Industrial Crops & Products 141 (2019) 111809

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Industrial Crops & Products

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Biocompatible protic ionic liquids-based microwave-assisted liquid-solid T

extraction of astaxanthin from Haematococcus pluvialis
⁎
Yunchang Fana, , Zeyu Niub, Chen Xua, Lei Yanga, Feiyang Chena, He Zhanga
a
College of Chemistry and Chemical Engineering, Henan Polytechnic University, Jiaozuo 454003, China
b
College of Food Science and Engineering, Central South University of Forestry and Technology, Changsha 410004, China

A R T I C LE I N FO A B S T R A C T

Keywords: Haematococcus pluvialis, a green microalga, can accumulate high level of astaxanthin which is regarded as a super
Biocompatible antioxidant extensively used in cosmetics, poultry, food, and pharmaceutical industries. The difficult point for
Protic ionic liquids (PILs) extraction of astaxanthin is how to break the thick resistant cell walls of Haematococcus pluvialis effectively. In
Astaxanthin this work, this difficulty was successfully solved by the biocompatible protic ionic liquids-based microwave-
Microwave-assisted liquid-solid extraction
assisted liquid-solid extraction (PILs-MALSE). Ethanolammonium caproate (EAC), a PIL used in here, can
(MALSE)
strongly dissolve mannan, one of the main components of the cell walls of Haematococcus pluvialis, which can
leave out the pretreatment process to disrupt the cell walls before extraction, thereby leading to fast extraction
(extraction time, 50 s). Furthermore, EAC has the obvious advantages over the imidazolium-based ILs, such as
low cost, easy synthesis, and excellent biocompatibility. The PILs-MALSE method is more effective for the ex-
traction of astaxanthin compared with conventional extraction techniques.

1. Introduction
Astaxanthin as a member of the carotenoid family has been ex-
tensively used in cosmetics, poultry, food, and pharmaceutical in-
dustries because of its excellent antioxidant activity. Its antioxidant
activity is shown to be approximately 10 times stronger than that of
other carotenoids (such as lutein, canthaxanthin, and β-carotene), and
500 times higher than that of vitamin E (Ambati et al., 2014; Sarada
et al., 2006; Ahmed et al., 2015). Growing evidence also shows that
astaxanthin has benefit effects on human health due to the potent an-
tioxidant activity, such as cancer prevention, inflammation protection,
and immune system boost (Ambati et al., 2014; Zhao et al., 2016; Alesci
et al., 2015). Furthermore, astaxanthin is also used as a pigmentation
source in aquaculture (Sarada et al., 2006), which are generally sub-
sequently consumed by humans.
Astaxanthin is usually produced synthetically or obtained from
natural sources, such as algae (Capelli et al., 2013; Ruen-ngam et al.,
2011), yeast (Barredo et al., 2017), shrimp byproducts (Li et al., 2017;
Zhang et al., 2014), marine plants (Lee and Row, 2016), etc. Recent
work suggests that natural astaxanthin is approximately 20 times more
potent at free radical elimination than the synthetic astaxanthin (free
radical inhibition per milligram astaxanthin, 0.59% (synthetic astax-
anthin) versus 12.34% (natural astaxanthin)) (Capelli et al., 2013).
Therefore, astaxanthin obtained from natural resources has received
much attention from the scientific community in recent years (Ruen-
ngam et al., 2011; Barredo et al., 2017; Li et al., 2017).
Haematococcus pluvialis, a unicellular green microalgae, is regarded
as a promising source of astaxanthin due to its ability to accumulate
high content of natural astaxanthin (Ruen-ngam et al., 2011; Molino
et al., 2018b; Wang et al., 2012). Unfortunately, the separation of as-
taxanthin from Haematococcus pluvialis is difficult due to the thick cell
walls of this alga (Ruen-ngam et al., 2011; Wang et al., 2012). For this
reason, various extraction methods, such as high-pressure CO2-based
extraction (Wang et al., 2012; Pan et al., 2012; Machmudah et al.,
2006), accelerated solvent extraction (ASE) (Molino et al., 2018b),
hydrothermal method (Cheng et al., 2017), Soxhlet extraction (Ruen-
ngam et al., 2011; Wang et al., 2012; Pan et al., 2012), and liquid-solid
extraction (Sarada et al., 2006; Desai et al., 2016) have been developed.
Among these methods, high-pressure CO2-based extraction is an eco-
friendly technique for the separation of valuable compounds from
natural matrices (Polanowska et al., 2019); ASE which uses extraction
solvents at high pressures and temperatures, is also an effective tech-
nique to extract high-added value compounds from natural resources
(Molino et al., 2018b). However, SFE and ASE must be operated at high
pressures, leading to high capital and operating costs (Temelli, 2009;
Giergielewicz-Możajska et al., 2001). Soxhlet extraction is usually time-
consuming and requires a large number of organic solvents (Ruen-ngam
et al., 2011; Wang et al., 2012). Liquid-solid extraction is a common

⁎
Corresponding author.
E-mail address: yunchangfan2009@163.com (Y. Fan).

https://doi.org/10.1016/j.indcrop.2019.111809
Received 24 June 2019; Received in revised form 12 August 2019; Accepted 22 September 2019
Available online 27 September 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
Y. Fan, et al.
used technique to extract astaxanthin from Haematococcus pluvialis.
However, a pretreatment procedure to disrupt the thick cell walls of
Haematococcus pluvialis is usually needed before extraction (Sarada
et al., 2006; Desai et al., 2016; Kim et al., 2016). In this context, Sarada
et al. (2006) used hydrochloric acid solution to disrupt the algal cell
walls, which facilitated the subsequent extraction of astaxanthin with
acetone (86–94% of astaxanthin were extracted from Haematococcus
pluvialis). Besides HCl, the new type of solvents, ionic liquids (ILs), have
also been used to destroy Haematococcus pluvialis cell walls due to their
excellent properties, such as good solubility for organic and inorganic
compounds, and high thermal and chemical stability (Desai et al., 2016;
Ventura et al., 2017; Liu et al., 2019, 2018; Choi et al., 2019; Lee and
Row, 2016). Desai et al. (2016) reported the use of ILs to treat the
Haematococcus pluvialis cells. It was found that the cell walls could be
partially disrupted by the ILs, resulting in the formation of tiny holes in
the cell walls and paving the way for the extraction solvent (ethyl
acetate) to penetrate and extract the astaxanthin. 1-Ethyl-3methylimi-
dazolium di-butylphosphate ([Emim] DBP) was the most effective IL for
the cell destruction. Choi et al., (2019) treated the Haematococcus plu-
vialis cells with dialkylimidazolium-based IL/water mixtures and it was
found that the cell walls were significantly torn and broken, thereby
facilitating subsequent extraction of astaxanthin by hexane. Huang
et al. (2018) used a hydrophobic solvent, dimethylaminocyclohexane
(DMCHA) to treat Haematococcus pluvialis cells. The treated cells ex-
hibited obvious morphological changes, showing rough, distorted, and
squashed cell surfaces. After extraction, the hydrophobic DMCHA was
converted into a hydrophilic IL, [DMCHAH+][HCO3−] via the addition
of water and CO2, which makes the lipid layer containing astaxanthin
float to the top of the aqueous solution and thus achieves the separation
of astaxanthin from the extraction system.
A very recent review has summarized the advances in various
techniques for the extraction of astaxanthin from Haematococcus plu-
vialis (Khoo et al., 2019).
Although many exciting results have been obtained using ILs to treat
Haematococcus pluvialis cells, challenges lie in the fact that the ILs used
in the reported work, such as dialkylimidazolium salt and [DMCHAH+]
[HCO3−] are moderately toxic and have poor biodegradability, which
may pose a serious threat to the environment and human health (Kumar
et al., 2011; Jordan and Gathergood, 2015; Nikinmaa, 2014; Vraneš
et al., 2016; Huang et al., 2018). Therefore, there is an urgent need to
develop more environmentally friendly and biocompatible ILs to solve
this issue.
In this work, protic ionic liquids (PILs) with biomolecules as raw
materials were synthesized and combined with more efficient extrac-
tion method, microwave-assisted liquid-solid extraction, to extract as-
taxanthin from Haematococcus pluvialis. The experimental parameters
affecting extraction efficiency were optimized and the toxicity of PILs
was also evaluated.
2. Experimental section
2.1. Materials
n-Caproic acid (99%), diethanolamine (99%), triethanolamine
(99%), n-octanol (99.5%), and astaxanthin (98%) were obtained from
Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China).
Ethanolamine (99%) was purchased from Energy Chemical Co.
(Shanghai, China). Acetylthiocholine iodide (98%), and 5,5′-dithiobis
(2-nitrobenzoic acid) (DTNB, 98%) were supplied by Tokyo Chemical
Industry Co., Ltd. (Tokyo, Japan). Acetylcholinesterase (AChE) from fly
head (200 U g−1) was purchased from Macklin Biochemical Co., Ltd.
(Shanghai, China). Silica gel with particle size of 200–300 mesh was
obtained from Qingdao Haiyang Chemical Co., Ltd. (Qingdao, China).
1-Ethyl-3-methylimidazolium ethylsulfate ([C2mim]ES, 99%) was sup-
plied by Chengjie Chemical Co., Ltd. (Shanghai, China). 1-Ethyl-3-me-
thylimidazolium di-butylphosphate ([C2mim]DBP, ≥97.0%), mannan
2
Industrial Crops & Products 141 (2019) 111809
from Saccharomyces cerevisiae, 4-nitroaniline (NA, ≥99%), and
Reichardt's dye (RD, 90%) were supplied by Sigma-Aldrich Co. (St.
Louis, USA). N,N-Diethyl-4-nitroaniline (DENA, 97%) was purchased
from Fluorochem Ltd. (Hadfield, UK). Cellulose microcrystalline (extra
pure, average particle size 90 μm) was obtained from Acros Organics
(Fair Lawn, NJ, USA). All other chemicals were of analytical grade
unless stated. Ultrapure water (18.2 MΩ cm) used in this work was
produced by an Aquapro purification system (Aquapro International
Co., Ltd., Dover, USA). Haematococcus pluvialis powder (100 mesh) was
obtained from Guangdong HaiRong Environmental Protection
Technology Co., Ltd. (Zhaoqing, China).
2.2. Methods
2.2.1. Synthesis of PILs
To synthesize ethanolammonium caproate (EAC), 0.1 mol of n-ca-
proic acid was added dropwise into 0.1 mol of ethanolamine under
stirring at 25 °C in a water bath to form a homogeneous liquid and EAC
was obtained as a light yellow liquid. Its chemical structure was iden-
tified by a proton nuclear magnetic resonance spectrometer (1H NMR,
model: Avance AV, 400 MHz, Bruker Biospin, Rheinstetten, Germany).
The chemical shifts (δ, ppm, solvent: DMSO-d6) were as follows:
0.828–0.863 (t, 3H), 1.194–1.266 (m, 4H), 1.421–1.456 (t, 2H),
1.951–1.989 (t, 2H), 2.731–2.757 (t, 2H), 3.499–3.525 (t, 2H), 6.811 (s,
4H).
The other two PILs, diethanolammonium caproate (DEAC) and
triethanolammonium caproate (TEAC) were prepared following a si-
milar way. DEAC was a light yellow liquid. The chemical shifts (δ) of 1H
NMR (400 MHz, DMSO-d6) were as follows: 0.820–0.855 (t, 3H),
1.183–1.275 (m, 4H), 1.407–1.480 (m, 2H), 1.982–2.020 (t, 2H),
2.860–2.872 (d, 4H), 3.579–3.606 (t, 4H), 7.323 (s, 4H). The PIL, TEAC
was a colorless liquid. The chemical shifts (δ) of 1H NMR (400 MHz,
DMSO-d6) were as follows: 0.849–0.883 (t, 3H), 1.225–1.308 (m, 4H),
1.459–1.532 (m, 2H), 2.130–2.167 (t, 2H), 2.680–2.709 (t, 6H),
3.480–3.509 (t, 6H), 6.478 (s, 4H).
The 1H NMR spectra of the three PILs are shown in Figs. S1–3 in the
Supplementary material. As can be seen from these figures, no obvious
impurity peaks are observed in the 1H NMR spectra.
The water contents in the three PILs were determined by a KF-1B
moisture meter (Benchuang Analytical Instrument Co., Ltd., Zibo,
China). It was found that the water contents of EAC, DEAC and TEAC
were 0.63%, 0.69% and 0.57%, respectively.
For clarity, the chemical structures of ILs used in this work and
astaxanthin are shown in Fig. 1.
2.2.2. PILs-based microwave-assisted liquid-solid extraction (PILs-MALSE)
The PILs-MALSE was conducted in a commercial microwave oven
(model G70F20CPIII-TK(W0), Guangdong Galanz Microwave Oven and
Electrical Appliances Manufacturing Co., Ltd., Foshan, China) with a
microwave irradiation frequency of 2.45 GHz and a maximum power of
700 W. Generally, 50 mg of Haematococcus pluvialis powder and a cer-
tain amount of PIL solution were added into a glass vessel, and this
mixture was heated by microwave irradiation. During the extraction
process, the glass vessel kept open, i.e., MAE was conducted in an open
mode. The studied ranges of PIL concentration, number of extraction,
microwave irradiation power, microwave irradiation time, and liquid-
solid ratio are 0–5.2 mol L–1, 1–5 times, 70–350 W, 20–110 s, and
3–30 g g–1, respectively. The selection of the experimental conditions
was decided by referring to the published work (Zhao et al., 2009;
Ruen-ngam et al., 2011; Bhan et al., 2017).
After extraction, the extract was diluted to 10 ml with ethanol. After
filtered by nylon membrane (pore size: 0.45 μm, Guodian Taoyuan
Medical and Chemical Instrument Factory, Haining, China), the re-
sultant extract was directly injected into a high performance liquid
chromatograph (HPLC, model, Agilent 1200; Agilent Technologies,
Santa Clara, CA, USA) equipped with a variable wavelength detector.
Y. Fan, et al.
Fig. 1. Chemical structures of ILs used in this work and astaxanthin. EAC, ethanolammonium caproate; DEAC, diethanolammonium caproate; TEAC, triethano-
lammonium caproate; [C2mim]ES, 1-ethyl-3-methylimidazolium ethylsulfate; [C2mim]DBP, 1-ethyl-3-methylimidazolium di-butylphosphate.
The chromatographic conditions referred to the reported literature
(Ruen-ngam et al., 2011; Pan et al., 2012) and were as follows: mobile
phase, the mixture of acetonitrile and 0.1% (v/v) acetic acid aqueous
solution (95:5, v/v) with a flow rate of 1.2 ml min–1; detection wave-
length, 476 nm; injection volume, 5.0 μL; separation column, ZORBAX
Eclipse XDB-C18 column (4.6 mm × 150 mm, 5 μm; Agilent Technolo-
gies); column temperature, 30 °C.
The extraction efficiency (E) of astaxanthin is expressed as the
weight ratio of astaxanthin to Haematococcus pluvialis powder.
The limit of blank (LoB, signal-to-noise ratio of 1.645 (S/
N = 1.645)), limit of detection (LoD, S/N = 3), and limit of quantifi-
cation (LoQ, S/N = 10) for astaxanthin analysis were calculated ac-
cording to the methods reported in literature (Şengül, 2016; Little,
2015) and it is found that they are 8.4 × 10−6 g L‒1 (LoB),
1.5 × 10‒5 g L‒1 (LoD), and 5.1 × 10‒5 g L‒1 (LoQ), respectively.
2.2.3. Soxhlet extraction without cell-wall disruption
Soxhlet extraction was carried out according to the procedures re-
ported in literature (Pan et al., 2012). Briefly, 0.5 g of Haematococcus
pluvialis powder was extracted by 150 ml of dichloromethane at 45 °C
until the color of the condensed solvent at the top of the apparatus
became colorless.
Additionally, Soxhlet extraction with acetone as solvent was also
conducted. The extraction procedures were similar to those described
above except that the extraction temperature was set at 60 °C (Ruen-
ngam et al., 2011).
2.2.4. Soxhlet extraction with cell-wall disruption
To disrupt the cell walls of Haematococcus pluvialis, 2.0 g of
Haematococcus pluvialis powder was mixed with 15 ml of HCl aqueous
solution (4.0 mol L−1) under stirring at 70 °C for 10 min and then the
Haematococcus pluvialis powder was washed with water several times to
remove HCl. Finally, the Haematococcus pluvialis powder treated by HCl
was dried to constant weight at 40 °C (Sarada et al., 2006). The Soxhlet
extraction for the HCl-treated Haematococcus pluvialis followed a similar
procedure as described above.
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Industrial Crops & Products 141 (2019) 111809
2.2.5. Determination of the hydrophobic parameters of the PILs
The hydrophobic parameters (the logarithm of n-octanol–water
partition coefficients (Pow), logPow) of the PILs used in this work were
measured according to the reported literature (Lee and Lee, 2009; Ropel
et al., 2005): the PIL solutions (1.0 mol L−1 for each) were prepared
with water that had been presaturated with n-octanol; the n-octanol
was also presaturated with water. In a typical procedure, 10 ml of a PIL
solution and 10 ml of n-octanol were mixed under stirring for 30 min at
25 °C and then phase separation was achieved by centrifugation. The
concentrations of PILs both in the n-octanol and water phases were
determined by a IC-2001 ion chromatograph (Tosoh Co., Tokyo, Japan)
equipped with a conductivity detector. The chromatographic conditions
were as follows: separation column, Super IC-Cation/P cation exchange
column (4.6 mm × 15 cm, Tosoh Co., Tokyo, Japan) ; column tem-
perature, 40 °C; mobile phase, the mixture of acetonitrile and metha-
nesulfonic acid aqueous solution (3.75 × 10‒3 mol L−1) (10% (v/v) of
acetonitrile) with a flow-rate of 0.6 ml min−1; injection volume, 30 μl
(before injection, both n-octanol and water phases were diluted 100
times with acetonitrile); detection mode, direct conductivity.
The Pow value is calculated by the following equation (Lee and Lee,
2009; Ropel et al., 2005):
Cn − octanol
Pow =
Cwater (1)
where Cn-octanol and Cwater are the concentrations of a PIL in the n-oc-
tanol and water phases, respectively.
2.2.6. Measurements of Kamlet‒Taft polarity parameters of the PILs
the Kamlet‒Taft polarity parameters, α (hydrogen bond donating
ability), β (hydrogen bond accepting ability), polarity (ET(30)), and π*
(dipolarity/polarisability) of the PILs used in this work were de-
termined according to the methods reported in literature (Hauru et al.,
2012). The detailed information was shown in the Supplementary
material (Section 2).
2.2.7. Acetylcholinesterase (AChE) inhibition assay
The inhibition of ILs on AChE activity was measured using the
colorimetric assay with acetylcholine iodide as the substrate and DTNB
Y. Fan, et al.
as the chromogenic reagent (Siopa et al., 2018; Stock et al., 2004): all
the solutions used were prepared with phosphate buffer (50 mmol L−1,
pH 8.0) and incubated at 25 °C for 15 min at least. To measure the
enzyme activity, 480 μL of phosphate buffer (50 mmol L−1, pH 8.0) was
mixed with 120 μL of AChE (2.5 U mL−1) and then the reaction was
initiated by the addition of acetylcholine iodide (1.2 mmol L−1, 360 μL)
and DTNB (3.03 mmol L−1, 2.24 mL). The hydrolysis of acetylthiocho-
line was monitored by measuring the absorbance (A) at a wavelength of
405 nm in 30-s intervals for 5 min. The enzyme activity was expressed
as ΔA0 min−1 obtained from the linear regression and the enzyme ac-
tivity in the presence of ILs was noted as ΔA min−1. Thus, the inhibition
level of ILs on AChE activity was calculated by the following equation
(Siopa et al., 2018):
ΔAmin-1
Inhibition percentage(%) = (1 - ) × 100
ΔA0min-1 (2)
The half maximal inhibitory concentration (IC50) refers to the IL
concentration that results in 50% reduction of the AChE activity.
2.2.8. Solubilities of cellulose and mannan in PILs
The solubilities of cellulose in PILs were determined via the method
described in literature (Zavrel et al., 2009): 5.0 g of a PIL was mixed
with 0.1 g of cellulose under stirring at 88 °C for 12 h. After cen-
trifugation, the PIL phase was withdrawn and the precipitation of the
dissolved cellulose was achieved by the addition of ethanol. The solu-
bilities of cellulose in PILs were defined as the ratio of the weight of
dissolved cellulose to the total weight of PILs and cellulose.
The solubilities of mannan in PILs at 88 °C were measured via a
similar way (Zhao et al., 2008): a PIL (0.2 g) was placed into a glass
tube and then heated to 88 °C. Mannan was added into the PIL carefully
at an increment of 7 mg. After stirring, the PIL solution should turn
clear before the addition of next increment. When some mannan is
insoluble and lasts for more than 12 h, it is considered that the sa-
turation limit is reached.
3. Results and discussion
3.1. The selection of PILs
In this work, EAC, DEAC, and TEAC were used to extract astax-
anthin and their extraction ability, shown in Fig. 2, follows the trend:
EEAC > EDEAC > ETEAC. It is known that Haematococcus pluvialis has a
thick cell wall which is mainly composed of cellulose and mannan
(Desai et al., 2016; Kim et al., 2016; Klochkova et al., 2013; Hagen
Fig. 2. Extraction efficiency of EAC, DEAC and TEAC for astaxanthin.
Microwave irradiation power, 210 W; liquid-solid ratio, 20 g g−1; microwave
irradiation time, 50 s; three extraction cycles. EAC, ethanolammonium
caproate; DEAC, diethanolammonium caproate; TEAC, triethanolammonium
caproate; E, extraction efficiency.
4
Industrial Crops & Products 141 (2019) 111809
et al., 2002). Therefore, the solubilities of cellulose and mannan in PILs
were measured and it is found that the solubilities of cellulose in EAC,
DEAC, and TEAC are 0.24%, 0.060%, and 0.070%, respectively; the
solubilities of mannan in EAC, DEAC, and TEAC are 59.2%, 36.5%, and
5.6%, respectively. It should be noted that the solubilities of cellulose in
PILs are lower than those in conventional imidazolium-based ILs
(Zakrzewska et al., 2010). The solubilities of mannan in ILs are not
available in the published literature, but Shen et al. (2011) studied the
solubilities of glucomannan which has a very similar structure to
mannan in the conventional imidazolium-based ILs, and found that the
imidazolium-based ILs could dissolve about 5% of glucomannan. Ob-
viously, the PILs, especially EAC has strong ability to dissolve mannan,
which may lead to the cell wall disruption and subsequent leaching of
astaxanthin. To confirm this, the morphologies of Haematococcus plu-
vialis cells before and after extraction were measured by a field-emis-
sion scanning electron microscope (FESEM, Quanta 250 FEG, Thermo
Fisher Scientific, Hillsboro, Oregon, USA). As shown in Fig. 3, after
extracted by PILs, Haematococcus pluvialis cells are wrinkled strongly
and there have cavities on the cell surfaces, indicating the cell wall of
Haematococcus pluvialis is effectively damaged by PILs.
Furthermore, the polarity (ET(30) values) of the three PILs was
measured and the results are listed in Table S1 in the Supplementary
material. As can be seen from Table S1, compared with EAC, DEAC and
TEAC have stronger polarity because they contain more hydroxyl
groups, making them more hydrophilic (logPow(EAC) = 0.71;
logPow(DEAC) = 0.51; logPow(TEAC) = 0.28). This means that the hy-
drophobic interaction between the PILs (DEAC and TEAC) and astax-
anthin is weaker, leading to lower extraction efficiency.
Since EAC exhibits highest extraction ability, it is selected as the
optimal extraction solvent and used in the following experiments.
3.2. Optimization of extraction conditions
The effect of the PIL concentration on the extraction efficiency is
illustrated in Fig. 4 (A): the extraction ability of water for astaxanthin is
rather poor. The increase in the PIL concentration leads to the increase
in the extraction efficiency with the PIL concentration ranging from
0.5 mol L−1 to 1.9 mol L−1. The extraction efficiency of astaxanthin
maintains steady in the PIL concentration range of 1.9–4.4 mol L−1 and
then increases slightly with improving PIL concentration up to
5.2 mol L−1. When pure PIL is adopted, its extraction ability for as-
taxanthin is close to that of 5.2 mol L−1 of PIL. Based on these results,
5.2 mol L−1 is regarded as the optimal PIL concentration.
The effect of the number of extraction cycles on the extraction ef-
ficiency is shown in Fig. 4 (B): when the extraction cycles exceed two
times, the extraction efficiency does not increase. Therefore, two ex-
traction cycles are selected as the optimal conditions.
As shown in Fig. 5(A), the extraction efficiency of astaxanthin in-
creases with the microwave irradiation power increasing from 70 W to
210 W and then decreases with further increasing the microwave irra-
diation power. It is clear that higher microwave irradiation power leads
to higher extraction temperature (Fig. 5(A)). Recently, numerous stu-
dies have shown that astaxanthin is a heat-sensitive compound and the
exposure to high temperatures can lead to its degradation (Ruen-ngam
et al., 2011; Pan et al., 2012; Zhou et al., 2019; Di Sanzo et al., 2018).
Therefore, the decrease in the extraction efficiency of astaxanthin at
higher microwave irradiation power can be attributed to the decom-
position of astaxanthin at higher extraction temperature. Given that
210 W can provide higher extraction efficiency for astaxanthin, it is
thus selected as the optimal microwave irradiation power. In addition,
the microwave irradiation power selected in this work is lower than
that of the acetone-based MALSE (720 W) (Ruen-ngam et al., 2011) and
higher than that of the MALSE using the mixture of ethanol and ethyl
acetate as extraction solvent (141 W) (Zhao et al., 2009). However, in
Zhao’s work, more extraction cycles are required (4 times), which is
more than the extraction cycles of this work (2 times), suggesting that
Y. Fan, et al.
Fig. 3. FESEM images of Haematococcus pluvialis powder before extraction (A) and extracted by EAC (B), DEAC (C) and TEAC (D). EAC, ethanolammonium caproate;
DEAC, diethanolammonium caproate; TEAC, triethanolammonium caproate.
the suggested PIL-based MALSE is more efficient.
The effect of microwave irradiation time on the extraction efficiency
is shown in Fig. 5(B), the extraction efficiency of astaxanthin increases
from 20 s to 50 s and keeps constant within 50 s to 70 s. While further
increasing microwave irradiation time is unfavorable, there appears an
obvious decrease in the extraction efficiency of astaxanthin (90 s and
110 s). It can be attributed to the higher extraction temperature caused
by longer microwave irradiation time. Based on these results, 50 s is
regarded as the optimal microwave irradiation time and used in the
following experiments. It is worth noting that the conventional solvent-
based MALSE requires a longer microwave irradiation time (83–300 s)
(Zhao et al., 2009; Ruen-ngam et al., 2011), suggesting that the PIL-
based MALSE is a faster extraction method.
To systematically investigate the effect of liquid-solid ratio on the
extraction of astaxanthin, additional experiments were conducted and
the experimental results are shown in Fig. 6. As can be seen, when the
liquid-solid ratio is greater than or equal to 10 g g−1, the extraction
efficiency of astaxanthin keeps almost constant. Therefore, 10 g g−1 is
selected as the optimal liquid-solid ratio.
Finally, a comparison on the MALSE conditions between this work
and the reported papers (Zhao et al., 2009; Ruen-ngam et al., 2011) was
conducted. The results listed in Table 1 indicate that compared with
conventional solvents, the use of PILs as extraction solvents has the
advantages of faster extraction, more efficient, and lower solvent con-
sumption.
3.3. Compared with reported methods
Soxhlet extraction is the conventional method to extract astaxanthin
from Haematococcus pluvialis (Ruen-ngam et al., 2011; Wang et al.,
2012; Pan et al., 2012). Therefore, the extraction performance of the
proposed PIL-based MALSE was compared with that of Soxhlet ex-
traction. The results illustrated in Fig. 7 show that without the dis-
ruption of cell wall, the extraction efficiency of Soxhlet extraction is
rather poor. After destroying the cell wall by 4.0 mol L−1 of HCl, the
extraction efficiency of Soxhlet extraction is close to that of the pro-
posed PIL-based MALSE. However, it should be noted that the treat-
ment of Haematococcus pluvialis with 4.0 mol L−1 of HCl leads to the
5
Industrial Crops & Products 141 (2019) 111809
significant loss of its weight (70.4%), which leads to a huge waste of
resources because Haematococcus pluvialis contains 25.69% of proteins,
6.30% of carbohydrates and 2.60% of lipids (Molino et al., 2018a).
Additionally, the imidazolium-based ILs, 1-ethyl-3-methylimidazo-
lium ethylsulfate ([C2mim]ES) (Praveenkumar et al., 2015) and
[C2mim]DBP (Desai et al., 2016) were also used to extract astaxanthin
from Haematococcus pluvialis or to permeabilise the Haematococcus
pluvialis cells before extraction. Fig. 7 shows that the extraction ability
of the two imidazolium-based ILs is lower than that of the proposed
PILs. Furthermore, various studies suggest that the commonly used
imidazolium-based ILs show moderate toxicity and poor biodegrad-
ability (Yan et al., 2012; Stock et al., 2004). Therefore, the bio-
compatibility of EAC, [C2mim]ES and [C2mim]DBP were also studied
via investigating their inhibition on the AChE activity. As shown in
Fig. 8, [C2mim]ES and [C2mim]DBP exhibit stronger inhibition on the
AChE activity (IC50([C2mim]ES) = 190.5 μmol L−1; IC50([C2mim]
DBP) = 307.6 μmol L−1), which is in good agreement with the ob-
servations reported in literature (Yan et al., 2012; Stock et al., 2004).
The PIL, EAC shows a weaker inhibition on the AChE activity
(IC50 = 3.9 × 104 μmol L−1), suggesting that EAC has better bio-
compatibility compared with imidazolium-based ILs.
3.4. The recyclability of PILs and the purification of astaxanthin
After extraction, the EAC phase was washed with ethyl acetate,
making astaxanthin enter ethyl acetate phase and then the recovered
EAC was dried at 70 °C for 2 h. As a result, EAC could be reused without
losing its extraction ability (Efresh EAC = 34.4 mg g−1; Erecycled
−1
EAC = 33.5 mg g ).
The ethyl acetate phase obtained above was distilled under vacuum
and the resultant extract contains free astaxanthin, astaxanthin
monoesters, and astaxanthin diesters (Ambati et al., 2014; Miao et al.,
2006). To obtain free astaxanthin, the extract was dissolved with me-
thanol and then the sodium hydroxide methanol solution was added to
make the final concentration of sodium hydroxide at 0.02 mol L−1. The
mixture was kept for 7 h in darkness under nitrogen atmosphere for
saponification of astaxanthin esters (Yuan and Chen, 2000, 1999). Fi-
nally, the saponified extract was separated by column chromatography.
Y. Fan, et al. Industrial Crops & Products 141 (2019) 111809

Fig. 5. Effect of microwave irradiation power (A) (liquid-solid ratio, 20 g g−1;

microwave irradiation time, 50 s; two extraction cycles; CEAC = 5.2 mol L−1)
and microwave irradiation time (B) (microwave irradiation power, 210 W; li-
quid-solid ratio, 20 g g−1; two extraction cycles; CEAC = 5.2 mol L−1) on the
extraction efficiency. EAC, ethanolammonium caproate; C, concentration; E,
extraction efficiency.
Fig. 4. Effect of the PIL concentration (A) (microwave irradiation power,
210 W; liquid-solid ratio, 20 g g−1; microwave irradiation time, 50 s; three
extraction cycles) and the number of extraction cycles (B) (microwave irra-
diation power, 210 W; liquid-solid ratio, 20 g g−1; microwave irradiation time,
50 s; CEAC = 5.2 mol L−1) on the extraction efficiency. EAC, ethanolammonium
caproate; IL, ionic liquid; C, concentration; E, extraction efficiency.

The mobile phase was the mixture of ethyl acetate and petroleum ether
(1:2, v/v). After evaporating the solvent under vacuum, the purity of
the obtained free astaxanthin was analyzed by the aforementioned
HPLC method and it is found to be 97.2%.

4. Conclusions

In this work, the biocompatible PILs were synthesized via acid-base

reaction and their ability to extract astaxanthin from Haematococcus
pluvialis with the aid of microwave was investigated. Experiments in-
dicated that EAC had good solubility for mannan, which led to the Fig. 6. Effect of liquid-solid ratio on the extraction efficiency. Microwave ir-
disruption of the cell wall and high astaxanthin extraction efficiency. radiation time, 50 s; two extraction cycles; CEAC = 5.2 mol L−1; microwave ir-
radiation power, 210 W. EAC, ethanolammonium caproate; C, concentration; E,
Furthermore, EAC showed weak inhibition on the AChE activity, sug-
extraction efficiency.
gesting that EAC was more biocompatible than the imidazolium-based
ILs. After extraction, EAC could be reused via back extraction with ethyl
acetate. Free astaxanthin was obtained by the saponification of the Funding
extract and high purity (97.2%) of free astaxanthin was achieved with
the aid of column chromatography. From the above, the PILs-based This research was financially supported by the Foundation of Henan
MALSE is a simple, effective, and environmentally benign method to province (No. 182102310728), the Program for Innovative Research
isolate astaxanthin from Haematococcus pluvialis. Team of Henan Polytechnic University (No. T2018-3) and the

6
Y. Fan, et al. Industrial Crops & Products 141 (2019) 111809

Ruen-ngam et al., (2011)

Zhao et al., (2009)
This work
Reference
Number of extraction

2
4
1

Fig. 7. Comparison of different methods on the extraction of astaxanthin. EAC,

ethanolammonium caproate; MALSE, microwave-assisted liquid-solid extrac-
tion; [C2mim]ES, 1-ethyl-3-methylimidazolium ethylsulfate; [C2mim]DBP, 1-
ethyl-3-methylimidazolium di-butylphosphate; E, extraction efficiency.
Liquid-solid ratio (g g−1)

41.4
79.1
10
Microwave time (s)

300
50
83
Microwave irradiation power (W)

MALSE, microwave-assisted liquid-solid extraction; EAC, ethanolammonium caproate.

Comparison on the MALSE conditions between this work and the reported papers.

Fig. 8. Inhibition of EAC, [C2mim]ES and [C2mim]DBP on the AChE activity.

[C2mim]ES, 1-ethyl-3-methylimidazolium ethylsulfate; [C2mim]DBP, 1-ethyl-3-
methylimidazolium di-butylphosphate; EAC, ethanolammonium caproate; IL,
ionic liquid; C, concentration; AChE, Acetylcholinesterase.
210
141
720

Fundamental Research Funds for the Universities of Henan Province

(NSFRF180415).

Appendix A. Supplementary data

Mixture of ethanol and ethyl acetate (2 : 1, v/v)

Supplementary material related to this article can be found, in the

online version, at doi:https://doi.org/10.1016/j.indcrop.2019.111809.

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