International Journal of

Molecular Sciences

Article
Fast-Acting and Receptor-Mediated Regulation of
Neuronal Signaling Pathways by Copaiba
Essential Oil
Yasuyo Urasaki 1 , Cody Beaumont 2 , Michelle Workman 2 , Jeffery N. Talbot 1 , David K. Hill 2
and Thuc T. Le 1, *
1 College of Pharmacy, Roseman University of Health Sciences, 10530 Discovery Drive, Las Vegas, NV 89135,
USA; [email protected] (Y.U.); [email protected] (J.N.T.)
2 dōTERRA International, LLC, 389 South 1300 West, Pleasant Grove, UT 84062, USA;
[email protected] (C.B.); [email protected] (M.W.); [email protected] (D.K.H.)
* Correspondence: [email protected]; Tel.: +1-702-802-2820




Received: 10 February 2020; Accepted: 24 March 2020; Published: 25 March 2020


Abstract: This study examined the biological activities of copaiba essential oil via measurement of its
effects on signaling pathways in the SH-SY5Y neuronal cell line. Nanofluidic proteomic technologies
were deployed to measure the phosphorylation of biomarker proteins within the signaling cascades.
Interestingly, copaiba essential oil upregulated the pI3K/Akt/mTOR, MAPK, and JAK/STAT signaling
pathways in neuronal cells. The effects of copaiba essential oil peaked at 30 min post-treatment, with a
half-maximal effective concentration (EC50 ) of approximately 80 ng/mL. Treatment with cannabinoid
receptor 2 (CB2) agonist AM1241 or the inverse agonist BML190 abrogated the regulatory effects of
copaiba essential oil on the pI3K/Akt/mTOR signaling pathway. Surprisingly, copaiba essential oil
also activated the apoptosis signaling pathway and reduced the viability of SH-SY5Y cells with an
EC50 of approximately 400 ng/mL. Furthermore, β-caryophyllene, a principal constituent of copaiba
essential oil, downregulated the pI3K/Akt/mTOR signaling pathway. Taken together, the findings
indicated that copaiba essential oil upregulated signaling pathways associated with cell metabolism,
growth, immunity, and apoptosis. The biological activities of copaiba essential oil were determined to
be fast acting, CB2 mediated, and dependent on multiple chemical constituents of the oil. Nanofluidic
proteomics provided a powerful means to assess the biological activities of copaiba essential oil.

Keywords: apoptosis; β-caryophyllene; capillary isoelectric focusing; copaiba essential oil;

nanofluidic proteomics; protein post-translational modification; neuronal signaling pathways;
pI3K/Akt/mTOR; JAK/STAT; MAPK
Int. J. Mol. Sci. 2020, 21, 2259 2 of 15

Recently, there has been immense interest in the therapeutic potential of copaiba essential oil and its
principal constituents [8].
The use of copaiba essential oil for drug development faces numerous challenges with regard to
quality control. Similar to those of many botanical extracts, the chemical profile of copaiba essential
oil varies depending on the botanical species, geography, soil composition, and rainfall index [2,8].
Furthermore, copaiba oleoresin is harvested through perforation of the trunk and collection of the
oleoresin that drips out. Commercially traded copaiba oleoresin is often a mixture of oleoresin
from many trees and species, which complicates the botanical species identification and reduces the
manufacturing precision of copaiba essential oil. A broad range of analytical methods coupled with
sophisticated chemometrics have been developed to identify chemical variations in copaiba essential
oil [8–11]. However, how such chemical variations affect the biological activities and the safety and
efficacy profiles of copaiba essential oil remains unclear.
In this study, the biological activities of copaiba essential oil were evaluated via measurement of
the effects of the essential oil on the signaling pathways in a cultured SH-SY5Y human neuroblastoma
cell line. The SH-SY5Y cell line is a model system for neuronal signaling research due to its conservation
of genes and pathways associated with disease pathogenesis [12]. Signaling pathways are composed
of series of protein kinases that relay extracellular stimuli to trigger cellular responses. Measurement
of the effects of copaiba essential oil on signaling pathways was chosen as a robust means of biological
activity assessment. The effects of copaiba essential oil on SH-SY5Y cells were characterized using
nanofluidic protein post-translational modification (PTM) profiling technologies, which measure
changes in the PTM profiles of signaling proteins [13,14]. Briefly, total protein extracts of SH-SY5Y cells
were separated by charge or size using capillary isoelectric focusing (cIEF) or Western immunoassays in
matrix-filled capillaries, respectively [15]. Changes in the isoelectric points or in the binding of primary
antibodies that recognize specific modification sites were used to measure protein PTMs. Previously,
nanofluidic protein PTM profiling had been deployed to identify aberrant signaling pathways in
diseased tissue biopsies [16–22] and to assess the quality of essential oils [23] and cannabidiol oils [24].
Here, nanofluidic protein PTM profiling was deployed to characterize the effects of copaiba essential
oil on neuronal signaling pathways.

2. Results

2.1. Chemical Composition of Copaiba Essential Oils

Copaiba essential oil was extracted by steam distillation of oleoresins collected from
Copaifera reticulata, Copaifera officinalis, Copaifera coriacea, and Copaifera langsdorffii. Ten copaiba essential
oil samples with different batch numbers were analyzed with gas chromatography coupled with mass
spectrometry (GC–MS) for their terpene compositions. Out of approximately 60 terpenes identified
in copaiba essential oil, eleven major terpenes are summarized in Figure 1A and Table 1, including
α-cubebene, α-copaene, β-elemene, β-caryophyllene, γ-elemene, α-bergamotene, α-humulene,
trans-cadina-1(6),4-diene, germacrene D, β-bisabolene, and ∆-cadinene. Collectively, these eleven
terpenes accounted for approximately 88% of the total terpenes in copaiba essential oil. Notably,
β-caryophyllene was the dominant terpene; on average, it constituted approximately 51% of the
total terpenes in copaiba essential oil (Figure 1B). Four other major terpenes, including α-copaene,
α-bergamotene, α-humulene, and germacrene D, collectively accounted for approximately 25% of the
total terpenes in copaiba essential oil (Table 1).
Int. J. Mol. Sci. 2020, 21, 2259 3 of 15
Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 15

A B
60

50

% Terpenes
40

30

20

10

Figure 1. Terpene composition of copaiba essential oil. (A) Distribution of 11 major terpenes in 10
Figure
different1.batches
Terpeneof composition of copaiba
copaiba essential essential
oil. Terpene oil. (A) Distribution
composition was analyzedofwith
11 major terpenes in 10
gas chromatography
different batches of copaiba essential oil. Terpene composition was analyzed
coupled with mass spectrometry. (B) Percentages of 11 major terpenes in copaiba essential with gas
oil. The
chromatography coupled with mass spectrometry. (B) Percentages of 11 major terpenes in
error bars represent the standard deviations across 10 different batches of copaiba essential oil. copaiba
essential oil. The error bars represent the standard deviations across 10 different batches of copaiba
essential oil. Table 1. Terpene composition of copaiba essential oil.

% Terpenes in Ten Different Batches of Copaiba Essential Oil

Table 1. Terpene composition of copaiba essential oil.
%, %, %, %, %, %, %, %, %, %,
Terpenes
180437 1722278 %182066A
Terpenes 51911 51912 Batches
in Ten Different 60326 of Copaiba
63800 Essential
55172 Oil 1924111 73599
α-Cubebene %,
1.51 %,
1.56 %,
1.33 1.35%, 1.33 %, 1.09 %, 1.73 %, 2.47 %, 1.31 %, 1.57 %,
Terpenes
α-Copaene 7.19
180437 7.43
1722278 5.97
182066A 7.73
51911 7.7451912 5.8360326 10.0963800 9.8455172 7.3619241118.24 73599
β-Elemene
α-Cubebene 1.67
1.51 1.44
1.56 1.28
1.33 1.591.35 1.591.33 1.23 1.09 1.7 1.73 1.56 2.47 1.56 1.31 1.45 1.57
β-Caryophyllene
α-Copaene 45.24
7.19 41.89
7.43 49.1
5.97 54.427.73 55 7.74 52.165.83 58.1810.09 47.099.84 54.42 7.36 49.07 8.24
γ-Elemene
β-Elemene 2.26
1.67 2.82
1.44 1.76
1.28 1.81.59 1.811.59 1.76 1.23 1.36 1.7 1.7 1.56 1.83 1.56 1.9 1.45
α-Bergamotene 7.19 8.51 8.3 6.66 6.72 7.68 6.17 7.12 6.67 8.07
β-Caryophyllene 45.24 41.89 49.1 54.42 55 52.16 58.18 47.09 54.42 49.07
α-Humulene 7.18 6.67 8.25 6.01 6.1 6.64 4.88 5.18 6.27 5.75
ɣ-Elemene 2.26 2.82 1.76 1.8 1.81 1.76 1.36 1.7 1.83 1.9
trans-Cadina-
α-Bergamotene 2.03
7.19 2.67
8.51 1.98
8.3 1.366.66 1.366.72 1.56 7.68 1.01 6.17 1.67 7.12 1.51 6.67 1.59 8.07
1(6),4-Diene
α-Humulene
Germacrene D 7.18
5.16 6.67
1.2 8.25
4.57 4.156.01 4.23 6.1 5.45 6.64 4.12 4.88 3.28 5.18 4.29 6.27 4.76 5.75
trans-Cadina-
β-Bisabolene 3.47 4.89 2.52 2.321.36 2.351.36 2.98 1.56 1.36 1.01 3.26 1.67 2.61 1.51 3.77 1.59
2.03 2.67 1.98
∆-Cadinene
1(6),4-Diene 2.78 3.88 2.3 2.14 2.14 2.5 1.64 3.17 2.36 2.75
Germacrene D 5.16 1.2 4.57 4.15 4.23 5.45 4.12 3.28 4.29 4.76
β-Bisabolene 3.47 4.89 2.52 2.32 2.35 2.98 1.36 3.26 2.61 3.77
2.2. Time-Dependent2.78
Δ-Cadinene Positive 3.88
Regulation2.3
of the pI3K/Akt/mTOR
2.14 2.14 Signaling
2.5 Pathway
1.64 3.17 2.36 2.75

One random copaiba essential oil batch was selected to study its effects on the pI3K/Akt/mTOR
2.2. Time-Dependent
signaling Positiveregulates
pathway, which Regulation of the pI3K/Akt/mTOR
neuronal Signalingand
cell growth, longevity, Pathway
energy metabolism [25,26].
Following the treatment
One random copaiba of essential
SH-SY5Yoil cells withwas
batch 100selected
ng/mL copaiba
to studyessential oil,
its effects ontotal
the cell extracts were
pI3K/Akt/mTOR
collected at specific time points and then assayed for protein PTM profiles.
signaling pathway, which regulates neuronal cell growth, longevity, and energy metabolism Specifically, the protein
[25,26].
PTM profiles
Following the of Akt1, Akt2,
treatment Akt3, mTOR
of SH-SY5Y and100
cells with p70S6K
ng/mL were measured
copaiba with
essential oil,either cIEF
total cell or Western
extracts were
immunoassays.
collected Using
at specific cIEF
time immunoassays,
points a singlefor
and then assayed primary
proteinantibody againstSpecifically,
PTM profiles. pan-Akt was sufficient
the protein
to resolve
PTM the of
profiles distribution
Akt1, Akt2, of Akt3,
Akt1, mTOR
Akt2, andandAkt3
p70S6Kphosphoisoforms
were measured (Figure 2A and
with either Figure
cIEF S1A,B).
or Western
Primary antibodies
immunoassays. against
Using cIEFAkt1, Akt2, or Akt3
immunoassays, were also
a single used,antibody
primary and theiragainst
phosphoisoform
pan-Akt was profiles were
sufficient
in agreement with those obtained with the pan-Akt primary antibody (Figure S2A–C).
to resolve the distribution of Akt1, Akt2, and Akt3 phosphoisoforms (Figures 2A and Figure S1A,B). In addition,
capillary antibodies
Primary Western immunoassays
against Akt1,were
Akt2,used to measure
or Akt3 the expression
were also used, and of mTOR
their (Ser2448) and profiles
phosphoisoform p70S6K
(Thr389)
were in phosphoisoforms
agreement with those (Figure 2B). Interestingly,
obtained the levelsprimary
with the pan-Akt of the phosphoisoforms
antibody (Figure of Akt1,
S2A-C).Akt2,
In
Akt3, mTOR, and p70S6K increased and peaked at 30 min after treatment with
addition, capillary Western immunoassays were used to measure the expression of mTOR (Ser2448) copaiba essential oil
before
and steadily
p70S6K declining
(Thr389) over the next 24(Figure
phosphoisoforms h (Figure 2C).
2B). Among the the
Interestingly, Aktlevels
isoforms,
of thecopaiba essential oil
phosphoisoforms
of Akt1, Akt2, Akt3, mTOR, and p70S6K increased and peaked at 30 min after treatment with copaiba
essential oil before steadily declining over the next 24 h (Figure 2C). Among the Akt isoforms, copaiba
Int. J. Mol. Sci. 2020, 21, 2259 4 of 15

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 15

induced greater effects on the phosphorylation of the Akt1 isoform than on the Akt2 and Akt3 isoforms.
essential oil induced greater effects on the phosphorylation of the Akt1 isoform than on the Akt2 and
Clearly, copaiba essential oil induced time-dependent positive regulation of the pI3K/Akt/mTOR
Akt3 isoforms. Clearly, copaiba essential oil induced time-dependent positive regulation of the
signaling pathway in the SH-SY5Y neuronal cell line.
pI3K/Akt/mTOR signaling pathway in the SH-SY5Y neuronal cell line.

A B
3000

ppAkt3

30 min
0 min; 30 min; 1 hr;

0 min

24 hr
16 hr
1 hr

4 hr
Chemiluminescence

2500 4 hr; 16 hr; 24 hr

pppppAkt1

pppAkt1

2000
ppppAkt1

ppAkt1

1500 mTOR
pppAkt3

ppAkt2/pAkt1

pAkt3
ppppppAkt1

1000

pAkt2
500

0 p-mTOR
(Ser2448)
5.00 5.25 5.50 5.75 6.00 6.25
pI
C
2.75
p-Akt1
Relative concentration

2.50 p70S6K
p-Akt2
2.25
p-Akt3
2.00
p-mTOR
1.75
p-p70S6K
1.50
p-p70S6K
1.25
(Thr389)
1.00
0.75
β-actin
0 4 8 12 16 20 24
Time after treatment (hr)

Figure2.2.Time-dependent
Figure Time-dependentpositive
positiveregulation
regulationof ofthe
thePI3K/Akt/mTOR
PI3K/Akt/mTORsignaling
signaling pathway
pathway by by copaiba
copaiba
essential oil. (A) Pan-Akt profiles and (B) expression levels of the mTOR
essential oil. (A) Pan-Akt profiles and (B) expression levels of the mTOR and p70S6Kand p70S6K phosphoisoforms
inphosphoisoforms
SH-SY5Y cells asinfunctions
SH-SY5Yof time
cells asafter treatment.
functions of time(C) Relative
after concentrations
treatment. (C) Relativeofconcentrations
the Akt1 (blue),of
Akt2 (orange),
the Akt1 Akt3
(blue), (gray),
Akt2 mTOR
(orange), (yellow),
Akt3 (gray),and p70S6K
mTOR (purple)
(yellow), andphosphoisoforms in SH-SY5Y cells
p70S6K (purple) phosphoisoforms
asinfunctions
SH-SY5Yofcells
timeasafter treatment.
functions Theafter
of time relative concentration
treatment. describes
The relative the fold change
concentration in a the
describes protein
fold
phosphoisoform
change in a proteinafter treatment compared
phosphoisoform to the control
after treatment condition.
compared The error
to the control bars areThe
condition. the error
standard
bars
deviations of six repeated
are the standard measurements
deviations of six repeated permeasurements
experimental condition. SH-SY5Y
per experimental cells were
condition. treated with
SH-SY5Y cells
100 ng/mL
were copaiba
treated with essential
100 ng/mL oil.copaiba essential oil.

2.3.
2.3.Dose-Dependent
Dose-DependentPositive
PositiveRegulation
Regulationofofthe
thepI3K/Akt/mTOR
pI3K/Akt/mTORSignaling
SignalingPathway
Pathway
Dose
Dosedependency
dependency assays were performed
assays were performedto tofurther
furtherexamine
examinethethe effects
effects of of copaiba
copaiba essential
essential oil
oil
on the pI3K/Akt/mTOR signaling pathway. SH-SY5Y cells were treated with serial dilutions of
on the pI3K/Akt/mTOR signaling pathway. SH-SY5Y cells were treated with serial dilutions of
copaiba essential oil for 30 min and then assayed for the protein PTM profiles. A positive
copaiba essential oil for 30 min and then assayed for the protein PTM profiles. A positive correlation correlation
between
betweenthe concentration
the concentration of copaiba essential
of copaiba oil and
essential oil the
andphosphorylation of Akt1,
the phosphorylation ofmTOR, and p70S6K
Akt1, mTOR, and
was observed (Figure 3A–C). The half-maximal effective concentration
p70S6K was observed (Figure 3A–C). The half-maximal effective concentration (EC 50 ) of copaiba
(EC50) ofessential
copaiba
oil for activation
essential oil for of the pI3K/Akt/mTOR
activation pathway was
of the pI3K/Akt/mTOR approximately
pathway 80 ng/mL. Dose
was approximately dependency
80 ng/mL. Dose
assays
dependency assays further confirmed the positive regulation of the pI3K/Akt/mTORbysignaling
further confirmed the positive regulation of the pI3K/Akt/mTOR signaling pathway copaiba
essential
pathwayoil.by copaiba essential oil.
Int. J. Mol. Sci. 2020, 21, 2259 5 of 15
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A B
ppAkt3 0 ng/ml 0 20 40 80 160 320 ng/ml

pppppAkt1

pppAkt1
20 ng/ml
Chemiluminescence

1000 40 ng/ml
80 ng/ml

ppppAkt1
160 ng/ml mTOR

ppAkt1
pppAkt3

pAkt3
ppAkt2/pAkt1
ppppppAkt1 320 ng/ml
500

pAkt2
p-mTOR
(Ser2448)
0
5.00 5.25 5.50 5.75 6.00 6.25
pI
C
2.50
p-Akt1 p70S6K
Relative concentration

2.25 p-Akt2
p-Akt3
2.00
p-mTOR
1.75 p-p70S6K

1.50 p-p70S6K
1.25 (Thr389)

1.00 β-actin
0.75
0 40 80 120 160 200 240 280 320
Copaiba (ng/ml)

Figure 3. Dose-dependent positive regulation of the PI3K/Akt/mTOR signaling pathway by copaiba

Figure 3. Dose-dependent positive regulation of the PI3K/Akt/mTOR signaling pathway by copaiba
essential oil. (A) Pan-Akt profiles and (B) expression levels of the mTOR and p70S6K phosphoisoforms
essential oil. (A) Pan-Akt profiles and (B) expression levels of the mTOR and p70S6K
as functions of copaiba essential oil dose. (C) Relative concentrations of the Akt1 (blue), Akt2 (orange),
phosphoisoforms as functions of copaiba essential oil dose. (C) Relative concentrations of the Akt1
Akt3 (gray), mTOR (yellow), and p70S6K (purple) phosphoisoforms as functions of copaiba essential
(blue), Akt2 (orange), Akt3 (gray), mTOR (yellow), and p70S6K (purple) phosphoisoforms as
oil dose. The relative concentration describes the fold change in a protein phosphoisoform after
functions of copaiba essential oil dose. The relative concentration describes the fold change in a
treatment compared to the control condition. The error bars are the standard deviations of six repeated
protein phosphoisoform after treatment compared to the control condition. The error bars are the
measurements per experimental
standard deviations condition.
of six repeated SH-SY5Y per
measurements cellsexperimental
were treated condition.
with various concentrations
SH-SY5Y of
cells were
copaiba essential oil for 30 min.
treated with various concentrations of copaiba essential oil for 30 min.
2.4. CB2-Mediated Regulation of the pI3K/Akt/mTOR Signaling Pathway
2.4. CB2-Mediated Regulation of the pI3K/Akt/mTOR Signaling Pathway
To examine the role of CB2 in the biological activities of copaiba essential oil, SH-SY5Y cells
To examine the role of CB2 in the biological activities of copaiba essential oil, SH-SY5Y cells were
were treated with copaiba essential oil in the presence of the CB2 agonist AM1241 [27] or the CB2
treated with copaiba essential oil in the presence of the CB2 agonist AM1241 [27] or the CB2 inverse
inverse agonist BML190 [28]. AM1241 and BML190 served as competitors to β-caryophyllene, a major
agonist BML190 [28]. AM1241 and BML190 served as competitors to β-caryophyllene, a major
constituent of copaiba essential oil, for CB2 binding. At 30 min after treatment with copaiba essential
constituent of copaiba essential oil, for CB2 binding. At 30 min after treatment with copaiba essential
oil, increased phosphorylation of Akt1, mTOR, and p70S6K was observed (Figure 4A–C). Treatment
oil, increased phosphorylation of Akt1, mTOR, and p70S6K was observed (Figure 4A–C). Treatment
with AM1241 or BML190 alone had no detectable effect on the phosphorylation of Akt1, mTOR, or
with AM1241 or BML190 alone had no detectable effect on the phosphorylation of Akt1, mTOR, or
p70S6K. Interestingly, treatment with copaiba essential oil together with AM1241 or BML190 abrogated
p70S6K. Interestingly, treatment with copaiba essential oil together with AM1241 or BML190
the positive effects of copaiba essential oil on the phosphorylation of Akt1, mTOR, and p70S6K.
abrogated the positive effects of copaiba essential oil on the phosphorylation of Akt1, mTOR, and
Our data indicated that CB2 mediated the effects of copaiba essential oil on the pI3K/Akt/mTOR
p70S6K. Our data indicated that CB2 mediated the effects of copaiba essential oil on the
signaling pathway.signaling pathway.
pI3K/Akt/mTOR
Int. J. Mol. Sci. 2020, 21, 2259 6 of 15
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A B

CPB/BML
ppAkt3 control

CPB/AM
AM1241

BML190
pppppAkt1

copaiba
Chemiluminescence

control
pppAkt1
copaiba
1000 AM1241

ppppAkt1
BML190

pppAkt3
CPB/AM

ppAkt1

ppAkt2/pAkt1

pAkt3
CPB/BML
ppppppAkt1

pAkt2
500 mTOR

0
5.00 5.25 5.50 5.75 6.00 6.25 p-mTOR
(Ser2448)
pI
C
2.25
* p-Akt1 p-Akt2
Relative concentration

p70S6K
2.00
p-Akt3 p-mTOR
1.75 * p-p70S6K
* *
1.50
*
p-p70S6K
1.25 (Thr389)

1.00
β-actin
0.75
Control Copaiba AM1241 BML190 CPB/AM CPB/BML

Figure 4. CB2 agonists abrogated the activation of the PI3K/Akt/mTOR signaling pathway by copaiba
essential oil. (A) Pan-Akt profiles and (B) expression levels of the mTOR and p70S6K phosphoisoforms
Figure 4. CB2 agonists abrogated the activation of the PI3K/Akt/mTOR signaling pathway by copaiba
in SH-SY5Y cells at 30 min following treatment with copaiba essential oil (100 ng/mL), AM1241
essential oil. (A) Pan-Akt profiles and (B) expression levels of the mTOR and p70S6K
(200 nM), BML190 (10 µM), 100 ng/mL copaiba essential oil together with 200 nM AM1241 (CPB/AM),
phosphoisoforms in SH-SY5Y cells at 30 min following treatment with copaiba essential oil (100
or 100 ng/mL copaiba essential oil together with 10 µM BML190 (CPB/BML). (C) Relative concentrations
ng/mL), AM1241 (200 nM), BML190 (10 µ M), 100 ng/mL copaiba essential oil together with 200 nM
of the Akt1 (blue), Akt2 (orange), Akt3 (gray), mTOR (yellow), and p70S6K (purple) phosphoisoforms
AM1241 (CPB/AM), or 100 ng/mL copaiba essential oil together with 10 µ M BML190 (CPB/BML). (C)
in SH-SY5Y cells at 30 min following treatment with copaiba essential oil, AM1241, BML190, CPB/AM,
Relative concentrations of the Akt1 (blue), Akt2 (orange), Akt3 (gray), mTOR (yellow), and p70S6K
or CPB/BML. The relative concentration describes the fold change in a protein phosphoisoform after
(purple) phosphoisoforms in SH-SY5Y cells at 30 min following treatment with copaiba essential oil,
treatment compared to the control condition. The error bars are the standard deviations of six repeated
AM1241, BML190, CPB/AM, or CPB/BML. The relative concentration describes the fold change in a
measurements per experimental condition. The asterisks indicate statistical significance for p ≤ 0.05
protein phosphoisoform after treatment compared to the control condition. The error bars are the
versus the control.
standard deviations of six repeated measurements per experimental condition. The asterisks indicate
statistical significance
2.5. Time-Dependent forRegulation
Positive p ≤ 0.05 versus
of thetheMAPK
control.
and JAK/STAT Signaling Pathways

2.5.Time-dependent positive
Time-Dependent Positive regulation
Regulation of theby copaiba
MAPK essential oil
and JAK/STAT was further
Signaling Pathwaysobserved for the
mitogen-activated protein kinase (MAPK) and JAK/STAT signaling pathways. On the one hand,
Time-dependent positive regulation by copaiba essential oil was further observed for the
the protein PTM profiles of MEK1, MEK2, and ERK1/2 were used to measure the signaling activity
mitogen-activated protein kinase (MAPK) and JAK/STAT signaling pathways. On the one hand, the
of the MAPK pathway, which regulates neuronal cell proliferation and differentiation [29]. On the
protein PTM profiles of MEK1, MEK2, and ERK1/2 were used to measure the signaling activity of the
other hand, the protein PTM profiles of STAT1, STAT3, and STAT5 were used to measure the signaling
MAPK pathway, which regulates neuronal cell proliferation and differentiation [29]. On the other
activity of the JAK/STAT pathway, which regulates neuronal innate immunity [30]. Following treatment
hand, the protein PTM profiles of STAT1, STAT3, and STAT5 were used to measure the signaling
with 100 ng/mL copaiba essential oil, the expression levels of the MEK1, MEK2, ERK1, and ERK2
activity of the JAK/STAT pathway, which regulates neuronal innate immunity [30]. Following
phosphoisoforms of the MAPK pathway and STAT1, STAT3, and STAT5 phosphoisoforms of the
treatment with 100 ng/mL copaiba essential oil, the expression levels of the MEK1, MEK2, ERK1, and
JAK/STAT pathway increased, peaked at 30 min, and then declined toward baseline levels at 24 h
ERK2 phosphoisoforms of the MAPK pathway and STAT1, STAT3, and STAT5 phosphoisoforms of
(Figure 5A–H). The
the JAK/STAT temporal
pathway regulatory
increased, peaked effects
at 30ofmin,
copaiba essential
and then oil on
declined the MAPK
toward and
baseline JAK/STAT
levels at 24
signaling pathways mirrored those on the pI3K/Akt/mTOR signaling pathway.
h (Figure 5 A–H). The temporal regulatory effects of copaiba essential oil on the MAPK and
JAK/STAT signaling pathways mirrored those on the pI3K/Akt/mTOR signaling pathway.
Int. J. Mol. Sci. 2020, 21, 2259 7 of 15
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A E
pMEK1 pSTAT1

Chemiluminescence
MEK1
Chemiluminescence

0 min 300 0 min

2000 30 min 30 min
24 hr 24 hr
200
STAT1
1000
100

0 0
5.00 5.25 5.50 5.75 6.00 6.25 5.00 5.25 5.50 5.75 6.00
pI pI
B F
pMEK2 STAT3
Chemiluminescence

Chemiluminescence
1500 0 min 0 min pSTAT3
30 min 200 30 min
24 hr 24 hr
1000

100
500
MEK2

0 0
5.00 5.25 5.50 5.75 6.00 6.25 5.00 5.25 5.50 5.75 6.00
pI pI
C G
ERK1 pSTAT5
Chemiluminescence

Chemiluminescence

10000 0 min 0 min

100
30 min ERK2 30 min
24 hr 24 hr
pERK1
ppERK2

5000 ppERK1 50
STAT5

pERK2
0 0
5.00 5.50 6.00 6.50 7.00 5.00 5.25 5.50 5.75 6.00
pI pI
D H
4.5
Relative concentration

4.5
Relative concentration

0 min 30 min 24 hr * 0 min 30 min 24 hr

*
3.5 3.5
*
2.5 2.5 *
* *
* * *
1.5 1.5

0.5 0.5
p-MEK1 p-MEK2 p-ERK1/2 p-STAT1 p-STAT3 p-STAT5

Figure 5. Time-dependent
Figure 5. activationofofthe
Time-dependent activation theMAPK
MAPKand andJAK/STAT
JAK/STATsignaling
signalingpathways
pathways byby copaiba
copaiba
essential
essential oil. (A–C) MEK1
oil. (A–C) MEK1 (A),(A),MEK2
MEK2(B),(B),and
andERK1/2
ERK1/2(C)(C)profiles
profilesasas functions
functions of of time
time after
after treatment.
treatment.
(D)
(D)Relative
Relativeconcentrations
concentrationsofofthe theMEK1,
MEK1,MEK,MEK,andandERK1/2
ERK1/2 phosphoisoforms
phosphoisoforms asas
a function
a function of of
time after
time
treatment. (E–H) STAT1 (E), STAT3 (F), and STAT5 (G) profiles as functions
after treatment. (E–H) STAT1 (E), STAT3 (F), and STAT5 (G) profiles as functions of time after of time after treatment.
(H) Relative(H)
treatment. concentrations of the STAT1,ofSTAT3,
Relative concentrations and STAT5
the STAT1, STAT3, phosphoisoforms as functions of time
and STAT5 phosphoisoforms as
after treatment.
functions of timeThe relative
after concentration
treatment. describes
The relative the fold change
concentration in athe
describes protein phosphoisoform
fold change in a proteinafter
treatment compared
phosphoisoform to treatment
after the controlcompared
condition.toThe
the error
controlbars are the standard
condition. The errordeviations
bars are the of six repeated
standard
measurements
deviations of sixper experimental
repeated condition.
measurements The asteriskscondition.
per experimental indicate statistical significance
The asterisks for p ≤ 0.05
indicate statistical
versus the control.
significance for p ≤SH-SY5Y
0.05 versuscellsthe
were treatedSH-SY5Y
control. with 100cells
ng/mL werecopaiba
treatedessential
with 100oil.ng/mL copaiba
essential oil.
2.6. Time-Dependent Activation of the Apoptosis Signaling Pathway
2.6. Time-Dependent Activation
Interestingly, treatment of the
with Apoptosis
copaiba Signaling
essential Pathway
oil also activated the apoptosis signaling pathway in
SH-SY5Y cells. Apoptosis
Interestingly, is awith
treatment programmed sequence
copaiba essential oilofalso
events that lead
activated to cell death.
the apoptosis The PTM
signaling profiles
pathway
of the Akt1, BID, caspase 3, and caspase 7 proteins were used to monitor the activity of
in SH-SY5Y cells. Apoptosis is a programmed sequence of events that lead to cell death. The PTM the apoptosis
profiles of the Akt1, BID, caspase 3, and caspase 7 proteins were used to monitor the activity of the
Int. J. Mol. Sci. 2020, 21, 2259 8 of 15

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 15

signaling pathway [31]. Following treatment with 100 ng/mL copaiba essential oil, the expression
apoptosis signaling pathway [31]. Following treatment with 100 ng/mL copaiba essential oil, the
levels of Akt1, BID, caspase 3 and caspase 7 phosphoisoforms increased significantly, peaked at 30 min,
expression levels of Akt1, BID, caspase 3 and caspase 7 phosphoisoforms increased significantly,
and then declined toward baseline levels at 24 h (Figure 6A–E). Consistently, treatment of SH-SY5Y
peaked at 30 min, and then declined toward baseline levels at 24 h (Figure 6A–E). Consistently,
cells with copaiba essential oil for 24 h reduced the viability of the cells with an EC50 of approximately
treatment of SH-SY5Y cells with copaiba essential oil for 24 h reduced the viability of the cells with
400
anng/mL
EC50 of(Figure 6F). The 400
approximately cytotoxic
ng/mLeffects
(Figure of 6F).
copaiba
The essential
cytotoxic oil are consistent
effects of copaibawith the reported
essential oil are
antiproliferative
consistent with activities of copaiba
the reported oleoresin activities
antiproliferative and its constituents on a broad
of copaiba oleoresin range
and of human cancer
its constituents on a
cell linesrange
broad [2,8].of human cancer cell lines [2,8].

A B
1000 pppAkt1 pBID

Chemiluminescence
0 min
Chemiluminescence

ppppAkt1

pppppAkt1 0 min
ppAkt1 30 min 30 min
24 hr 1000 24 hr
ppppppAkt1

500
500
pAkt1

Akt1 BID
0 0
5.00 5.25 5.50 5.75 6.00 6.25 4.75 5.00 5.25 5.50
pI pI
C D
1500 pCaspase 3
Chemiluminescence

pCaspase 7
Chemiluminescence

0 min 0 min
150
30 min 30 min
1000 24 hr 24 hr
100 Caspase 7

500 50
Caspase 3
0 0
5.00 5.25 5.50 5.75 6.00 6.25 5.00 5.25 5.50 5.75 6.00 6.25
pI pI
E F
5.5 120
Relative concentration

0 min 30 min 24 hr * 100

Cell viability (%)

4.5
80
3.5
* 60
2.5
*
* 40
1.5 * 20
0.5 0
p-Akt1 p-BID p-Cas3 p-Cas7 1 10 100 1000 10000 100000
Copaiba (ng/ml)

Figure 6. Time-dependent positive regulation of the apoptosis signaling pathway by copaiba essential
Figure 6. Time-dependent positive regulation of the apoptosis signaling pathway by copaiba essential
oil. (A–D) Akt1 (A), BID (B), caspase 3 (C), and caspase 7 (D) profiles as functions of time after
oil. (A-D) Akt1 (A), BID (B), caspase 3 (C), and caspase 7 (D) profiles as functions of time after
treatment of SH-SY5Y cells with 100 ng/mL copaiba essential oil. (E) Relative concentrations of p-Akt1,
treatment of SH-SY5Y cells with 100 ng/mL copaiba essential oil. (E) Relative concentrations of p-
caspase 3, and caspase 7 as functions of time after treatment with 100 ng/mL copaiba essential oil for
Akt1, caspase 3, and caspase 7 as functions of time after treatment with 100 ng/mL copaiba essential
0 min (blue), 30 min (orange), and 24 h (gray). (F) Viability percentages of SH-SY5Y cells as functions
oil for 0 min (blue), 30 min (orange), and 24 h (gray). (F) Viability percentages of SH-SY5Y cells as
of the concentrations of copaiba essential oil. The relative concentration describes the fold change in
functions of the concentrations of copaiba essential oil. The relative concentration describes the fold
a protein phosphoisoform after treatment compared to the control condition. The error bars are the
change in a protein phosphoisoform after treatment compared to the control condition. The error bars
standard deviations of six repeated measurements per experimental condition. The asterisks indicate
are the standard deviations of six repeated measurements per experimental condition. The asterisks
statistical significance for p ≤ 0.05 versus the control.
indicate statistical significance for p ≤ 0.05 versus the control.
3. Discussion
3. Discussion
In this study, we found that copaiba essential oil positively regulated multiple signaling pathways
In this
in neuronal study,
cells we found
in a time- that copaiba essential
and dose-dependent manner. oil
Thepositively
signaling regulated multiple signaling
pathways positively regulated
pathways in neuronal cells in a time- and dose-dependent manner. The signaling
by copaiba essential oil included the pI3K/Akt/mTOR, MAPK, and JAK/STAT pathways, whichpathways positively
regulate
regulated
neuronal by copaiba essential
metabolism, oil included
proliferation the pI3K/Akt/mTOR,
and immunity, respectively. MAPK, and JAK/STAT
The positive pathways,
regulatory effects of
which regulate neuronal metabolism, proliferation and immunity, respectively. The positive
copaiba essential oil peaked at 30 min with an EC50 of approximately 80 ng/mL and were mediated in
regulatory effects of copaiba essential oil peaked at 30 min with an EC50 of approximately 80 ng/mL
Int. J. Mol. Sci. 2020, 21, 2259 9 of 15

part by CB2. The positive effects of copaiba essential oil on neuronal signaling pathways were consistent
with its reported functions in metabolism [32], wound healing [33–36], and anti-inflammation [37–41].
Interestingly, copaiba essential oil also activated the apoptosis signaling pathway in a time-dependent
manner and reduced the viability of neuronal cells with an EC50 of approximately 400 ng/mL. This
observation is consistent with the reported anticancer effects of copaiba essential oil [42,43]. Future
evaluation of the therapeutic potential of copaiba essential oil should take into consideration its
toxicity profile.
Notably, we observed differential regulation of signaling pathways by copaiba essential oil in
different cell types. Previously, we have reported that copaiba essential oil upregulates the MAPK and
JAK/STAT signaling pathways in the HepG2 hepatocyte cell line [23]. The positive regulatory effects of
copaiba essential oil on the MAPK and JAK/STAT signaling pathways were conserved in the SH-SY5Y
neuronal cell line. In contrast, we found that copaiba essential oil had opposite regulatory effects on
the pI3K/Akt/mTOR signaling pathway in the two different cell lines, causing activation in SH-SY5Y
cells and suppression in HepG2 cells. The precise mechanism underlying this differential regulation in
different cell types is unclear. However, we found that the composition of Akt isoforms was different
between liver and brain cells. In SH-SY5Y neuronal cells, all three Akt isoforms, Akt1, Akt2, and
Akt3 were present with 40%, 10%, and 50% distributions, respectively. In contrast, in HepG2 liver
cells, Akt3 was completely absent, and Akt1 and Akt2 were present with 90% and 10% distributions,
respectively (Figure S3). Akt isoforms have both overlapping and distinctive functional specificity
and tissue distribution [44]. Akt1 regulates cell survival, growth, and proliferation; Akt2 regulates
glucose metabolism; and Akt3 regulates neuronal development. In particular, Akt3 also regulates ion
and cell volume homeostasis via the WNK1/SGK1 signaling pathway [45,46]. It is plausible that Akt3
is responsible for the differential regulation of the pI3K/Akt/mTOR signaling pathway in liver and
brain cells, although further investigation is warranted.
Furthermore, we continued to observe differential effects on cell signaling pathways between
copaiba essential oil and its principal constituent, β-caryophyllene. We have previously reported
opposite regulatory effects of copaiba essential oil and β-caryophyllene on the JAK/STAT signaling
pathway in HepG2 liver cells [23]. In this study, we found that treatment with isolated
β-caryophyllene negatively regulated the pI3K/Akt/mTOR signaling pathway in a dose-dependent
manner (Figure S4A–C). Isolated β-caryophyllene reduced the viability of SH-SY5Y cells with an
EC50 of approximately 1.25 µg/mL, which might contribute to the observed toxicity profile of copaiba
essential oil (Figure S4D). Interestingly, direct interaction with CB2 by β-caryophyllene, AM1241,
or BML190 was insufficient to activate the pI3K/Akt/mTOR signaling pathway in neuronal cells
(Figures S4A–C and S5A,B). The opposite effects of β-caryophyllene and copaiba essential oil on the
pI3K/Akt/mTOR signaling pathway suggest a collective behavior of copaiba essential oil resulting
from complex interactions of its many constituents. Indeed, evidence from the literature indicates that
synergy between multiple constituents of copaiba essential oil is critical to the biological effects of
the oil [47,48]. In addition to synergism, antagonism of chemical compounds also occurs in botanical
extracts [49]. Future studies on individual terpenes or combinations of terpenes in copaiba essential oil
could lead to in-depth understanding of their synergistic, potentiative, or antagonistic effects.
Finally, the effects of copaiba essential oil on neuronal signaling pathways described herein
provide critical information for future design of therapeutic applications. First, copaiba essential oil
exhibited a fast-acting mechanism, exerting maximal effects on neuronal signaling pathways within
30 min of treatment. Identification of this unique characteristic could assist in selection of dosing
frequency and evaluation of the pharmacokinetics of copaiba essential oil. Second, the effects of
copaiba essential oil on neuronal signaling pathways were mediated by CB2. This observation is
consistent with the existing literature and supports the suitability of copaiba essential oil for pain relief
and anti-inflammatory therapeutic applications [32]. Third, copaiba essential oil exerted differential
effects on the pI3K/Akt/mTOR signaling pathway in different cell types. This novel observation
suggests that systematic characterization of the effects of copaiba essential oil in various tissues is
Int. J. Mol. Sci. 2020, 21, 2259 10 of 15

necessary to select the most suitable route of administration. Finally, nanofluidic protein PTM profiling
permitted precise and quantitative measurement of the effects of copaiba essential oil on neuronal
signaling pathways. Future integration of nanofluidic protein PTM profiling and chemical composition
analysis could improve the quality control of copaiba essential oil by identifying permissible chemical
variations that do not affect its biological activities. The proven capabilities of nanofluidic protein
PTM profiling technology, including its ultrasensitivity, high reproducibility, high throughput and
automatic operation [14,15], will be invaluable for preclinical quality testing and clinical evaluation of
the pharmacodynamics of copaiba essential oil.

4. Materials and Methods

4.1. Copaiba Essential Oil

Ten copaiba essential oil batches with different batch numbers were obtained from dōTERRA
International (Pleasant Grove, UT, USA). The terpene compositions of the copaiba essential oil batches
are listed in Table 1. Copaiba essential oil with batch number 180437 was randomly selected to study
the effects of this oil on neuronal signaling pathways.

4.2. GC–MS Method for Copaiba Essential Oil Analysis

The chemical composition of copaiba essential oil was analyzed using a gas chromatograph
(Agilent 6890N, Santa Clara, CA, USA) coupled with a mass spectrometer (Agilent 5973 Network).
The samples were loaded with an automatic injector (Agilent 7683). Sample preparation consisted of
dissolving 0.02 mL of copaiba essential oil in 1.0 mL of hexane. Each sample injection was repeated
three times. An Agilent DB-5MS capillary column with a length and diameter of 30 m and 0.25 mm,
respectively, was used. The stationary phase film thickness was 0.25 µm. The flow rate of the carrier
gas, nitrogen, was 1.2 mL/min. The temperatures of the injector, ion source, and quadrupole were
250 ◦ C, 230 ◦ C, and 150 ◦ C, respectively. Samples of 3 µL were injected in split mode (40:1). The
analyses were carried out in scan mode over an m/z range of 40–500. The time of analyte allocation was
52 min. The column temperature program was 40 ◦ C for 3 min followed by 80 ◦ C for 2 min, 120 ◦ C for
5 min, 200 ◦ C for 2 min, and 250 ◦ C for another 2 min. ChemStation software was used to collect and
process the data. The compounds in the samples were identified by comparing the mass spectra with
standard spectra from the NIST 2014 library. Retention indices were determined. Compounds with
mass spectra showing more than 95% conformity with the standard library spectra were considered.
To confirm the identification, the retention indices of the analyzed compounds were compared with
the literature. The relative content percentages of the analyzed compounds were based on the peak
area of the total ionic current of all compounds present in each sample.

4.3. Cell Line and Culture Conditions

SH-SY5Y cells (cat. no. CRL2266, American Type Culture Collection, Manassas, VA, USA) were
cultured in a 1:1 mixture of Eagle’s minimum essential medium (cat. no. 302003, ATCC) and F12
medium (cat. no. 302004, ATCC) supplemented with 10% fetal bovine serum (cat. no. SH30088.03, GE
Healthcare Life Sciences, Pittsburgh, PA, USA).

4.4. Treatment Conditions

SH-SY5Y cells were grown in culture medium to approximately 70% confluence. The culture
medium was replaced with culture media premixed with copaiba essential oil (100 ng/mL) or serial
dilutions of copaiba essential oil, the CB2 agonist AM1241 (200 nM, Selleckchem, Houston, TX, USA),
the CB2 inverse agonist BML190 (10 µM, Selleckchem), copaiba essential oil (100 ng/mL) and AM1241
(200 nM), or copaiba essential oil (100 ng/mL) and BML190 (10 µM). The incubation time in the
new culture medium was used as the time after treatment. AM1241 (Ki = 3.4 nM) and BML190
(Ki = 435 nM) were used as competitors to β-caryophyllene (Ki = 155 nM) for CB2 binding [4,28,50,51].
Int. J. Mol. Sci. 2020, 21, 2259 11 of 15

The concentrations of AM1241 at 200 nM and BML190 at 10 µM were chosen to match or exceed that of
β-caryophyllene at approximately 250 nM in 100 ng/mL copaiba essential oil. Treatment of SH-SY5Y
cells with broad ranges of concentrations of AM1241 (0.1 nM – 10 µM) and BML190 (10 nM – 100 µM)
were also evaluated [28,50,51]. Data on selected doses of AM1241 and BML190 are presented in the
Figure S5.

4.5. Preparation of Cell Lysates

Approximately one million SH-SY5Y cells were incubated on ice for 10 min with 60 µL of lysis
buffer (cat. no. 040-764, ProteinSimple, Santa Clara, CA, USA), sonicated for 5 s 4 times, mixed by
rotation for 2 h at 4 ◦ C, and centrifuged at 12,000 rpm in an Eppendorf 5430R microfuge for 20 min at
4 ◦ C. The supernatant was collected as the cell lysate. The total protein concentration in the cell lysate
was determined with a Bradford protein assay and adjusted to a final concentration of 0.3 µg/µL with
the separation gradients (cat. no. Premix G2, pH 5–8 or pH 3–10, ProteinSimple) for charge-based
cIEF immunoassays or to 0.4 µg/µL with denaturing buffers (cat. no. PS-ST01EZ or PS-ST03EZ,
ProteinSimple) for size-based Western immunoassays.

4.6. Antibodies and Biomarker Proteins

The primary and secondary antibodies and biomarker proteins and their functions are listed in
the Tables S1 and S2.

4.7. cIEF Immunoassays

Approximately 400 nanoliters of SH-SY5Y cell lysates in separation gradients were loaded into
individual capillaries of the 96-capillary NanoPro 1000 system (ProteinSimple). Isoelectric focusing was
performed at 15 mW for 50 min. The proteins were cross-linked to the capillary walls using ultraviolet
irradiation for 80 s. Primary and secondary antibodies were sequentially introduced with incubation
times of 120 and 60 min, respectively. Following incubation with chemiluminescence detection agents,
the proteins were detected with an average exposure time of 240 s. Hsp70 was used as a loading control.
All cIEF immunoassays were performed in triplicate for each protein, and duplicate experiments were
performed per treatment condition, producing six repeated measurements per protein.

4.8. Capillary Western Immunoassays

Cell lysates in denaturing buffers were denatured at 95 ◦ C for 5 min, and then transferred to
assay plates (cat. no. SM-W004 or SM-W008, ProteinSimple) preloaded with blocking reagents, wash
buffer, primary and secondary antibodies, and chemiluminescent substrates. Sized-based protein
separation and detection in capillaries were performed using the default protocols of the Jess system
(ProteinSimple). β-Actin was used as a loading control. All capillary Western immunoassays were
performed in triplicate for each protein, and duplicate experiments were performed per treatment
condition, producing six repeated measurements per protein.

4.9. Data Analysis

Assignment of pI values to protein phosphoisoforms was based on the literature and our own
published data [18,23,24,52–56]. Protein expression levels were quantified using the Compass software
from ProteinSimple. The expression levels of protein phosphoisoforms were adjusted with loading
controls for both charge-based and size-based immunoassays, and further adjusted with the expression
levels of total proteins for size-based immunoassays.

4.10. Cell Proliferation Assays

MTS assays (cat. no. G3581, Promega, Madison, WI, USA) were performed according to
manufacturer’s protocols. MTS signals were detected with a multimode microplate reader (Synergy 2
Int. J. Mol. Sci. 2020, 21, 2259 12 of 15

BioTek, Winooki, VT, USA). All MTS assays were performed in triplicate with duplicate experiments
per treatment condition, producing six repeated measurements per treatment condition.

4.11. Statistical Analysis

The data are presented as the mean values ± standard deviations across six repeated measurements.
Statistical significance was calculated with Student’s t-test and thresholded at p ≤ 0.05 versus the control.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/7/2259/

s1. The following are available online: Supplemental Figure S1, Akt isoform identification in SH-SY5Y cell lysates
using capillary isoelectric focusing (cIEF) immunoassays; Supplemental Figure S2, Short-term positive regulation of
Akt isoform phosphorylation by copaiba essential oil; Supplemental Figure S3, Akt isoform identification in HepG2
cell lysates using capillary isoelectric focusing (cIEF) immunoassays; Supplemental Figure S4, Dose-dependent
negative regulation of the PI3K/Akt/mTOR signaling pathway by β-caryophyllene; Supplemental Figure S5,
Pan-Akt profiles of SH-SY5Y cells as functions of treatment with CB2 agonists; Supplemental Table S1, List of
primary and secondary antibodies; Supplemental Table S2, List of biomarker proteins and their functions.
Author Contributions: Conceptualization, T.T.L., D.K.H., C.B., Y.U. and J.N.T.; Methodology (nanofluidic
proteomics), T.T.L. and Y.U.; Methodology (GC–MS), C.B. and M.W.; Validation, T.T.L., Y.U., C.B. and M.W.;
Formal analysis, T.T.L. and Y.U.; Investigation, T.T.L. and Y.U.; Resources, T.T.L.; Data curation, Y.U. and M.W.;
Writing—original draft preparation, T.T.L. and Y.U.; Writing—review and editing, D.K.H., C.B., M.W. and J.N.T.;
Supervision, T.T.L.; Project administration, T.T.L.; Funding acquisition, T.T.L., J.N.T., C.B. and D.K.H. All authors
have read and agreed to the published version of the manuscript.
Funding: This research received institutional funding support from the Roseman University of Health Sciences
and a research grant from dōTERRA International.
Acknowledgments: The authors thank Russel J. Osguthorpe and Nicole Stevens (dōTERRA International) for
stimulating discussions.
Conflicts of Interest: C.B., M.W. and D.K.H. are employees of dōTERRA International, a company that produces
and sells essential oils. D.K.H. and C.B. participated in the initial conceptualization of the project. C.B. and M.W.
analyzed the chemical composition of copaiba essential oil using a GC–MS method with good laboratory practice
(GLP) compliance.

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