Research Article

Received: 17 May 2022 Revised: 27 July 2022 Accepted article published: 29 July 2022 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.7202

Surfactant-assisted extraction of Melaleuca

alternifolia (tea tree) oil by hydrodistillation
and its application in microemulsion
Thuy-Vi Vo,a Thi-Huyen Truongb and Bing-Hung Chenb*

Abstract
BACKGROUND: The surfactant-assisted extraction of essential oils from Melaleuca alternifolia foliage, also known as tea tree oil
(TTO), was systematically studied with the design of experiments (DoE) aiming to improve the extraction of TTO. Specifically,
the optimal parameters of hydrodistillation process were obtained with the response surface methodology (RSM) based on a
central composite design (CCD).
RESULTS: The concept of enhanced TTO extraction with the renewable nonionic Triton CG-110 surfactant was proven. An opti-
mal extraction yield of TTO was predicted by the RSM model at 6.71 wt% under the following conditions: (i) 597 mg L−1 Triton
CG-110 as liquid extractant, (ii) a ratio of liquid extractant/desiccated leaf at 25.4 mL g−1, and (iii) 140 min for the extraction
time. The presence of 650 mg L−1 Triton CG-110 in the extractant could increase the extraction rate of TTO by 17.5%, compared
to that without surfactant. The forecast of the DPPH antioxidant activity of TTO by RSM was in good accordance with the mea-
sured values. Various microemulsion formulations of TTO with Triton CG-110 were developed and reported.
CONCLUSION: The presence of a proper surfactant in the liquid extractant can improve the extraction of essential oils, not only
in the form of yield but also in the rate of extraction. No trace of surfactant could be found in the produced essential oil because
of the relatively higher boiling point and the lower volatility of the surfactant. The RSM was successfully applied to obtain the
optimal extraction parameters for tea tree oil by the surfactant-enhanced hydrodistillation process.
© 2022 Society of Chemical Industry (SCI).

Keywords: extraction; tea tree oil (TTO); response surface methodology (RSM); antioxidant activity (DPPH assay); surfactant;
hydrodistillation

INTRODUCTION
A growing tendency to use natural products in cosmetics, aroma-
therapy, medicines, and foods has led to the increased production
of essential oils.1,2 Particularly, tea tree oil (TTO) extracted from
Melaleuca alternifolia is often marketed as a remarkable medicinal
product with applications in antibacterial, antifungal, antiviral,
antiprotozoal, and anti-inflammatory activities.1-4 Melaleuca alter-
nifolia, commonly known as tea tree, is a narrow-leaved tree of the
Myrtacea family and native to Australia. The vital usage of tea tree
oil can be found in the topical treatment of acne and other derma-
tological diseases, as well as fungal infectious diseases.4 In gen-
eral, tea tree oil is considered relatively safe for topical
applications.4
Tea tree oil (TTO) is composed of more than 100 compounds,
mainly monoterpenes, and their alcohol derivatives.4,5 Out of
the many ingredients in TTO, terpinen-4-ol is the most abundant
constituent and is generally deemed as the main contributor to
the antimicrobial property of TTO.6-8 In contrast, 1,8-cineole (euca-
lyptol) is regarded as an undesirable compound because it is an
irritant and a sensitizer to skin and mucous membranes at a
higher dosage.8 As a result, the quality of TTO commercially avail-
able must be strictly regulated and is, thus, specified in the inter-
national standard ISO 4730:2017.9 Specifically, ISO 4730 stipulates
that the concentration of terpinen-4-ol in TTO should fall within
the range from 35% to 48%, while the maximum concentration
of 1,8-cineole should not exceed 10%.9
Various methods to extract the biologically active plant-derived
essential oils have been practically implemented. For instance,
solvent extraction, hydrodistillation/steam distillation, supercriti-
cal fluid extraction (SFE), and microwave-assisted extraction
(MAE) have been widely considered for the extraction of nonvol-
atile compounds (e.g. fatty oil or phenolic compounds).4,10-18 In
particular, hydrodistillation is deemed one of the more economic
methods to produce essential oils with high purity.4,10-13 How-
ever, this method still suffers from drawbacks, such as a lower
extraction efficiency and a longer extraction time. As a result,
scientists and engineers are researching ways to reduce the
* Correspondence to: B-H Chen, Department of Chemical Engineering, National
Cheng Kung University, Tainan, 70101, Taiwan. E-mail: bkchen@ncku.edu.tw;
bhchen@alumni.rice.edu
a Faculty of Chemical Technology, Ho Chi Minh City University of Food Industry,
Ho Chi Minh City, Vietnam
b Department of Chemical Engineering, National Cheng Kung University,
Tainan, Taiwan
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 www.soci.org
extraction time while increasing the extraction yield in the hydro-
distillation process.
Surfactants are amphiphilic and, thus, can reduce the interfacial
tension between two immiscible phases.2,19 As such, the apparent
solubility of hydrophobic essential oils in water could be greatly
improved by solubilization with surfactant aggregates in aqueous
solution, resulting in a microemulsion.2,19 Likewise, hydrophobic oil
can be easily emulsified and lifted off the solid substrate that is
immersed in a surfactant bath; this process is commonly known as
detergency.2 Therefore, the presence of a proper surfactant in an
aqueous extractant was assumed to help the mobilization and quick
release of oil molecules from plant tissues, such as elaioplasts, into
liquid extractant phases. Accordingly, the extraction yield and kinet-
ics of essential oils could be enhanced in such a surfactant-assisted
hydrodistillation process. For example, the extraction yield of Salvia
officinalis L. essential oil by hydrodistillation was enhanced in pres-
ence of Tween 20, a common nonionic surfactant that is also known
as polysorbate 20.14 Briefly, the extraction yield was increased from
1.5% to 1.8% with Tween 20, in contrast to a trivial effect with the
use of hydrophobic Span 80 nonionic surfactant. That is, the hydro-
philicity of surfactants could impose a substantial influence on the
extraction of essential oils by hydrodistillation. It is known that hydro-
phobic surfactants, like Span 80, are practically insoluble in water and
do not form micelles in aqueous solutions to help the solubilization
of hydrophobic essential oils. Moreover, the improved extraction of
essential oils using hydrophilic surfactants in the hydrodistillation
process was likely attributable to the enhanced mass-transfer of
essential oils via solubilization and emulsification by surfactants.2
So far there are limited reports available in the open literature
concerning the surfactant-assisted hydrodistillation of essential
oils, and even less that focus on tea tree oil.14,17 Hence, the aim
for this work is to develop an optimal extraction process of tea
tree oil with a surfactant-assisted hydrothermal treatment. The
surfactant selected in this work was Triton CG-110, a type of alkyl
polyglucoside (APG) surfactant. APGs are synthesized from the
glycosation of plant-derived sugars, usually glucose derivatives,
with fatty alcohols. In general, APGs are renewable nonionic sur-
factants that are known for their superior sustainability and biode-
gradability without having any adverse environmental
impact.20-22
As mentioned above, surfactant-assisted hydrodistillation of tea
tree oil was attempted and studied in this work. The Design of
Experiments (DoE) strategy was adopted to systematically opti-
mize the factors relevant to the hydrodistillation process of tea
tree oil. Specifically speaking, the response surface methodology
(RSM) with the central composite design (CCD) was employed to
evaluate the effects of all factors in the hydrodistillation of TTO
to give the optimal process parameters from the minimum set
of experiments required. RSM has been widely applied in the
extraction of essential oils using a variety of methods, including
microwave-assisted extraction, supercritical CO2 extraction, and
ultrasonic-assisted extraction.11-13 For example, the supercritical
CO2 extraction process of Eucalyptus leaf oil was improved with
RSM; in it, three independent factors (i.e. extraction temperature,
extraction time, and CO2 pressure) were adopted.16 The extrac-
tion yield of Phaleria macrocarpa seed oil by the conventional n-
hexane extraction method was optimized by using RSM with the
CCD.11 Three independent factors, including extraction tempera-
ture, solvent-to-feed ratio, and extraction time, were investi-
gated.11 Furthermore, the Box–Behnken design was successfully
applied to optimize the microwave-assisted extraction of Cur-
cuma longa L. oil (curcuma root).12
2
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T-V Vo, T-H Truong, B-H Chen
The effects of process parameters, including the concentration
of Triton CG-110 in the extractant, the ratio of liquid extractant/
desiccated leaf, and the extraction time, on the surfactant-
assisted hydrodistillation of TTO were investigated to give an
improved extraction yield of TTO. The antioxidant activity of
TTO, determined by DPPH assay, was studied as well. To explore
the potential applications of TTO in cosmetics and medicinal
formulations,2 microemulsions formulated with TTO and Triton
CG-110 were attempted. Additionally, the physicochemical prop-
erties of TTO-formulated microemulsion were evaluated and
reported.
MATERIALS AND METHODS
Materials
Leaves of Melaleuca alternifolia, commonly known as tea tree,
were collected from a local farm in Tainan City, Taiwan, and dried
in an air-conditioned room at 25 °C for 10 days. A period of 7–
10 days was found to be enough to desiccate fresh leaves. Nota-
bly, twigs of tea tree contained very little oil and were, thus, dis-
carded away. Triton CG-110, one type of nonionic alkyl
polyglucoside (APG) surfactant, was purchased from Sigma-
Aldrich. According to the supplier, Triton CG-110 is a mixture of
58–62% D-glucopyranose, oligomeric, decyl octyl glycoside, and
38.0–42.0% water. The critical micelle concentration (CMC) of Tri-
ton CG-110 is 1748 ppm, while the cloud point of 1 wt% Triton
CG-110 is greater than 100 °C. Particularly, the water content in
the Triton CG-110 used in this work was 39.65 wt%, as determined
with TGA.
Nine major components of TTO, used as standards for GC-quan-
tification, were purchased from Sigma-Aldrich, Acros Organics,
and Alfa Aesar. These components included γ-terpinene (97%),
p-cymene (99%), terpinolene (≥ 85%), ⊍-pinene (98%), (R)-
limonene (97%), terpinen-4-ol (≥ 95%), ⊍-terpineol (97%),
⊍-terpinene (≥ 95%), and 1,8-cineole (99%). Propylene glycol,
absolute ethanol, n-decane, 2, 20 -diphenyl−1-picrylhydrazyl
(DPPH, 95%), and L(+)-ascorbic acid (≥ 99%) were acquired from
Avantor J.T. Baker, Alfa Aesar, and Riedel-de Haën.
All chemicals were of reagent grade and used as received. Deio-
nized water from a Millipore Milli-Q ultra-purification system with
a resistivity greater than 18.2 MΩ·cm was used in the sample
preparation.
Optimization of hydrodistillation process with the
response surface methodology
The extraction process of tea tree oil by hydrodistillation in the
Clevenger system was described in detail elsewhere.23 In this
work, the Design of Experiments (DoE) strategy was applied to
optimize the hydrodistillation process. The response surface
methodology (RSM) with the central composite design (CCD)
was employed to obtain the optimal extraction conditions. Based
on our preliminary experiments, the three independent variables
taking part in the determination of TTO yield were: (i) the surfac-
tant concentration (mg L−1) in liquid extractant, (ii) the ratio of liq-
uid extractant/solid raw material (mL g−1 desiccated leaf), and
(iii) the extraction time (min). The three levels of independent vari-
ables, respectively coded as −1, 0, and + 1, as along with their cor-
responding physical values, are listed in Table 1. In brief, 20 sets of
experiments, i.e. 8 (23) factorial points + 6 (2 × 3) axial points + 1
central point (1 × 6, in sextuplicate), were performed for the
face-centered CCD (⊍ = ±1) and tabulated in Table 2. Notably,
the hydrodistillation experiments with the same set of process
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Surfactant-assisted extraction of Melaleuca alternifolia (tea tree) www.soci.org

Table 1. Coded levels of independent variables/experimental factors and their corresponding values adopted in this study

Coded level/Actual value

Factor Unit Symbol −1 0 +1

−1
Triton CG-110 concentration mg L A 300 650 1000
Liquid/solid raw material ratio — B 15 22.5 30
Extraction time min C 90 120 150

Table 2. Central composite design (CCD) with three independent variables and the corresponding empirical measurements in extraction yield and
antioxidant activity

Triton CG-110 (A) Liquid/material ratio (B) Extraction time (C)

−1 −1
Expt. No. Uncoded (mg L ) Coded Uncoded (mL g ) Coded Coded (min) Uncoded Yield (wt%) Antioxidant activity (%)

1 1000 1 30 1 150 1 5.98 ± 0.15 41.72 ± 0.20

2 650 0 22.5 0 150 1 6.66 ± 0.21 46.86 ± 0.22
3 300 −1 15 −1 90 −1 5.74 ± 0.14 29.07 ± 0.12
4 650 0 22.5 0 120 0 6.64 ± 0.19 50.51 ± 0.16
5 300 −1 30 1 90 −1 5.98 ± 0.13 36.36 ± 0.36
6 650 0 22.5 0 120 0 6.68 ± 0.18 49.49 ± 0.18
7 300 −1 30 1 150 1 6.27 ± 0.15 36.97 ± 0.22
8 650 0 30 1 120 0 6.55 ± 0.26 50.11 ± 0.12
9 650 0 22.5 0 90 −1 6.36 ± 0.16 43.01 ± 0.14
10 650 0 15 −1 120 0 6.17 ± 0.26 43.40 ± 0.13
11 1000 1 15 −1 90 −1 5.41 ± 0.41 30.76 ± 0.20
12 300 −1 22.5 0 120 0 6.23 ± 0.17 43.88 ± 0.30
13 650 0 22.5 0 120 0 6.65 ± 0.44 50.44 ± 0.33
14 300 −1 15 −1 150 1 5.94 ± 0.26 37.98 ± 0.26
15 650 0 22.5 0 120 0 6.67 ± 0.39 50.33 ± 0.18
16 650 0 22.5 0 120 0 6.65 ± 0.43 49.98 ± 0.41
17 1000 1 30 1 90 −1 5.82 ± 0.20 35.18 ± 0.21
18 1000 1 15 −1 150 1 5.55 ± 0.54 40.40 ± 0.34
19 1000 1 22.5 0 120 0 5.98 ± 0.35 45.86 ± 0.32
20 650 0 22.5 0 120 0 6.65 ± 0.49 50.21 ± 0.40

parameters, corresponding to the same point on the
experimental-design box, were performed at least in triplicate.
The hydrodistillation process was conducted by placing 10 g of
dried tea tree leaves in the Clevenger system. The extraction yield
of TTO was defined as grams of essential oil extracted per 100 g of
dried leaves used, while the antioxidant activity of TTO deter-
mined by DPPH assay was measured with 5 wt% TTO in ethanol,
according to the procedures given in Section 2.5. The quadratic
regression models were set up to simulate the correlation
between the extraction yield/antioxidant activity of TTO and the
experimental factors, expressed as below:
y ¼ ⊎0 þ ⊎ 1 ·A þ ⊎ 2 ·B þ ⊎3 ·C þ ⊎11 ·A2 þ ⊎22 ·B2
þ ⊎33 ·C 2 þ ⊎ 12 ·A·B þ ⊎ 13 ·A·C þ ⊎ 23 ·B·C
where y is either the extraction yield of TTO (in wt%, or equiva-
lently g oil extracted per 100 g desiccated leaves) or the DPPH
antioxidant activity (AOA) of TTO (%); ⊎0 is the intercept; ⊎1, ⊎2,
and ⊎3 are coefficients of the linear effect; ⊎12, ⊎13, and ⊎23 are coef-
ficients of cross interactions; ⊎11, ⊎22, and ⊎33 are the coefficients of
quadratic terms; A, B, and C are the coded independent variables
listed in Table 2.
Analysis composition of tea tree oil
Both gas chromatography coupled to time-of-flight mass spectrom-
etry (GC-TOF-MS) and gas chromatography coupled with flame ion-
ization detector (GC-FID) instruments were utilized to characterize
and quantify the components of tea tree oil. The GC-TOF-MS instru-
ment consisted of an Agilent GC 6890 N and a TOF detector
(Pegasus III TOF-MS system), installed with the separation column
(DB-35MS Ultra Inert, 30 m × 0.25 mm × 0.25 μm). The temperature
program of this GC–TOF-MS was initially set at 100 °C for the first
minute, then increased to 310 °C at 8 °C·min−1, and finally kept at
310 °C for 10 min. Helium with a flowrate at 1 mL·min−1 was used
as the carrier gas in the GC-TOF-MS. The operation parameters for
ð1Þ the mass spectrometer are: (i) the electron impact mode (EI) at
70 eV, and (ii) the temperature of the ion source at 230 °C.
The quantification of the nine major ingredients of tea tree oil was
conducted by using the GC-FID (Model GC-2014, Shimadzu, Japan).
The samples were spiked with 300 mg L−1 n-decane as an internal
standard to provide better quantitative analyses of these in-house
hydrodistilled TTO samples. The HP-5 capillary column
(30 m × 0.25 mm × 0.25 μm) was installed as the separation col-
umn. Tea tree oil diluted with ethanol by 100× (w/w) was automati-
cally injected into GC-FID with an AOC-20i Auto Injector (Shimadzu,
Japan) with the split mode (1:100) using ultra nitrogen at 1 mL·min−1
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 www.soci.org T-V Vo, T-H Truong, B-H Chen

as the carrier gas. The temperature profile of the GC-FID was pro-
grammed to increase from 70 °C initially to 95 °C by 10 °C min−1,
then further to 105 °C at 1 °C min−1, and finally to reach 300 °C at
25 °C min−1 and on hold for at least 15 min. Standard solutions of
the major constituents of tea tree oil for GC-FID quantification were
prepared in ethanol by mixing these components from their pure
stocks. The concentration of each component in the standard solu-
tion was controlled in the range from 50 to 7000 mg L−1.
Formulation and characterization of microemulsions
The preparation procedure of O/W microemulsions composed of tea
tree oil and mixed surfactants was given in our previous report.23 The
mixed surfactant, noted as Smix, is made of Triton CG-110 in 3 parts and
propylene glycol (PPG) in 1 part by mass. The concentration of tea tree
oil was fixed at 1 wt% or 3 wt% in all microemulsions prepared, which
were further characterized with their aggregate sizes by the dynamic
light scattering (Zetasizer Nano ZS, Malvern Instrument Ltd., UK), the
pH values, and the viscosity measured with Ostwald viscometers. Fur-
thermore, the shelf-stability of the microemulsions was monitored
mostly by the change in the aggregate sizes over a period of 1 month.
DPPH free radical scavenging assay by UV–Vis machine
The antioxidant activities of pure TTO, the microemulsions, and the
nine TTO components were evaluated using 2, 2 - diphenyl-
1-picrylhydrazyl free radical (DPPH•) scavenging method through
discoloration of purple DPPH solution. In detail, 2.0 mL of sample
were mixed with 2.0 mL of 0.15 mM DPPH• solution in ethanol vig-
orously by vortex at room temperature. Decreasing of the DPPH•
absorbance with reaction time was recorded by UV/Vis spectropho-
tometer (CT-2200/2300/2400 spectrophotometer, Germany). The
DPPH antioxidant activity expressed as percentage of DPPH• inhibi-
tion was calculated from absorbance at 518 nm after reacting in the
dark for 30 min; this is shown as Eqn (2), below. The 20 mg L−1 solu-
tion of ascorbic acid was used as the reference standard.
AB −AS
Antioxidant activity% = ×100 ð2Þ
AB
where AB is the absorbance of blank, and AS is the absorbance of
the sample or ascorbic acid.
Statistics
The analysis of variance (ANOVA) was performed to evaluate the
experimental results that are expressed with the mean and the
standard deviation (N ≥ 3). The Design-Expert software was
applied for the CCD experimental design, especially in the analysis
of the experimental results and the prediction of the responses.
RESULTS
Hydrodistillation process of tea tree oil
Hydrodistillation process predicted with RSM
Preliminary experiments were conducted to determine the suit-
able ranges for the independent variables, which are given in
Table 1. The factors investigated included the concentration of
Triton CG-110 in extractant (A), the ratio of the extractant-to-
desiccated leaves of tea tree (B), and the extraction time (C).
Notably, the independent variables for RSM were preferably
normalized and coded from −1 to 1 and denoted as A, B, and C
(Table 1). The central point of the design, namely zeros for all
coded variables, corresponds to 650 mg L−1 for Triton CG-110 in
extractant (i.e. A = 0), 22.5 mL g−1 for the ratio of extranet-to-
desiccated leaves (i.e. B = 0), and 120 min for extraction process
(i.e. C = 0). Table 2 summarized the extraction yield and the DPPH
antioxidant activity of TTO carried out under the conditions spec-
ified according to the central composite design (CCD).
The regression models attained for the response factors (i.e. the
extraction yield and the antioxidant activity (AOA) of TTO mea-
sured by DPPH assay) were shown as Eqns (3) and (4), respectively:
Yield ðwt%Þ ¼ 6:6307 – 0:1410 A þ 0:1783 B þ 0:1086 C ð3Þ
þ 0:0325 A·B – 0:0245 A·C
þ 0:0135 B·C –0:4840 A2 –0:2255 B2 – 0:0830 C 2
AOAð%Þ ¼ 50:19 þ 0:9665 A þ 1:87 B þ 2:96 C –0:0680 A·B ð4Þ
þ 0:8321 A·C – 1:430B·C –5:36 A –3:47 B –5:29 C
2 2 2
The significance of these three factors and their interactions, as
well as the quality of the model, were examined with ANOVA
(Table 3). In brief, both regression models obtained from RSM

Table 3. Statistical analysis on the extraction yield and DPPH antioxidant activity of TTO

Extraction yield (wt%) DPPH antioxidant activity (%)

Mean square F-value P-value Mean Square F-value P-value

Model 0.3507 202.99 <0.0001 98.84 80.5 < 0.0001

A 0.1988 115.06 <0.0001 9.34 7.61 0.0202
B 0.3179 183.99 <0.0001 35.04 28.54 0.0003
C 0.1179 68.26 <0.0001 87.32 71.12 < 0.0001
AB 0.0085 4.89 0.0514 0.037 0.0302 0.8656
AC 0.0048 2.78 0.1265 5.54 4.51 0.0596
BC 0.0015 0.8438 0.3799 16.28 13.26 0.0045
A2 0.6441 372.77 <0.0001 78.96 64.31 < 0.0001
B2 0.1486 86.00 <0.0001 33.18 27.03 0.0004
C2 0.0189 10.95 0.0079 77 62.71 < 0.0001
R2 0.9946 0.9864
Adj-R2 0.9897 0.9741
Coef. of variation (%) 0.667 2.57
Adequate precision 42.44 26.25
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are adequate to predict the extraction yield and the antioxidant
activity of TTO, as the p-values and F-values of both regression
models are less than 0.0001 and far greater than the critical
F-values, FCritical(0.025, 9, 10) = 3.779, respectively. In the model,
expressed in Eqn (3), the linear terms (A, B, C) and the quadratic
terms (A2, B2, and C2) are all significant, in contrast to the insignif-
icant effects by cross-product terms (AB, AC, and BC), as judged by
their p-values revealed in Table 3.
The reliability and the accuracy of the model for describing
the relationship between the independent variables and the
response factors were checked with the coefficient of determi-
nation (R2), the adjusted R2, and the coefficient of variation
(CV) (Table 3). The R2 and the adjusted R2 values for the extrac-
tion yield of TTO were as high as 0.9946 and 0.9897, while those
for the DPPH antioxidant activity of TTO were 0.9864 and
0.9741, respectively (Table 3). Not only was the R2 close to
one, but also the very small difference between the R2 and the
adjusted R2 for both response factors indicates the good fits of
both empirical models. Particularly, the high reproducibility of
both regression models was confirmed with the small coeffi-
cients of variation (CV): 0.67% and 2.57% for the extraction yield
and the antioxidant activity, respectively. In addition, the large
values of the adequate precision of both models implied the
superior applicability of both models established over the
design space (Table 3).
In summary, the extraction yield and the DPPH antioxidant
activity (AOA) of TTO could be further simplified as follows:
Yieldðwt%Þ ¼ 6:6307 –0:141 A þ 0:1783 B
þ 0:1086 C –0:484 A –0:2255 B –0:083 C
2 2
AOAð%Þ ¼ 50:19 þ 0:9665 A þ 1:87 B
þ 2:96 C – 1:43 B·C –5:36 A2 –3:47 B2 – 5:29 C 2
The graphic presentations of the obtained regression models for
the extraction yield and the antioxidant activity of TTO were dis-
played in Figs 1 and 2, respectively. Figure 1 showed the 3D
response surfaces of the extraction yield of TTO under the influ-
ences of the factors studied. In general, with Triton CG-110 at
650 mg L−1 (i.e. A = 0), the extraction yield was gradually
improved with an increasing ratio of liquid extractant/dried leaves
and with an extended extraction time (Fig. 1(a)). This could be
ascribed to the improved osmosis and mobilization of TTO from
the foliage with more liquid extractant over a sufficiently long
course of hydrodistillation. An indiscernible enhancement was
found at a really extended period of extraction (e.g. over 130–
150 min), while a slight decrease in yield was noticed as the liq-
uid/material ratio reached more than 24.5 mL g−1 leaf.
Besides this, the effects of the Triton CG-110 concentration in
the extractant and the ratio of liquid extractant/dried leaves on
the extraction yield of TTO over 120 min as the preset extraction
time were presented in Fig. 1(c). A yield of 5.42% to 6.68% was
found. The maximum yield coincidently occurred with
600 mg L−1 Triton CG-110. As previously mentioned, Triton CG-
110 is a nonionic surfactant, leading to the reduced surface ten-
sion between water phase and oil phase and, thus, promoting
the wetting on the surface of tea tree leaves. Hence, the surfactant
made the imbibition of extractant into plant tissues easier and,
therefore, facilitated bringing oil molecules out. Therefore, the
amount of essential oil extracted could be improved. However,
with too much surfactant in the extractant, foaming could
become a concern, hindering the escape of the vapor phase con-
taining essential oils from the boiled liquid extractant. As a result,
the extraction of TTO could be diminished.
Likewise, the effects of these three factors on the DPPH antiox-
idant activity of the extracted TTO were studied and shown in
Fig. 2. As for the reference, L-ascorbic acid at 20 ppm showed
excellent scavenging activity (69.5%). In addition, the antioxidant
ð5Þ activity of TTO was inferred to be mainly caused by
2 monoterpenes,24 i.e. the main components of TTO. In general, all
three factors played a vital role in the extraction yield of TTO
and, consequently, a similar trend would be expected in the anti-
ð6Þ oxidant activity (AOA) of the extracted TTO.
In regard to the extraction yield of TTO, the optimal conditions
predicted with either Eqns (3) or (5) were summarized collectively
as follows: (i) 597 mg L−1 Triton CG-110, (ii) ratio of liquid extrac-
tant/solid raw material at 25.4, and (iii) 140 min for extraction time
(i.e. with coded values at A = −0.15, B = 0.39, and C = 0.67). Note-
worthily, the optimal extraction yield of TTO was expected to be
6.71 wt%, which is comparable with those reported earlier in the

(a) With A=0 ([Triton] = 650mg/L) (b) With B=0 [Ratio = 22.5] (c) With C=0 (Time = 120min)

6.8 6.8
6.8
6.6 6.6
6.6
6.4 6.4 6.4
6.2 6.2 6.2
6 6
Yield (%)
Yield (%)

6
Yield (%)

5.8 5.8 5.8

5.6 5.6 5.6
5.4 5.4 5.4

1000 90 1000 15
150 30 900 100 900
140 27 800 18
110 800
130 24 700 700 21
120 120
600 600 24
110 21 500 130
C: Time (min) 100 18 B: Liquid/material ratio A: Concentration of 400 140 C: Time (min) A: Concentration of 500400 27 B: Liquid/material ratio
90 15 APG 300 150 APG 300 30

Figure 1. Response surface showing the influences of factors studied, the concentration of Triton CG-110 [A], the liquid extractant/desiccated leaf ratio
[B], and the extraction time [C], on the extraction yield of tea tree oil (TTO): (a) With A = 0 ([Triton CG-110] = 650 mg L−1); (b) With B = 0 [Ratio = 22.5];
(c) With C = 0 (Time = 120 min).
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 www.soci.org T-V Vo, T-H Truong, B-H Chen

(a) With A=0 ([Triton] = 650mg/L) (b) With B=0 [Ratio = 22.5] (c) With C=0 (Time = 120min)

55 55 55

50 50 50

45 45 45

40 40 40

DPPH (%)
DPPH (%)

DPPH (%)
35 35
35

30 30
30

25 25
25

150 1000
30 1000
150 30 27 900
140 140 900 800
27 800
130 130 24 700
24 120 700
120 600 21 600
110 21 110 500 A: Concentration of APG
C: Time (min) B: Liquid/material ratio C: Time (min) 500 A: Concentration of APG B: Liquid/material ratio 18
100 18 100 400 400
90 15 90 300 15 300

Figure 2. Response surface showing the influences of factors studied, the concentration of Triton CG-110 [A], the liquid extractant/desiccated leaf ratio
[B], and the extraction time [C], on the DPPH antioxidant activity (DPPH %) of extracted tea tree oil (TTO): (a) With A = 0 ([Triton CG-110] = 650 mg L−1);
(b) With B = 0 [Ratio = 22.5]; (c) With C = 0 (Time = 120 min).

literature.10 For instance, the yield of oil from tea tree foliage an extraction yield of TTO in the presence of 650 mg L−1 Triton
grown in Australia was found to be between 42 and 63 mg g−1 CG-110 with an extraction time of 120 min and a ratio of liquid
leaf by steam distillation for tea trees with low and high contents extractant/desiccated leaves at 22.5 was found empirically at
of oil, respectively.10 The antioxidant activity (AOA) of TTO was 6.7 wt%, in contrast to 5.7 wt% for the same conditions but with-
forecast to be near 48.23% under the conditions to have the max- out surfactant being added into the extractant solution (Fig. 3).
imal extraction of TTO, i.e. the conditions predicted by either That is, the hydrodistillation extraction of TTO was enhanced by
Eqns (3) or (5). However, if the maximal antioxidant activity of ca. 17.5% with 650 mg L−1 Triton CG-110 solution as an
TTO is desired, the regression model, shown as Eqns (4) or (6), extractant.
would suggest that the hydrodistillation process of TTO should In addition to more TTO being harnessed from tea tree foliage,
be performed at (i) 689 mg L−1 Triton CG-110, (ii) 24.1 for the ratio the extraction of TTO also proceeded more quickly with Triton
of liquid extractant/solid raw material, and (iii) 128 min for extrac- CG-110 present in extractant. The kinetics mechanism of the
tion time (i.e. at A = 0.11, B = 0.21 & C = 0.26). Thus, the maximal surfactant-assisted hydrodistillation could be explained on
DPPH antioxidant activity of TTO would be expected as 50.82%. In account of the hydrodiffusion process. The hydrodiffusion pro-
brief, the optimal conditions for the surfactant-enhanced hydro- cess allows the extract, namely the essential oils and the hot
distillation of tea tree oil are different and depend on whether it extractant, to diffuse out through plant tissues at the solid–liquid
is desirable to have the maximal extraction yield of TTO or the interface. The surfactant-containing extractant will not only accel-
most DPPH antioxidant activity of TTO. erate the diffusion rate but also the solubilization capacity of
essential oils by lowering the surface tension at the solid–liquid
Validation of the RSM regression models interface. As shown in Fig. 3, the extraction kinetics of TTO could
Validation of the RSM regression models was performed under be fit well with the model based on the mass balance of oil mole-
conditions resembling the optimum obtained based on the cules through the interface between a pseudo solid state made of
extraction yield of TTO. All experiments were conducted in quin- tea tree leaves and liquid extractant:25
tuplicate. The empirical results, along with the experimental
conditions, were given in Table 4. In general, the regression d


V  C Oil =k '  a  C Oil
L −C
Oil
ð7Þ
models predicted well the extraction yield of TTO and the DPPH dt
antioxidant activity (AOA) of TTO. For example, the empirical
extraction yield of TTO for Experiment No. 3 was (6.69 ± 0.54) where COil is the concentration of tea tree oil in the extractant
wt%, which is close to the optimum at 6.686 wt%, as predicted phase, CLOil the TTO concentration on leaves, V the volume of
by the RSM regression model, i.e. Eqn (5). Similarly, the DPPH extractant, t the extraction time, a the overall surface area of
antioxidant activity of TTO obtained from this very set of exper- tea tree foliage, and k’ the mass transfer coefficient. If the satu-
iments was found as (50.68 ± 0.24) %, which is also in the vicinity ration concentration of tea tree oil in the extractant phase is rel-
of the predicted value at 50.73%. In summary, both regression atively small in comparison to that of tea tree oil in leaves (CLOil),
models obtained from the Design of Experiments could provide CLOil could assumedly be a pseudo-constant during the extrac-
well-predicted results for the extraction yield and the DPPH anti- tion process. The concentration of TTO in the liquid extractant
oxidant activity of tea tree oil under different extraction could be further expressed as the following exponential
conditions. function:



Extraction kinetics of tea tree oil COil =C Oil
L  1−e
−kt
, while k = k ’  a =V: ð8Þ
The effect of surfactants present in the extractant on the extrac-
tion kinetics of tea tree oil was investigated as well. The presence The amount of tea tree oil extracted into liquid extractant would
of surfactants, either the Tween 20 or Triton CG-110 evaluated in be equal to COil‧V. With the constant volume of extractant (V)
this work, could improve the extraction yield of TTO. For example, and constant mass of tea tree leaves (Wleaf) used in the
6

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Surfactant-assisted extraction of Melaleuca alternifolia (tea tree) www.soci.org

Table 4. Validation of the RSM regression models with confirmation experiments

Expt Triton CG- Liquid/Solid Time Predicted Real Predicted

No. 110 (mg L−1) ratio (min) yielda (%) yield (%) AOAa (%) Real AOA (%)

1 608.6 24.0 128 6.68 6.60 ± 0.20 50.55 49.56 ± 0.33

2 624.5 24.3 126 6.68 6.68 ± 0.36 50.65 50.69 ± 0.21
3 645.5 24.5 128 6.69 6.69 ± 0.54 50.73 50.68 ± 0.24
4 651.5 25.1 126 6.68 6.36 ± 0.33 50.74 50.70 ± 0.22
5 666.7 24.3 129 6.67 6.46 ± 0.33 50.78 50.89 ± 0.41
a
Indicated the values predicted by Eqns (5) and (6), respectively.

7 sage oil. It is worth noting that Tween 20 was attempted in this

-1
k1=0.065 min ; R =0.995 2 work as well. A high dose of Tween 20 at 3000 mg L−1 as an
6
extractant could increase the mass-transfer coefficients of TTO
to 0.067 min−1. The maximal extraction yield was enhanced by
only 2.8%. In brief, a longer extraction time (t) generally gives a
5 k2=0.054 min-1; R2=0.976
higher extraction yield (Fig. 3). Explicitly, a sufficient extraction
Yield (wt%)

time was required for the extractant to be effectively imbibed into

4 the elaioplasts of the cells in the leaves to mobilize TTO molecules
and for TTO molecules to meander out of leaves.
3 Similarly, Triton CG-110 was chosen because it is a hydrophilic
plant-derived surfactant that is generally regarded as sustainable.
2 It is our conjecture that this hydrophilic Triton CG-110 would work
well, like the hydrophilic Tween 20, in the hydrodistillation pro-
cess of TTO. Triton CG-110 molecules could reduce the surface
1 650 ppm Triton CG110 tension between the aqueous extractant phase and the oil phase
No Surfactant and, thus, they could promote wetting on the surface of tea tree
0 foliage. Hence, the permeation of the surfactant solution into
0 20 40 60 80 100 120 140 160 tight plant tissues becomes easier. In return, the mass transfer of
Time (min) oil molecules out of the plant cells happened more quickly.14

Figure 3. Effect of Triton CG-110 surfactant on the extraction kinetics of
tea tree oil (TTO).
experiments, the extraction yield of tea tree oil (Y), based on the
mass of dried leaves used, could be written as follows:

−kt

These nine components made up 89.1% of the hydrodistilled TTO,
W  C  V =W leaf ∝ C ∝ 1−e
Y=MOil
Oil Oil
where MwOil is the averaged molecular weight of tea tree oil and is
treated as a constant. The extraction yield of tea tree oil (Y) could
be further simplified as follows:

was in good compliance with ISO 4730. The content of 1,8-cineol in
Y =Y 0 ‧ 1−e−kt
where Y0 is the maximum extraction yield of tea tree oil.
Concurrently, the effect of the surfactant on the enhanced extrac-
tion kinetics could be observed with the apparent mass-transfer coef-
ficients (k), which were obtained as 0.065 min−1 and 0.054 min−1,
respectively, for hydrodistillation extraction with 650 mg L−1 Triton
CG-110 and without Triton CG-110 present in the liquid extractant.
The enhancement in the hydrodistillation process of essential
oils by nonionic surfactants could be found elsewhere in the open
literature. For instance, both Tween 20 and Span 80, common
food-grade nonionic surfactants, were studied for their effects
on the hydrodistillation extraction of sage (Salvia officinalis L.)
essential oil.14 However, only the hydrophilic Tween 20 surfactant
was found to have the capability to increase the extraction yield of
Characterization of tea tree oil
According to GC-TOF-MS analysis, the in-house hydrodistilled tea tree
oil was found to contain 36 discernible compounds (in Fig. 4(a)). Nine
major constituents in tea tree oil were qualitatively identified with GC-
TOF-MS and successfully quantified by the GC-FID analysis using n-
decane as an internal standard, as given in our previous report.23
ð9Þ
while the remaining 10.9% was composed on sabinene, ⊎-pinene,
cadinene, globulol, and cubenol (identified with the information avail-
able in the NIST library for GC-TOF-MS). Of the different components
in TTO, terpinen-4-ol was the most abundant. Particularly, terpinen-
4-ol represented 47.4% of the TTO garnered in this work, and this
ð10Þ TTO was only 3.6%, which is lower than the threshold of 10% that is
cautiously emphasized in ISO 4730. Other constituents in TTO were
⊍-pinene (2.5%), ⊍-terpinene (8.6%), p-cymene (2.3%), limonene
(1.3%), γ-terpinene (16.1%), terpinolene (2.6%), and ⊍-terpineol
(4.7%). In conclusion, the quality of the in-house hydrodistilled TTO
was acceptable according to the specifications of ISO 4730.
The absence of Triton CG-110 surfactant in the obtained tea tree
oil was confirmed with the GC-TOF-MS analyses (Fig. 4(b)). Inter-
estingly, four isomers of decyl octyl glycoside, appearing from
21 min to 24 min (Fig. 4(b)), were recognized in Triton CG-110
by GC–MS. The insert showed the mass spectrum of the com-
pound eluted out at 20.51 min that was identified as one isomer
of decyl octyl glycoside. However, by comparing Fig. 4(a), (b), no
detectable peak of decyl octyl glycoside was found in the GC-
TOF-MS chromatograms of the hydrodistilled TTO. That is, the
7

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 www.soci.org
Figure 4. GC–MS chromatograms of (a) the hydrodistilled tea tree oil and (b) Triton CG-110 surfactant. The inset is the mass spectrum of the compound
corresponding to the peak at 20.51 min.
Triton CG-110 surfactant present in the surfactant-enhanced
hydrodistillation process would not exist in the produced TTO.
Additionally, the HPLC analyses further confirmed that no detect-
able levels of surfactants, from either Triton GC-110 or Tween
20, were present in the hydrodistilled tea tree oil. It is possible that
surfactant molecules are much less volatile than essential oils and,
thus, will not go to vapor phase during the hydrodistillation pro-
cess; this vapor phase is later condensed into hydrosol/essential
oils.
Application of tea tree oil in aqueous microemulsions with
Triton CG-110
Microemulsions have found applications in various fields, such as
personal care products, medicines, etc. In this work, the aqueous
microemulsions of the in-house hydrodistilled tea tree oil were
prepared with Triton CG-110 as the surfactant and propylene gly-
col (PPG) as the co-surfactant, according to the phase diagrams
previously established with TTO, Triton CG-110, and PPG.23 The
8
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T-V Vo, T-H Truong, B-H Chen
TTO-microemulsions prepared were examined for their physico-
chemical properties and explored for their potential applications,
mainly in the DPPH antioxidant activity.
Table 5 showed the formulation of microemulsions and their
physicochemical properties in pH, viscosity, and aggregate sizes.
Particularly, all the pH values of all microemulsions prepared ran-
ged from 4.8 to 5.0, which is suitable for topical applications.26 The
viscosities of these microemulsions are near that of water, ranging
from 0.95 cp to 2.64 cp. In general, microemulsions with more sur-
factants tend to be more viscous.
The shelf stability of microemulsions was monitored with the
change in their aggregate sizes being measured with dynamic
light scattering (DLS). Immediately after preparation, the average
aggregate sizes of microemulsions were found to be between
9.3 nm and 19.5 nm. Alternatively, they could be regarded as oil-
swollen micelles.19,27,28 Aggregates in both MD1 and MD3 grew
with time over 1 month of storage. In MD1 and MD3, the mass
ratio of TTO-to-mixed surfactant (Smix) formulated in microemul-
J Chem Technol Biotechnol 2022
Surfactant-assisted extraction of Melaleuca alternifolia (tea tree)
Table 5. Properties of microemulsions (MD1, MD2, and MD3)
Sample TTO (wt%) Smix (wt%) Viscosity (cp)
MD1 1 2.3 0.95 ± 0.01
MD2 1 9 1.18 ± 0.01
MD3 3 7 1.36 ± 0.03
Smix stands for a mixture of Triton CG-110 and propylene glycol (PPG) in a mass ratio of Triton CG-110/PPG = 3/1 (w/w).
sions remained the same, at 2.3. The rest of MD1 and MD3 were
merely deionized water. However, their long-term shelf-stability
was quite different. The average particle size of MD1 increased
to 181 nm from 19.5 nm after 1 month, indicating the possibility
of forming an emulsion.28 MD3 was bluish after 1 month in stor-
age, as the average particle size increased from 17.8 nm to
27.4 nm. In contrast, MD2 showed a very stable trend in aggregate
size over the same storage period. In terms of aggregate sizes,
MD2 remained quite stable, ranging from 9.3 nm initially to
10.4 nm after a month. Visual observation of these microemul-
sions revealed that no precipitation nor any discernible phase-
change occurred during the month-long storage. Hence, the
MD2 microemulsion formulation would be suitable for further
studies in applications such as cosmetics.
The DPPH antioxidant activity (AOA) of MD2 microemulsion was
found empirically at 55%, in contrast to 69.5% for that of
20 ppm L-ascorbic acid. Further measurement of the antioxidant
activity for each component of microemulsion MD2 gave 15%
for 1 wt% TTO in ethanol and 41% for 9 wt% mixed surfactant
(Smix) solution. Besides this, no discernible antioxidant activity
was observed in 2.25 wt% propylene glycol (PPG). In brief, a
TTO-microemulsion could still retain the antioxidant activity of
its constituents without deviation.
DISCUSSION
The concept of surfactant-enhanced extraction of tea tree oil by
hydrodistillation was proven in this work. That is, the presence
of suitable surfactants, such as Triton CG-110, in the extractant
solution could facilitate the extraction process of tea tree oil by
hydrodistillation, not only in the maximum extraction capacity
but also in faster kinetics of extraction. However, there are only
a few types of surfactants so far reported in the open literature
for application in the extraction of essential oils. Additionally, this
does not guarantee that more oil can be extracted with more sur-
factants being added to the extractant. One possible cause for the
inferior extraction yield could be the foaming of the extractant
solution. The persistent foam and froth of the boiled extractant
solution likely prevents the vapor (rich in essential oils) from leav-
ing the liquid extractant. Thus, the extraction yield of TTO would
be jeopardized. Coincidently, the empirical extraction yield of
TTO was relatively lower in those observed with significant foam-
ing in the hydrodistillation system. Thus, foaming resulting from
vigorous bubbling and boiling of extractant solutions should be
preferably avoided during the hydrodistillation process.
In general, ionic surfactants tend to foam more than nonionic
surfactants, and hydrophobic surfactants are more inclined
toward foaming more than hydrophilic surfactants.19 Therefore,
hydrophilic nonionic surfactants are more suitable for the
surfactant-assisted hydrodistillation process. In addition, the
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www.soci.org
Particle size (nm)
pH After preparation After 1 month
4.95 19.5 ± 0.1 180.8 ± 3.6
4.86 9.3 ± 0.1 10.4 ± 0.1
4.87 17.8 ± 0.1 27.4 ± 0.1
concentration of nonionic surfactants to be formulated in the liq-
uid extractant would be of grave concern. The concentration of
surfactant does not only affect the solubilization of essential oil,
but also the foaming. For a specific surfactant, the foam height,
which is generally considered an indicator of foaming, commonly
increases with an increase in surfactant concentration below the
CMC.19 In this work, foaming was observed in the hydrodistilla-
tion; meanwhile, the hydrophilic Triton CG-110 present in the
extractant solution was more than 1500 mg L−1, but slightly less
than its CMC at 1748 ppm. The cloud point of 1 wt% Triton
CG-110 is greater than the boiling point of water, revealing the
hydrophilic nature of Triton CG-110. The optimal concentration
of Triton CG-110 in extractant solution determined from RSM
based on the extraction yield of TTO was ca. 600 mg L−1, lower
than its CMC. Remarkably, though Triton CG-110 is categorized
as a nonionic surfactant by the manufacturer, Triton CG-110 com-
mercially available does contain low levels of electrolytes as stabi-
lizers during synthesis and storage. Hence, it would behave as a
weak electrolyte and result in a weak acidic surfactant solution,
instead of a neutral solution, which is evidently shown in the pH
values from 4.87 to 4.95 in Table 5.
In all, surfactant-assisted hydrodistillation is a simple method
similar to conventional hydrodistillation. It could improve the
extraction of essential oils, not only in the extraction yield but also
in the rate of extraction. Furthermore, it could be applicable on an
industrial scale due to its lower production cost and easier scale-
up in production process, compared to modern methods, such
as SFE and MAE. However, its main disadvantage could result from
the foaming in surfactant-containing extractants that prevents
the vaporization of essential oils and, thus, inhibits the separation
of essential oil from extractants. Therefore, the selection of proper
surfactants and operation conditions is pivotal to the success of
the surfactant-assisted hydrodistillation.
In summary, both types of surfactants and their concentrations
in the extractant solution must be taken into account together for
the enhanced extraction of essential oils by hydrodistillation. As
shown by this work, the Design of Experiments (DOE) strategy
could be successfully applied with RSM to optimize the
surfactant-enhanced hydrodistillation process of tea tree oil.
In addition to the extraction yield, a correlation function based
on RSM could fit well with the measured DPPH antioxidant activity
of TTO (Eqn (3)). This empirical equation could be further deduced
to obtain the optimal parameters of the TTO extraction process,
should the antioxidant activity of TTO be mainly concerned,
instead of the maximal extraction yield of TTO. Notably, it is likely
that the optimal conditions predicted by RSM based on either the
extraction yield or the antioxidant activity could be seemingly dif-
ferent. For example, to maximize the DPPH antioxidant activity of
the hydrodistilled TTO, Eqn (6) gave the optimal conditions as fol-
lows: (i) 689 mg L−1 Triton CG-110 in extractant, (ii) 24.1 as the
9
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 www.soci.org
mass ratio of extractant-to-desiccated foliage, and (iii) 128 min for
process time. This optimal condition is quite different from that
used to obtain the maximal extraction yield of TTO. Explicitly,
the optimal conditions for the extraction yield and the antioxidant
activity of TTO predicted by Eqns (5) and (6) are different. This is
mainly attributable to the different contributions of each constit-
uent of the essential oil to the antioxidant activity, as it is known
that the essential oil consists of various compounds.
CONCLUSIONS
The response surface methodology based on a central composite
design was utilized to attain the optimal extraction parameters of
tea tree oil by the surfactant-enhanced hydrodistillation process.
As a result, the optimal condition for the maximal extraction yield
was collectively determined as follows: (i) 597 mg L−1 Triton CG-
110 as liquid extractant, (ii) a feed of 1 g dried tea tree leaves in
25.4 mL liquid extractant, and (iii) 140 min for the extraction time.
The RSM model predicted an optimal extraction yield of tea tree
oil at 6.71 wt%, while the DPPH antioxidant activity of the
obtained tea tree oil was forecast as 48.2% under the same condi-
tions. Empirically, the extraction yield and the DPPH antioxidant
activity of TTO from confirmation experiments were found to be
6.69% ± 0.54% and 50.68% ± 0.24%, respectively, and were in
good accordance with those predicted by RSM. With 650 mg L−1
Triton CG-110, the first-order rate constant for the extraction
kinetics of TTO was 0.065 min−1, compared to 0.054 min−1 for
that without a surfactant present in the extractant. That is, the
presence of 650 mg L−1 Triton CG-110 in the extractant improved
the extraction yield of TTO by 17.5%, compared to that without
surfactant present. The microemulsion made of 1 wt% TTO and
9 wt% mixed surfactant (6.7 wt% Triton CG-110 and 2.3 wt%
PPG) was stable for at least over a month in storage, while the
average aggregate size of this microemulsion varied insignifi-
cantly from 9.3 nm to 10.4 nm over a period of 1 month in storage.
ACKNOWLEDGEMENTS
This work was financially supported by the Ministry of Science and
Technology of Taiwan (MoST 109-2221-E-006-088-MY3). We are
also grateful for the instrumental support from the Core Facility
Center of National Cheng Kung University (MoST 110-2731-M-
006-001).
DECLARATIONS OF COMPETING INTERESTS
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.
REFERENCES
1 Battisti MA, Caon T and de Campos AM, A short review on the antimi-
crobial micro- and nanoparticles loaded with Melaleuca alternifolia
essential oil. J Drug Delivery Sci Technol 63:102283 (2021). https://
doi.org/10.1016/j.jddst.2020.102283.
2 Thakur D, Kaur G, Puri A and Nanda R, Therapeutic potential of essen-
tial oil-based microemulsions: reviewing state-of-the-art. Curr Drug
Delivery 18:1218–1233 (2021). https://doi.org/10.2174/156720181
8666210217161240.
3 Lam NS, Long X, Su XZ and Lu F, Melaleuca alternifolia (tea tree) oil and
its monoterpene constituents in treating protozoan and helminthic
infections. Biomed Pharmacother 130:110624 (2020). https://doi.org/
10.1016/j.biopha.2020.110624.
10
wileyonlinelibrary.com/jctb © 2022 Society of Chemical Industry (SCI).
T-V Vo, T-H Truong, B-H Chen
4 Lee CJ, Chen LW, Chen LG, Chang TL, Huang CW, Huang MC et al., Cor-
relations of the components of tea tree oil with its antibacterial
effects and skin irritation. J Food Drug Anal 21:169–176 (2013).
https://doi.org/10.1016/j.jfda.2013.05.007.
5 Brophy JJ, Davies NW, Southwell IA, Stiff IA and Williams LR, Gas chro-
matographic quality control for oil of Melaleuca terpinen-4-ol type
(Australian tea tree). J Agric Food Chem 37:1330–1335 (1989).
https://doi.org/10.1021/jf00089a027.
6 Li FL, Peng C, Yuan K, Xu QW and Song H, Deterpenation of tea tree oil by
liquid-liquid extraction with hexalkylguanidinium ionic liquid. J Mol Liq
339:117048 (2021). https://doi.org/10.1016/j.molliq.2021.117048.
7 Mondello F, De Bernardis F, Girolamo A, Cassone AA and Salvatore G, In
vivo activity of terpinen-4-ol, the main bioactive component of Mel-
aleuca alternifolia Cheel (tea tree) oil against azole-susceptible and-
resistant human pathogenic Candida species. BMC Infect Dis 6:158
(2006). https://doi.org/10.1186/1471-2334-6-158.
8 Southwell IA, Freeman S and Rubel D, Skin irritancy of tea tree oil.
J Essent Oil Res 9:47–52 (1997). https://doi.org/10.1080/10412905.
1997.9700713.
9 International Organization for Standardization, ISO 4730:2017 Essential
oil of Melaleuca, terpinen-4-ol type (Tea Tree oil). https://www.iso.
org/standard/69082.html.
10 Gomes PGC, Veloso AF, Maynard IFN, Marques MN, de Souza RL,
Pereira MM et al., Integrative process to extract chlorophyll and
purify rosmarinic acid from rosemary leaves (Rosmarinus officialis).
J Chem Technol Biotechnol 95:1503–1510 (2020). https://doi.org/10.
1002/jctb.6343.
11 Azmir J, Zaidul I, Rahman M, Sharif K, Sahena F, Jahurul M et al., Optimi-
zation of oil yield of Phaleria macrocarpa seed using response sur-
face methodology and its fatty acids constituents. Ind Crops Prod
52:405–412 (2014). https://doi.org/10.1016/j.indcrop.2013.11.009.
12 Fernández-Marín R, Fernandes S, Andrés MA and Labidi J, Microwave-
assisted extraction of Curcuma longa L. oil: optimization, chemical
structure and composition, antioxidant activity and comparison
with conventional Soxhlet extraction. Molecules 26:1516 (2021).
https://doi.org/10.3390/molecules26061516.
13 Chen F, Zu Y and Yang L, A novel approach for isolation of essential oil
from fresh leaves of Magnolia sieboldii using microwave-assisted
simultaneous distillation and extraction. Sep Purif Technol 154:271–
280 (2015). https://doi.org/10.1016/j.seppur.2015.09.066.
14 Charchari S and Abdelli M, Enhanced extraction by hydrodistillation of
sage (Salvia officinalis L.) essential oil using water solutions of non-
ionic surfactants. J Essent Oil Bear Plants 17:1094–1099 (2014).
https://doi.org/10.1080/0972060X.2014.935041.
15 Wong V, Wyllie S, Cornwell C and Tronson D, Supercritical fluid extraction
(SFE) of monoterpenes from the leaves of Melaleuca alternifolia (Tea
Tree). Molecules 6:92103 (2001). https://doi.org/10.3390/60100092.
16 Zhao S and Zhang D, Supercritical CO2 extraction of Eucalyptus leaves
oil and comparison with Soxhlet extraction and hydro-distillation
methods. Sep Purif Technol 133:443–451 (2014). https://doi.org/10.
1016/j.seppur.2014.07.018.
17 Essien SO, Young B and Baroutian S, The antibacterial and anti-
proliferative ability of kānuka, Kunzea ericoides, leaf extracts
obtained by subcritical water extraction. J Chem Technol Biotechnol
96:1308–1315 (2021). https://doi.org/10.1002/jctb.6647.
18 Negro V, Ruggeri B, Mancini G and Fino D, Recovery of D-limonene
through moderate temperature extraction and pyrolytic products
from orange peels. J Chem Technol Biotechnol 92:1186–1191
(2017). https://doi.org/10.1002/jctb.5107.
19 Rosen MJ and Kunjappu JT, Surfactants and Interfacial Phenomena.
John Wiley & Sons, Hoboken, New Jersey (2012).
20 Balzer D, Alkylpolyglucosides, their physico-chemical properties and
their uses. Tenside, Surfactants, Deterg 28:419–427 (1991). https://
doi.org/10.1515/tsd-1991-280610.
21 Kühn AV and Neubert RH, Characterization of mixtures of alkyl poly-
glycosides (plantacare) by liquid chromatography-electrospray
ionization quadrupole time-of-flight mass spectrometry. Pharma
Res 21:2347–2353 (2004). https://doi.org/10.1007/s11095-004-
7688-0.
22 Savic S, Lukic M, Jaksic I, Reichl S, Tamburic S and Müller-Goymann C,
An alkyl polyglucoside-mixed emulsifier as stabilizer of emulsion
systems: the influence of colloidal structure on emulsions skin
hydration potential. J Colloid Interface Sci 358:182–191 (2011).
https://doi.org/10.1016/j.jcis.2011.02.049.
J Chem Technol Biotechnol 2022
Surfactant-assisted extraction of Melaleuca alternifolia (tea tree)
23 Vo TV, Chou YY and Chen BH, Preparation of microemulsion from an
alkyl polyglucoside surfactant and tea tree oil. Molecules 26:1971
(2021). https://doi.org/10.3390/molecules26071971.
24 Choi HS, Song HS, Ukeda H and Sawamura M, Radical-scavenging
activities of citrus essential oils and their components: detection
using 1, 1-diphenyl-2-picrylhydrazyl. J Agric Food Chem 48:4156–
4161 (2000). https://doi.org/10.1021/jf000227d.
25 Santos SB, Martins MA, Caneschi AL, PRM A and Reis Coimbra JS, Kinet-
ics and thermodynamics of oil extraction from Jatropha curcas
L. using ethanol as a solvent. Int J Chem Eng 2015:871236 (2015).
https://doi.org/10.1155/2015/871236.
26 Lambers H, Piessens S, Bloem A, Pronk H and Finkel P, Natural skin sur-
face pH is on average below 5, which is beneficial for its resident
J Chem Technol Biotechnol 2022 © 2022 Society of Chemical Industry (SCI).
www.soci.org
flora. Int J Cosmet Sci 28:359–370 (2006). https://doi.org/10.1111/j.
1467-2494.2006.00344.x.
27 Kim S, Ng WK, Shen S, Dong Y and Tan RB, Phase behavior, microstruc-
ture transition, and antiradical activity of sucrose laurate/propylene
glycol/the essential oil of Melaleuca alternifolia/water microemul-
sions. Colloids Surf, A 348:289–297 (2009). https://doi.org/10.1016/j.
colsurfa.2009.07.043.
28 Lins RF, Lustri WR, Minharro S, Alonso A and de Sousa Neto D, On the
formation, physicochemical properties and antibacterial activity of
colloidal systems containing tea tree (Melaleuca alternifolia) oil. Col-
loids Surf, A 497:271–279 (2016). https://doi.org/10.1016/j.colsurfa.
2016.02.024.
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