Effect of ultrasonic waves on the stability of all-trans lutein and its degradation kinetics

Article abstract of DOI:10.1016/j.ultsonch.2015.04.020

Abstract Ultrasound treatment has been widely applied in the extraction of biologically active compounds including carotenoids. However, there are few reports on their effects on the stability of these compounds. In the present study, the stability of all-trans lutein, one of the carotenoids, was investigated under the action of ultrasound. Results showed that ultrasound induced the isomerization of all-trans lutein to its isomers, namely to 13-cis lutein, 13′-cis lutein, 9-cis lutein and 9′-cis lutein as analyzed by HPLC coupled with DAD and LC-MS; and the percentage of the isomerization increased with increasing both ultrasonic frequency and power. The stability of all-trans lutein in dichloromethane was worst among multiple kinds of solvents. Interestingly, the retention rate of all-trans lutein improved as the temperature increased, which runs counter to the Arrhenius law. Under ultrasound irradiation, the degradation mechanism might be different with various temperatures, the degradation of all-trans lutein followed first-order kinetics at 20 °C, while second-order kinetics was followed at 30-50 °C. As the ultrasonic reaction time prolonged, lutein epoxidation nearly occurred. Those results presented here emphasized that UAE techniques should be carefully used in the extraction of all-trans lutein.

Full text of DOI:10.1016/j.ultsonch.2015.04.020

Accepted Manuscript
Effect of ultrasonic waves on the stability of all-trans lutein and its degradation
kinetics
Jiang-Feng Song, Da-Jing Li, Hui-Li Pang, Chun-Quan Liu
PII:
S1350-4177(15)00113-3
DOI:
http://dx.doi.org/10.1016/j.ultsonch.2015.04.020
ULTSON 2843
Reference:
Ultrasonics Sonochemistry
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Received Date:
Revised Date:
Accepted Date:
31 July 2014
2 April 2015
18 April 2015
Please cite this article as: J-F. Song, D-J. Li, H-L. Pang, C-Q. Liu, Effect of ultrasonic waves on the stability of all-
trans lutein and its degradation kinetics, Ultrasonics Sonochemistry (2015), doi: http://dx.doi.org/10.1016/
j.ultsonch.2015.04.020
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Effect of ultrasonic waves on the stability of
all-trans lutein and its degradation kinetics
Jiang-Feng Song a, b, Da-Jing Li a, c ∗, Hui-Li Pangc, Chun-Quan Liu a
a Institute of Farm Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing
210014, China
b
National Research and Development Center for Vegetable Processing, Nanjing 210014,
China
c College of Forestry, Northeast Forestry University, Harbin 150040, China
Abstract
Ultrasound treatment has been widely applied in the extraction of biologically
active compounds including carotenoids. However, there are few reports on
their effects on the stability of these compounds. In the present study, the
stability of all-trans lutein, one of the carotenoids, was investigated under the
action of ultrasound. Results showed that ultrasound induced the isomerization
of all-trans lutein to its isomers, namely to 13-cis lutein, 13’-cis lutein, 9-cis
lutein and 9’-cis lutein as analyzed by HPLC coupled with DAD and LC-MS;
and the percentage of the isomerization increased with increasing both
∗ Corresponding author. Tel.: +86 25 8439 1255; fax: +86 25 8439 1570.
E-mail address: songjiangfeng102@163.com (J.F.Song); lidajing@163.com (D.J. Li).

ultrasonic frequency and power. The stability of all-trans lutein in
dichloromethane was worst among multiple kinds of solvents. Interestingly,
the retention rate of all-trans lutein improved as the temperature increased,
which runs counter to the Arrhenius law. Under ultrasound irradiation, the
degradation mechanism might be different with various temperatures, the
o
degradation of all-trans lutein followed first-order kinetics at 20 C, while
o
second-order kinetics was followed at 30-50 C. As the ultrasonic reaction
time prolonged, lutein epoxidation nearly occurred. Those results presented
here emphasized that UAE techniques should be carefully used in the
extraction of all-trans lutein.
Keywords: Ultrasonic wave
Stability
All-trans lutein
Degradation
1. Introduction
Carotenoids continue to be a fast-moving topic of considerable interest
because of their important functions, including serving as food colourant for
humans and preventing heart disease, cancer and other diseases [1].
Carotenoids also have several kinds of important biological activities, such as

radical scavenging, singlet oxygen trapping and other protective activities [2,
3]. Carotenoids form a large isoprenoid family; the non-oxygenated
carotenoids are called carotenes (e. g. α- and β-carotene), whereas their
oxygenated derivatives are explicitly specified as xanthophylles (e. g. lutein,
zeaxanthin and β-cryptoxanthin). Lutein, a xanthophyll of a carotenoid, is
anticipated as a therapeutic product to prevent human eye diseases. Findings
show that ingestion of lutein containing foods or supplements results in
increased macular pigment optical density, and may help to improve visual
function in patients suffering from two common eye diseases of ageing,
cataract and age-related macular degeneration (AMD) [4, 5], moreover, it has
been reported in epidemiological studies that lutein reduces the risk of some
chronic diseases such as colon cancer, breast cancer, type 2 diabetes, and heart
disease [6].
In recent years, ultrasound waves have been widely used to extract
carotenoids from natural sources [7-10]. The ultrasound treatment can enhance
extraction, decrease the temperature required and reduce the time of extraction.
Ultrasound generates the growth of bubbles inside liquids causing the
cavitation phenomenon to occur. This phenomenon results in enhanced mass
transfer. Nevertheless, ultrasonic wave was less efficient than conventional
techniques in certain cases where oxidation and degradation of active
compounds occurred under prolonged sonication [11, 12]. The interaction

between the highly reactive hydroxyl radicals (resulted from sonication) and
the active compounds might be responsible for the degradation.
Lutein mainly exist in trans-isomer forms in fruits and vegetables, which
are polyene compounds with many conjugate double bonds, and are sensitive
to light, oxygen and heat [13], so the structure of all-trans lutein is prone to
damage during extraction and fruits and vegetables processing. Although the
stability of lutein has always been a subject of intense research [14], to the
best of our knowledge, there are no data on the effect of ultrasonic waves on
the stability of all-trans lutein and its lutein stereoisomers. Little information is
available involving sonochemical studies of the structure and property changes
of other carotenoids. Zhao et al. [11] found that all-trans astaxanthin in a
model system degraded to unidentified colorless compounds under ultrasound
treatment. The degradation increased by increasing both treatment time and
ultrasonic power. Sun et al. [9] indicated that the relatively low temperature of
ultrasound assisted extraction (UAE) could limit the degradation of all-trans
β-carotene during extraction compared to the conventional extraction, low
extraction efficiency and degradation during UAE may occur if conditions
were not optimized. The literature mentioned above only preliminarily
explored the stability of some other carotenoids under special conditions of
ultrasound treatment, so far, the study on the stability of all-trans lutein under
ultrasound treatment remains unclear.

The objective of this study was to determine the effects of different
ultrasound treatments on the stability of all-trans lutein. This study may help
us to effectively control the extraction outcome, reduce degradation, and may
establish a better understanding of the mechanism of ultrasound-assisted
extraction of all-trans lutein.
2. Materials and methods
2.1. Materials
All-trans lutein with high purity 92.3% was obtained from laboratory-made
samples. Methanol, acetonitrile (ACN) and methylene chloride of HPLC grade
were purchased from Tedia (Fairfield, OH, USA), Ethanol, ethyl acetate,
tetrahydrofuran, toluene, methylene chloride and acetone of AR grade were
purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China).
Nitrogen, with purity of 99.99%, was purchased from Nanjing Wenda Special
Gas Co., Ltd. (Nanjing, China).
2.2. Ultrasonic treatment
Ultrasonic treatments were performed on a Kunshan KQ-300GVDV
ultrasonic cleaner (Kunshan Ultrasonic Instruments Ltd. Co., China) with
frequencies of 45, 80 and 100 kHz and a maximum input power of up to 300
W. Actual power dissipated was calculated by measuring the rise in
temperature of a fixed amount of water (3000 mL) in the ultrasonic bath for a

given time [15]. The conical flask with a flat bottom diameter of 8.1 cm
mentioned below was just located in the cleaning bath under the ultrasound
source to reduce the experimental errors caused by uneven power transfer and
the surface of reactants was slightly lower than the level of water in the
cleaning bath. Ice bathing was replenished as needed to control the
temperature of the reaction solution. A reflux condenser was attached to the
conical flask. The conical flask was sealed and purged with nitrogen to ensure
an inert atmosphere for the reaction vessel.
20 mg all-trans lutein was placed in a 250 mL conical flask and 20 mL
solvent was added. The mixture was submitted to ultrasonic irradiation under
nitrogen atmosphere for a certain period of time, and thereafter, the solvent
was evaporated. The residue was taken-up in 5 mL acetone; the test solution
was filtered through a 0.45 µm microporous membrane. After that, the samples
were stored in a dark room at 4 oC until the analysis of lutein composition. All
operations were carried under dark conditions.
2.3. Experiment design
Firstly, the effects of commonly usedorganic solvents (ethanol, ethyl acetate,
tetrahydrofuran, toluene, dichloromethane), ultrasonic frequencies (45, 80,
100 kHz) and ultrasonic powers (120, 180, 240, 300 W) on the stability of
lutein were investigated under a ultrasonic irradiation time for 2.5 h where
nearby all the lutein would be completely extracted in sample matrix. Then

thermal degradation kinetics of lutein was studied under optimal ultrasonic
conditions with minimum all-trans lutein loss, and further tests were also
conducted to investigate the products of degradation by prolonged ultrasonic
duration up to 192 h.
2.4. HPLC analysis
For HPLC, YMC Carotenoid C30 column (4.6 mm × 250 mm i.d., 5µm,
YMC, Wilmington, NC) and HPLC (Agilent 1200 series, USA) was
performed. Elution was carried out using solvent dichloromethane, ACN and
methanol (20:30:50, V/V/V) at a flow rate of 1.0 mL/min and the eluents were
examined by UV light at a wave-length of 450 nm.
For MS, experiments were carried out on an Agilent 1290 Infinity
LC/Agilent Technologies 6460 MS. The positive ion mode (APCI) was used
to detect lutein and its isomers, with total ion current (TIC) scanning range
50-1200 m/z, corona current 4µA, capillary voltage 4500V and nitrogen as
nebulizer gas (purity 99.9% and flow rate 4 L/min) and vaporizer temperature
at 350 oC.
Lutein and its isomers were identified through the HPLC retention time,
UV-visible absorption maxima, peak ratio III/II and electron ionization and
chemical ionization mass spectroscopy (EIMS and CIMS) fragmentation
patterns of these compounds combined with their previously published
UV-visible spectra [16-18].

The relative concentrations of lutein and its isomers were performed using
the calibration curve of all-trans lutein (Sigma-Aldrich).
2.5. All-trans lutein degradation kinetics under ultrasonic waves
Kinetic experimental data were presented in the dimensionless form C/C0 at
different ultrasonic time intervals t, where C is the all-trans lutein
concentration and C0 is the amount of all-trans lutein placed initially in the
vials.
The reaction order is evaluated by trial-and-error procedures according to
Berrios et al. [19]. If the reaction order is zero, C and t is liner, otherwise, if
the degradation kinetics follows first or second-order model, ln(C/C0) or 1/C
has a linear relationship with t. The kinetic types were expressed by the
following equations,
C=－k0t+C0
(1)
(2)
(3)
ln(C/C0) =k1t
1/C= k2t + 1/C0
Where k0, k1, k2 is the kinetic constant.
3. Results and discussion
3.1. Isomer identification
Fig.1a showed the chromatogram of all-trans lutein in dichloromethane.
After sonication for 2.5 h at 50 oC, four kinds of lutein cis isomers were

finally formed as shown in Fig.1b with Peak 1, 2, 4 and 5. After sonication
for 24 h at 50 oC, three new kinds of lutein cis isomers and epoxides were
formed as shown in Fig.1c. The identification of these compounds (Peak 1, 2,
4 and 5) was based on the HPLC retention time, UV-visible absorption
maxima, peak ratio III/II and electron ionization and chemical ionization mass
spectroscopy (EIMS and CIMS) fragmentation patterns, which made it
possible for direct detection and identification of native geometric isomers of
lutein (data not shown).
3.2. Effect of solvent type on the stability of all-trans lutein
Five different solvents (ethanol, ethyl acetate, toluene, tetrahydrofuran, and
dichloromethane) were screened for investigating the stability of all-trans
lutein under ultrasonic treatment (ultrasonic frequency of 45 kHz, ultrasonic
power of 240 W, temperature of 30 oC) for 2.5 h, the change of the C/C0 value
of all-trans lutein was shown in Fig.2. Higher C/C0 value of all-trans lutein was
observed under ultrasound in the five organic solvents compared to the
identical conditions without ultrasonic treatment. And all-trans lutein showed
different extents of degradation, the stability of all-trans lutein in
dichloromethane was worst, while a similar degradation was observed for
ethanol, ethyl acetate, toluene and tetrahydrofuran solvents. Similar results
were observed by Sun et al. [9], where the dichloromethane hardly had any
extractability in the ultrasonic condition on the extraction of the all-trans

β-carotene from citrus peels; the results suggested that the lower extraction
yield in dichloromethane under ultrasonic condition might be attributed to the
degradation of the all-trans β-carotene extracted. Variation of the solvent
affected the stability of all-trans lutein, and also influenced the impact of
ultrasound effect. The vapor pressure and kinematic viscosity effects on
cavitational and bubble collapse intensity and diffusional rates of solvents into
the gaseous cavity might be the crucial factors [20].
The change of the C/C0 value of lutein cis isomers was shown in Fig.3.
13-cis lutein, 13’-cis lutein and 9-cis lutein in any organic solvents were all
produced under ultrasonic treatment, and 13-cis lutein was found to be the
major cis isomer. In contrast, C/C0 of 13-cis lutein in dichloromethane was
lower, while the C/C0 of 9-cis lutein and 13’-cis lutein was higher; it indicated
that ultrasonic waves might cause stronger chemical effects in
dichloromethane than that in ethanol, and which resulted in a faster rate of
degradation of 13-cis lutein. The subsequent degradation of 13-cis lutein
might proceed faster than its formation [21, 22].
According to our experiments, dichloromethane caused obvious degradation
of all-trans lutein under ultrasound treatment, which could be selected as a
control to take further investigation on the stability of all-trans lutein under
ultrasonic conditions, and ethanol, generally recognized as a safe (GRAS)
solvent, could be considered as an alternative solvent for use in ultrasound

extraction of all-trans lutein. So both dichloromethane and ethanol were
selected as reaction solvents.
3.3. Effect of ultrasonic frequency on the stability of all-trans lutein
Fig.4 showed the effect of different ultrasonic frequencies (45, 80 and 100
kHz) on the stability of all-trans lutein under ultrasonic treatment (ultrasonic
power of 240 W, temperature of 30 oC) for 2.5 h. It could be seen that C/C0 of
all-trans lutein in both ethanol and dichloromethane under ultrasonic
frequency of 80 kHz and 100 kHz was lower than that of 45 kHz, so the
concentration of all-trans lutein gradually decreased with increased ultrasonic
frequency. The retention rate of all-trans lutein in ethanol under ultrasonic
frequency of 45 kHz and 100 kHz was 0.93 and 0.88, respectively, which was
consistent with the results of Ye et al. [8]. They found that under ultrasound
assistance at the frequency of 20-50 kHz, the structure of carotenoids was not
destroyed, the heat effect of 25 kHz was stronger than that of 20 kHz and
accelerated the speed at which particles diffuse. The formation of
sonochemical effect requires a bubble of a certain size. But as frequency
increases, the production of cavitation becomes less likely, there was no
sufficient time for the bubble to grow large enough to collapse totally.
The change of the C/C0 value of lutein cis isomers was shown in Fig.5.
13-cis lutein was the major isomer in both ethanol and dichloromethane under
the ultrasonic treatments with different ultrasonic frequencies. 9-cis lutein and

13’-cis lutein had lower C/C0 values, but there was no significant difference
(P<0.05) among all tested ultrasonic frequencies. Only 9’-cis lutein in
dichloromethane under ultrasonic treatment with ultrasonic frequency of 100
kHz was produced, which indicated that 9’-cis lutein was less susceptible to
formation than the other lutein cis isomers.
3.4. Effect of ultrasonic power on the stability of all-trans lutein
Fig.6 showed the effect of different ultrasonic powers (120, 180, 240 and
300 W) on the stability of all-trans lutein under ultrasonic treatment
(ultrasonic frequency of 45 kHz, temperature of 30 oC) for 2.5 h. To estimate
the actual input power being used, power dissipation was calculated based on
calorimetric study. It was observed that power dissipated in the reaction vessel
was 36.6, 50.4, 61.8 and 73.7 W, respectively. Variation in the power
dissipated suggested that there was a variation in caviation intensity [15]. The
C/C0 of all-trans lutein decreased gradually with ultrasonic power from 120 to
300 W, it indicated that cavitation bubbles were easily generated by ultrasonic
treatment in a short time, and collapsed more violently with increasing
ultrasonic power, then the formed cavitation bubbles increased the shear field
and improved the rate of degradation. This was similar to that described in the
literature. Zhao et al. [11] found that the ultrasound degraded all-trans
astaxanthin to unidentified colorless compound (s) as suggested by HPLC
analysis and UV/vis measurements, and the degradation likewise increased as

both treatment time and ultrasonic power increased from 100 to 600 W. Qiao
et al. [23] investigated the sonochemical effects on seven free phenolic acids
under ultrasound treatment in a model system, and found that it is easier to
form cavitation bubbles and the bubbles collapse more violently with
increasing ultrasonic power output in the range 45-225 W. Meanwhile, higher
the ultrasonic power was, the difference of the retention rate of all-trans lutein
between ethanol and dichloromethane was larger, and more violent cavitation
effect in dichloromethane under sonication produced.
The change of the C/C0 valueof lutein cis isomers was shown in Fig.7. Total
retention rate of lutein cis isomers ascent, in the meantime the all-trans lutein
decreased as the ultrasonic power increased, it suggested that the occurrence
of cis-trans isomerization during ultrasound.
3.5. Degradation reaction order of all-trans lutein under ultrasonic
treatment
Fig.8 showed that the effect of different temperatures (20, 30, 40 and 50 oC)
on the stability of all-trans lutein under ultrasonic frequency of 45 kHz, power
of 240 W, and reaction time of 2.5 h with ethanol or dichloromethane as a
solvent. It could be seen that as the temperature of the solution increased, the
value of C/C0 grew rapidly, the reaction rate of all-trans lutein gradually
decreased. It suggested that there was more likely transient (vaporous)
cavitation occurring. This is confirmed by the decrease in the reaction rate

with increase in temperature. A possible explanation of the temperature effect
at 45 kHz would thus be that the vapour which enters the bubble during its
formation cushions the collapse of the bubble, the temperature of the hotspot
formed by the cavity collapse decreases [24, 25]. As a result, the rate
decreases as bulk temperature increases. Contrary to vaporous cavitation, the
effective Arrhenius activation energy should not be negative in this case. So
lower effect of ultrasound at higher temperatures has been attributed to the
cushioning effect of vapour contained inside the bubble during cavitation.
Table 1 summarizes the results of correlation coefficient (R) of zero-order,
first-order and second-order kinetic model under ultrasonic treatment at
different temperatures. According to the trial-and-error procedure, the kinetics
of all-trans lutein degradation under ultrasonic treatment at 20 oC followed a
first-order reaction with correlation R1 above 0.985, while a second-order
reaction with R2 above 0.986 was followed at 30, 40 and 50 oC, respectively.
The difference of reaction order indicated that degradation mechanism of
all-trans lutein was different under different temperatures. The reason might be
o
that under the ultrasound at 20 C, the cavitation bubble for sonochemical
reaction is the dominant factor on all-trans lutein degradation, but as the
temperature increased, thermal effects enhanced, which could play a role
combined with cavitation. Several studies about the kinetics of carotenoids
under different treatments have been reported in the literature. Sun et al. [26]

found that degradation kinetics of all-trans β-carotene under ultrasound fitted
first-order reaction at -5 to 15 oC, and fitted second-order reaction at 25 oC.
Henry et al. [27] reported that the oxidative degradation reaction of the
carotenoids such as all-trans β-carotene, 9-cis β-carotene, lycopene and lutein
in safflower seed oil follows a first-order kinetic model during heat treatment
at 75, 85 and 95 oC. Ahmed et al. [28] investigated the degradation kinetics of
carotenoids and visual color of papaya puree at selected temperatures (70 to
105 oC), and found that degradation of carotenoids and visual color followed
first order reaction kinetics. Dependence of the rate constant followed the
Arrhenius relationship.
The corresponding initial degradation rate constant (k) of all-trans lutein
were presented in Table 2. In both ethanol and dichloromethane, k was found
to decrease markedly and quickly as the increasing temperatures under
ultrasonic treatments, it could be shown that in the 20-50 oC temperature range,
the degradation rate in ethanol increased by 5.5-fold.
3.6. Newly formed compounds during prolonged ultrasonic treatment
The stability of all-trans lutein was further studied by prolonged ultrasound
o
(ultrasonic frequency of 45 kHz, power of 240 W, temperature of 50 C,
solvent of dichloromethane or ethanol) for 192 h. From Fig.9, it could be seen
that the C/C0 value of all-trans lutein decreased markedly (P<0.05), after 144 h,
the retention rate of all-trans lutein was very low. During the ultrasonic

treatment, in addition to 13-cis lutein and 13’-cis lutein, 15-cis lutein,
5,6-epoxy lutein and 5,8-epoxy lutein were newly formed compounds, both
9-cis lutein and 9’-cis lutein were not detected (Fig.1c). The 13’-cis lutein and
13-cis lutein were the major isomers and 15-cis lutein was present at low
levels. It meant numerous degradation products of all-trans lutein were formed
during exposure to the prolonged experimental conditions, and the trans-to-cis
conversion rate was lower in lutein or that cis isomers of lutein are perhaps
less stable and more reactive. 13-cis lutein might be more readily formed from
all-trans lutein than 9-cis lutein during ultrasound because of the low
activation energy [29]; meanwhile 9-cis lutein underwent fast degradation as
soon as they were generated from all-trans lutein. The formation of 5, 6-epoxy
lutein and 5, 8-epoxy lutein indicated lutein epoxidation occurring under
ultrasonic wave combined with thermal effect [30, 31].
4. Conclusion
This study demonstrated that ultrasound had significant effects on the
stability of all-trans lutein. And the effects were pronouncedly different with
various ultrasonic conditions. The stability of all-trans lutein in
dichloromethane was worst. Under various ultrasonic treatments at 20-50 oC
for 2.5 h, the all-trans lutein produced the same isomers, namely 13-cis lutein,
13’-cis lutein, 9-cis lutein and 9’-cis lutein, and 9’-cis lutein was ultimately

formed in designated time. With increased ultrasonic frequency and power, the
retention rate of all-trans lutein gradually decreased in both ethanol and
dichloromethane, meanwhile an identifiable upward change trend of isomers
was generally similar. Interestingly, the retention rate of all-trans lutein
improved as the temperature increased, which runs counter to the Arrhenius
law. Under ultrasound, the degradation mechanism might be different under
o
various temperatures, at 20 C, the degradation of all-trans lutein followed
first-order kinetics, while fitted second order at 30-50 oC. As the ultrasonic
reaction time prolonged, lutein epoxidation nearly occurred. All the results
emphasize the careful application of both ultrasound-assisted extraction
techniques in the extraction of lutein or other unstable biological molecules.
Acknowledgements
The research was financially supported by the Jiangsu Agricultural Science
and Technology Innovation Fund [Project No. CX14 (2055)].
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(c)
Fig.1. Chromatogram of all-trans lutein in dichloromethane before (a) and

after 2.5 h (b), 24 h (c) of ultrasonic treatment at 50 oC. Peak 1,13-cis
lutein; peak 2, 13’-cis lutein; peak 3, all-trans lutein; peak 4, 9-cis lutein;
peak 5, 9’-cis lutein; peak 6, 5, 6 - epoxy lutein; peak 7, 5, 8 -epoxy lutein;
peak 8, 15-cis lutein.

Fig.2. Effect of solvent type on the stability of all-trans lutein under ultrasonic
treatment (ultrasonic frequency of 45 kHz, ultrasonic power of 240 W,
temperature of 30 oC, reaction time of 2.5 h).

Fig.3. Effect of solvent type on the stability of lutein cis isomers under
ultrasonic treatment (ultrasonic frequency of 45 kHz, ultrasonic power of 240
W, temperature of 30 oC, reaction time of 2.5 h).

Fig.4 Effect of ultrasonic frequency on the stability of all-trans lutein under
o
ultrasonic treatment (ultrasonic power of 240 W, temperature of 30 C,
reaction time of 2.5 h).

Ethanol
Dichloromethane
Fig.5. Effect of ultrasonic frequency on the stability of lutein cis isomers
under ultrasonic treatment (ultrasonic power of 240 W, temperature of 30 oC,
reaction time of 2.5 h).

Fig.6. Effect of ultrasonic power on the stability of all-trans lutein under
ultrasonic treatment (ultrasonic frequency of 45 kHz, temperature of 30 oC,
reaction time of 2.5 h).