chemically induced edema in the peritoneum and ear in then 10C/min to 250C, and finally left at 250C for 5 min. The
rats and reduces leukocyte migration, in vitro.16 In mice, a injection port temperature was 260C, while that of the detector
combination of thyme and oregano oils reduces overt symp- was 250C (Split ratio: 1/60). The carrier gas was helium with a
toms, damage to the colon, and expression of proinflammatory flow rate of 1.0 mL/min. The analyzed essential oil volume was
cytokines associated with a model of colitis.17 While progress 1 lL. Percentages of the constituents were calculated by elec-
has been made in understanding the role of thyme oil in re- tronic integration. Retention indices were calculated for separate
ducing inflammation in whole-animal models, studies in which compounds relative to NIST Mass Spectral Library 5, 6. The MS
leukocyte migration may be observed in vivo are not possible in conditions were as follows: ionization voltage, 70 eV; ion source
rodent models. Establishing the appropriate dosage and effects temperature, 150C; and electron ionization mass spectra were
of thyme oil on inflammation in zebrafish, where leukocyte acquired over the mass range 50–550 m/z. The thyme oil (lot no.
migration may be observed in vivo,4 will provide the basis for 30220001), serving as the experimental reagent, was obtained
future determination of the mechanisms of action for the anti- from the doTERRA company in Pleasant Grove, Utah. These
inflammatory effects of thyme oil. specifications were similar to the methods used in similar re-
Extracts of T. vulgaris contain two major bioactive, mono- search intended to determine the composition of essential oils.22
terpene components, carvacrol (4-isopropyl-2-methylphenol)
Downloaded by North Carolina Agr & Tech State Univ from www.liebertpub.com at 05/30/18. For personal use only.
Before injury, embryos were anesthetized with 0.016% compound intervals from doTERRA, as seen in Table 1. All
Tricaine to allow proper handling and ability to induce an of the stated compounds were identified in the same relative
adequate injury. Individual fish were placed on a sterile 4% concentrations as was reported. Discrepancies can be related
agar dissecting pad. With the use of a dissecting microscope, to GC-MS primarily serving as a qualitative technique and
tails were clipped with sterile 31 gauge needles and fish were not being modified for quantitative analysis in this study. The
returned to preinjury solutions in the 28C incubator for 6 h. compounds present in the highest concentrations were thy-
mol and p-cymene. Of all 15 compounds present in the thyme
Sudan black staining and imaging oil preparation, thymol and p-cymene have been shown to be
the most toxic.25–27
Six hours postinjury, the fish were fixed overnight with a
4% paraformaldehyde solution and then stained using a Su-
Effects of thyme oil on zebrafish embryo survival
dan black neutrophil staining method.23 Embryo images were
generated by using a Nikon LABOPHOT-2 compound mi- The survival of embryos treated with 0.005% (v/v),
croscope and a Jenoptik ProgResC3 imaging system. 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
Neutrophil counts and statistical analysis in a rapid decline in embryonic survival, with only 60% of
embryos alive at 4 hpf, 20% alive at 9 hpf, and complete
Neutrophil counts were made by three separate individuals
lethality by 24 hpf. Reducing the dosage by fivefold, to
through single-blind analysis, in which two of the three in-
0.001%, resulted in embryonic death that was delayed and
dividuals were unaware of the treatments given before each
reduced, with 77% survival at 72 hpf. This level of lethality
injury. Numbers generated from the three separate counts
was similar to levels observed with the diluent, DMSO,
were averaged to achieve the final neutrophil count on each
which had a survival rate of 81% at 72 hpf. The 0.0005%
picture.
thyme oil concentration displayed a slight decline beginning
Statistical analysis of neutrophil migration was performed
at 9 hpf, but a final survival rate of 88% at 72 hpf. A solution
on average counts generated by single-blind analysis by
of 0.005% (v/v) coconut oil was used as an oil control and
Student’s t-test using Minitab software (Minitab, Inc., State
minimal lethality was observed, with a survival rate of 93% at
College, PA). A 95% confidence interval was used for
72 hpf.
ANOVA analysis.
Malformation effects of thyme oil on 72 hpf zebrafish
Results
When embryos were treated immediately postfertilization
Chemical analysis of thyme oil components
with 0.005% thyme oil, embryonic death occurred rapidly, with
The composition of the thyme oil used in this study was a 40% death rate within 4 hpf, compared to 0%, 9%, and 3%
examined by GC-MS and compared to compound types and with DMSO and 0.005% coconut oil, 0.001% thyme oil, and
concentrations provided by the manufacturer’s (doTERRA) 0.0005% thyme oil, respectively. Before 4 hpf, no structural
handbook.24 Relative concentrations of 15 compounds from abnormalities were present; however, a developmental delay
NIST Mass Spectral Library were compared to the stated was observed in the 0.005% group, in which development
4 POLEDNIK, KOCH, AND FELZIEN
Downloaded by North Carolina Agr & Tech State Univ from www.liebertpub.com at 05/30/18. For personal use only.
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 dosage caused pericardial edema and a developmental delay
malformation of the head, tail, or heart, scoliosis, deformity observed by decreased pigmentation and premature yolk size
of yolk, and growth retardation.28 Figure 2 shows images of and shape. The frequency of these observations varied in
72 hpf embryos treated at 48 hpf with DMSO, 0.005% co- individual experiments, with rates at 20%, 37%, and 78% in
conut oil, 0.001% thyme oil, and 0.0005% thyme oil. Treat- three separate experiments. These abnormalities were not
ment at 48 hpf with 0.005% thyme oil resulted in 100% observed in embryos treated with DMSO, 0.005% coconut
lethality by 72 hpf. In some embryos, the 0.001% thyme oil oil, or 0.0005% thyme oil. Because of the edema present in
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 72 hpf with 0.001% thyme oil (mean: 13.31, p < 0.05) and
three authors counted neutrophils through a single-blind ap- 0.0005% thyme oil (mean: 11.67, p < 0.05).
proach, in which only one author was aware of the treatments.
Data from the four experiments shown in Figure 6 were
Discussion
pooled to provide a large enough sample size for statistical
analysis. Figure 8 summarizes the interval number of neu- The composition of essential oils has been shown to differ
trophils seen in each treatment with pooled data. Significant due to the type of plant, harvest time, and extraction tech-
differences in the number of neutrophils present at the wound niques. This study utilized a complete thyme oil preparation
were seen for both thyme oil treatments. There was a sig- rather than individual components to mimic standard usage
nificant reduction in neutrophil migration in the fish treated at of thyme oil as an anti-inflammatory homeopathic treatment.
8 POLEDNIK, KOCH, AND FELZIEN
in clutches when fewer embryos were hatching at the time of controlling glucocorticoid receptor function. In fact, the car-
treatment. Pericardial edema was frequently seen in embryos vacrol component of thyme oil has been shown to act as an
that remained unhatched at the 72 hpf time point, suggesting agonist to the nuclear receptor family, peroxisome proliferator-
that the effect may be caused when embryos are less mature at activated receptors.33 Others have also shown that injection
the time of treatment, as indicated by the absence of hatching of cortisol in zebrafish embryos caused pericardial edema
at 48 hpf. The enlargement of the pericardium may be due to at 48 hpf and lower resting and stress-induced heart rates at
tissue swelling and/or structural weakening. Importantly, these 72 hpf.34 Genes involved in heart function and development
malformations were often occurring while the embryos were in were repressed in the presence of cortisol, suggesting a possible
chorion, suggesting that the oil components were capable of specific effect on heart development and function. Determining
crossing the embryonic barrier. This is in agreement with the whether carvacrol and other related compounds in thyme oil
hatching literature and may have contributed to the delayed may work in a similar manner to cortisol, through regulation of
growth of the embryos. a variety of genes involved in reducing inflammation and
In 72 hpf embryos undergoing short-term treatment with regulating the development and/or function of the circulatory
0.005% thyme oil, all embryos died as early as 50 min. In system, will be important.
embryos that were either recently dead or dying, blood Considering possible nonspecific connections between the
pooling near the anterior portion of the yolk sac was observed effects on the heart and the inflammatory response is also
in 60%–80% of the embryos treated with 0.005% thyme oil. important. Pericardial edema and reduced heart rate likely
Others have shown similar toxic effects by a variety of drugs decrease blood flow and could contribute to a reduction in
on zebrafish development, including heart edema, belly neutrophil infiltration. It is unlikely, however, that these issues
edema, and vessel weakening.28 It is unclear as to the specific are the only reason for the reduction, as we did not observe a
effects on the heart structure, and further studies, involving positive correlation between reduced heart rate and decreased
necropsy and blood vessel staining, would be necessary to neutrophil infiltration. While 0.0005% thyme oil caused an
determine whether the vessels were weakened by the oil, overall 31% decrease in neutrophil migration to the injury site
pressure was too high due to swelling, or tissue necrotic ef- and a 20% decrease in heart rate, 0.001% thyme oil caused a
fects disrupted the heart structure. 21% decrease in neutrophil migration to the injury site and
Using the dosages determined by viability experimentation, a 51% decrease in the heart rate. In addition, we did not see a
the effects of thyme oil on inflammation were examined. While connection between the timing and appearance of pericardial
an anti-inflammatory effect was observed in the presence of edema, and the timing and reduction of neutrophil infiltration.
0.001% and 0.0005% thyme oil, the effect was incomplete. Only when embryos were treated at 48 hpf with 0.001% thyme
Treatment with 0.0005% and 0.001% thyme oil resulted in a oil did we see evidence of pericardial edema, and effects were
31% and 21% reduction in neutrophil infiltration, respectively. not observed until 72 hpf. Our inflammation study was per-
To our knowledge, this is the first in vivo evidence that thyme formed beginning at 72 hpf, and embryos were exposed for a
oil reduced wound-induced inflammation. Our evidence is total of 8 h to thyme oil treatment before fixation and staining
consistent with previous experiments. At much higher con- at 80 hpf. Pericardial edema seemed specific to treatment at
centrations in mouse and human macrophages, thyme oil 48 hpf, possibly due to developmental events occurring at that
significantly decreased the production of proinflammatory cy- time. Taken together, our results support a dosage of 0.0005%
tokines, resulting in anti-inflammatory effects.17,19 This effect thyme oil as the dosage with minimal negative effects on the
on gene expression regulation resembles the mechanism of heart and development, and maximal effects on neutrophil
action of cortisol, which regulates cytokine expression by infiltration, a critical finding for future studies.
10 POLEDNIK, KOCH, AND FELZIEN
While it is likely that the thyme oil compounds that act as phil directional migration and basal random motility in a
irritants are cytotoxic for zebrafish cells, the mechanism of zebrafish injury-induced inflammation model. J Immunol
thyme oil toxicity is still unknown. Thyme oil has been 2014;192:5998–6008.
shown to induce apoptosis in squamous cell oral cancers.29 9. Bakkali F, Averbeck S, Averbeck D, Idaomar M. Biolo-
Thus, examining whether zebrafish cells undergo apoptotic gical effects of essential oils—a review. Food Chem Tox-
versus necrotic death in the presence of thyme oil will be icol 2008;46:446–475.
important to address. Studies on phagocytic cell infiltration 10. Cavanagh HMA, Wilkinson JM. Biological activities of
during zebrafish tail injury has demonstrated that the de- lavender essential oil. Phytother Res 2002;16:301–308.
crease in these cells over time is due to a combination of 11. Cox SD, Mann CM, Markham JL, Bell HC, Gustafson JE,
reentry into the bloodstream and apoptosis.35 Use of the Warmington JR, et al. The mode of antimicrobial action of
the essential oil of Melaleuca alternifolia (tea tree oil). J
transgenic fish line with fluorescently labeled neutrophils,
Appl Microbiol 2000;88:170–175.
Tg(mpx:GFP), to monitor neutrophil migration into tissues
12. Imelouane B, Amhamdi H, Wathelet J-P, Ankit M, Kheded
as well as reentry into the blood stream over time will be an K, Elbachiri A. Chemical composition and antimicrobial
important next step in this work. Examining the mechanisms activity of essential oil of thyme (Thymus vulgaris) from
for cell death causing cytotoxicity as well as possible cell Eastern Morocco. Int J Agric Biol 2009;11:205–208.
death resulting in a reduction in neutrophils recruited during 13. Poulin Y. Practical approach to the hormonal treatment of
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 acne. J Cutan Med Surg 2004;8(Suppl 4):16–21.
mechanisms of action of thyme oil. 14. Aeschbach R, Löliger J, Scott BC, Murcia A, Butler J,
Halliwell B, et al. Antioxidant actions of thymol, carvacrol,
Acknowledgments 6-gingerol, zingerone and hydroxytyrosol. Food Chem
Toxicol 1994;32:31–36.
We would like to thank Elizabeth Evans and Aaron Bossert
15. Vigo E, Cepeda A, Gualillo O, Perez-Fernandez R. In vitro
for their assistance with zebrafish care and maintenance,
anti-inflammatory effect of eucalyptus globulus and Thy-
IACUC approval and oversight, and development of the mus vulgaris: nitric oxide inhibition in J774A.1 murine
needle injury model; Paula Morehouse and Dale Harak for macrophages. J Pharm Pharmacol 2004;56:257–263.
their assistance with GC-MS analysis of thyme oil; Samantha 16. Fachini-Queiroz FC, Kummer R, Estevão-Silva CF, Car-
Zavertnik and Kanaporn Temrutrinit for their contributions to valho MDDB, Cunha JM, Grespan R, et al. Effects of
the development of the needle injury model; and Joanna thymol and carvacrol, constituents of Thymus vulgaris L.
Cielocha and Aaron Bossert for their assistance with imaging essential oil, on the inflammatory response. Evid Based
and assembly of figures. Complement Alternat Med 2012;2012:657026.
17. Bukovská A, Čikoš Š, Juhás Š, Il’ková G, Rehák P, Koppel
Disclosure Statement J. Effects of a combination of thyme and oregano essential
oils on TNBS-induced colitis in mice. Mediators Inflamm
No competing financial interests exist.
2007;2007:23296.
18. Gholijani N, Gharagozloo M, Farjadian S, Amirghofran Z.
References Modulatory effects of thymol and carvacrol on inflamma-
1. Kiecolt-glaser JK, Mcguire L, Robles TF, Glaser R. Emo- tory transcription factors in lipopolysaccharide-treated
tions, morbidity, and mortality: new perspectives from macrophages. J Immunotoxicol 2016;13:157–164.
psychoneuroimmunology. Depression 2002;53:83–107. 19. Ocaña A, Reglero G. Effects of thyme extract oils (from
2. Ershler WB, Keller ET. Age-associated increased interleukin- Thymus vulgaris, Thymus zygis, and Thymus hyemalis) on
6 gene expression, late-life diseases, and frailty. Annu Rev cytokine production and gene expression of oxLDL-
Med 2000;51:245–270. stimulated THP-1-macrophages. J Obes 2012;2012:104706.
3. Lee SJ, Umano K, Shibamoto T, Lee KG. Identification of 20. Ali S, van Mil HGJ, Richardson MK. Large-scale assess-
volatile components in basil (Ocimum basilicum L.) and ment of the zebrafish embryo as a possible predictive model
thyme leaves (Thymus Vulgaris L.) and their antioxidant in toxicity testing. PLoS One 2011;6:e21076.
properties. Food Chem 2005;91:131–137. 21. Westerfield: The Zebrafish Book. A Guide for the La-
4. Trede NS, Langenau DM, Traver D, Look AT, Zon LI. The boratory Use of Zebrafish, 5th ed. University of Oregon,
use of zebrafish to understand immunity. Immunity 2004; Eugene, OR, 2000.
20:367–379. 22. Cosentino S, Tuberoso CIG, Pisano B, Satta M, Mascia V,
5. Essawi T, Srour M. Screening of some palestinian medic- Arzedi E, et al. In vitro antimicrobial activity and chemical
inal plants for antibacterial activity. J Ethnopharmacol composition of sardinian thymus essential oils. Lett Appl
2000;70:343–349. Microbiol 1999;29:130–135.
6. Liao YF, Chiou M-CH, Tsai J, Wen C-C, Wang Y-H, 23. Le Guyader D, Redd MJ, Colucci-Guyon E, Murayama E,
Cheng C-C, et al. Resveratrol treatment attenuates the Kissa K, Briolat V, et al. Origins and unconventional be-
wound-induced inflammation in zebrafish larvae through havior of neutrophils in developing zebrafish. Blood 2008;
the suppression of myeloperoxidase expression. J Food 111:132–141.
Drug Anal 2011;19:167–173. 24. DoTERRA: Modern Essentials: A Contemporary Guide to
7. Hoodless LJ, Lucas CD, Duffin R, Denvir MA, Haslett C, the Therapeutic Use of Essential Oils, 4th ed. Aroma Tools,
Tucker CS, et al. Genetic and pharmacological inhibition of Pleasant Grove, UT, 2012.
CDK9 drives neutrophil apoptosis to resolve inflammation 25. Li XJ, Yang YJ, Li YS, Zhang WK, Tang HB. a-pinene,
in zebrafish in vivo. Sci Rep 2016;5:36980. linalool, and 1-octanol contribute to the topical anti-
8. Yan B, Han P, Pan L, Lu W, Xiong J, Zhang M, et al. Il-1 inflammatory and analgesic activities of frankincense by
and reactive oxygen species differentially regulate neutro- inhibiting COX-2. J Ethnopharmacol 2016;179:22–26.
THYME OIL EFFECTS ON VIABILITY AND INFLAMMATION 11
26. Ramalho TR, Oliveira MT, Lima AL, Bezerra-Santos CR, 32. Zu Y, Yu H, Liang L, Fu Y, Efferth T, Liu X, et al. Ac-
Piuvezam MR. Gamma-terpinene modulates acute in- tivities of ten essential oils towards Propionibacterium
flammatory response in mice. Planta Med 2015;81:1248– acnes and PC-3, A-549 and MCF-7 cancer cells. Molecules
1254. 2010;15:3200–3210.
27. Games E, Guerreiro M, Santana FR, Pinheiro NM, de 33. Hotta M, Nakata R, Katsukawa M, Hori K, Takahashi S,
Oliveira EA, Lopes FD, et al. Structurally related mono- Inoue H. Carvacrol, a component of thyme oil, activates
terpenes p-cymene, carvacrol and thymol isolated from PPARa and c and suppresses COX-2 expression. J Lipid
essential oil from leaves of Lippia sidoides Cham. (Ver- Res 2010;51:132–139.
benaceae) protect mice against elastase-induced emphy- 34. Nesan D, Vijayan MM. Embryo exposure to elevated cor-
sema. Molecules 2016;21:E1390. tisol level leads to cardiac performance dysfunction in
28. Lee SH, Kang JW, Lin T, Lee JE, Jin DI. Teratogenic zebrafish. Mol Cell Endocrinol 2012;363:85–91.
potential of antiepileptic drugs in the zebrafish model. 35. Zhang N, Wang W, Li W, Liu C, Chen Y, Yang Q, et al.
Biomed Res Int 2013;2013:726478. Inhibition of 11b-HSD2 expression by triclosan via in-
29. Dai W, Sun C, Huang S, Zhou Q. Carvacrol suppresses duction of apoptosis in human placental syncytiotropho-
proliferation and invasion in human oral squamous cell blasts. J Clin Endocrinol Metab 2015;100:E542–E549.
carcinoma. Onco Targets Ther 2016;9:2297–2304.
30. Ordas A, Raterink R, Cunningham F, Jansen H, Wiweger Address correspondence to:
Downloaded by North Carolina Agr & Tech State Univ from www.liebertpub.com at 05/30/18. For personal use only.