Ultrasonics Sonochemistry 20 (2013) 1026–1032

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Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultson

Effects of various factors of ultrasonic treatment on the extraction yield

of all-trans-lycopene from red grapefruit (Citrus paradise Macf.)
Yuan Xu, Siyi Pan ⇑
College of Food Science and Technology, Huazhong Agricultural University, No.1, Shizishan Street, Hongshan District, Wuhan, Hubei 430070, PR China

a r t i c l e i n f o a b s t r a c t

Article history: The effects of various factors, including the extraction time, temperature, solvent/material ratio, the
Received 17 October 2012 ultrasonic intensity and duty cycle of ultrasonic irradiation on the extraction yield of all-trans-lycopene
Received in revised form 16 January 2013 from red grapefruit by ultrasound-assisted extraction (UAE) were investigated. In comparison with con-
Accepted 17 January 2013
ventional solvent extraction (CSE), UAE showed a pronounced greater extraction yield and reduced
Available online 26 January 2013
extraction time effectively with a peak value at 30 min. The extraction yield was significantly influenced
by temperature and the optimum condition was 30 °C. The extraction yield increased with increasing of
Keywords:
solvent/material ratio until equilibrium was arrived at the optimal ratio of 3:1 (mL/g). The extraction
Ultrasound-assisted extraction
Pulsed ultrasound
yield increased first and then decreased with an increase in ultrasonic intensity. The extraction yield
All-trans-lycopene of UAE increased with the increase of duty cycle, whereas pulsed ultrasound with proper intervals was
Red grapefruit more efficient than continuous ultrasonication. The degradation via isomerisation of all-trans-lycopene
Extraction yield under ultrasonic treatment was also observed with the formation of 9,130 -di-cis-, 9,13-di-cis-, 15-cis-,
Isomerisation 13-cis- and 9-cis-lycopene isomers which were tentatively identified by HPLC-PAD.
Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction conjugated double bonds makes lycopene particularly susceptible

Lycopene, a predominant carotenoid, is a natural pigment
responsible for the red hue in many plants and has received great
interest because of the prominent biological activities in recent
years. Red grapefruit (Citrus paradise Macf.), which has a character-
istic red color in the peel and flesh, is a typical lycopene-pigmented
citrus [1] with good market in the world. In recent years, red grape-
fruits and their products have attracted more interest because of
the nutritional properties [2] such as antioxidative activity, anti-
atherogenic and anticarcinogenic [3–5] roles attributed to the exis-
tence of lycopene.
Lycopene is an acyclic carotenoid with 11 conjugated carbon–
carbon double bonds occurring in various geometrical isomers
and naturally present in all-trans-isomer in most higher plants
and microorganism [6]. The high degree of conjugation and ex-
treme hydrophobicity confer strong antioxidant property to the
surface of human lymphoid cells [7], making lycopene one of the
most potent antioxidants. It gives the health protection against
various chronic diseases including certain cancer, coronary heart
disease, and degenerative eye diseases [8–10], and exhibits tumor
suppression activity which has received attention in its pharma-
ceutical potential [11]. Whereas, the structure of a long chain of
⇑ Corresponding author. Tel.: +86 027 87283778; fax: +86 027 87288373.
E-mail addresses: xuyuan0804@163.com (Y. Xu), siyipan@yahoo.com.cn,
pansiyi@mail.hzau.edu.cn (S. Pan).
to degradation, which mostly results in the formation of cis-iso-
mers with a reduction of bioactivity potency [12–14]. Lycopene
can be easily isomerised under the influence of excess heat and
light during extraction, processing or storage. Chen et al. [13] ob-
served that the increase in light irradiation intensity and tempera-
ture favoured the degradation and isomerisation of lycopene.
Mayer-Miebach et al. [15] found that thermal treatment above
100 °C initiated isomerisation of all-trans-lycopene and resulted
in a significant increase in 9-cis-lycopene. Consequently, the adop-
tion of suitable technique or processing condition for stabilizing
trans-lycopene during extraction and storage is important criteria
for process optimization and bioactivity preservation.
The utilization of this bioeffect product depends on its extrac-
tion from natural materials commonly via conventional solvent
extraction (CSE) and supercritical fluid (CO2) extraction (SCFE).
CSE consumes large quantities of solvents and long extraction time
with poor efficiency, whereas SCFE endows with a remarkable sol-
vating capacity which is safe and simple but the drawbacks are
high energy input and more expensive equipment.
In recent years, the application of ultrasound sonication has
showed up great expectations in plant extraction field with prom-
ising results. As an innovative technology, ultrasound-assisted
extraction (UAE) could enhance heat and mass transfer by disrupt-
ing the matrix cell walls to promote the release of bioactive com-
pounds [16]. A variety of nutritional materials such as phenolics,
saponins, polysaccharides, and carotenoids have been extracted

1350-4177/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ultsonch.2013.01.006
 Y. Xu, S. Pan / Ultrasonics Sonochemistry 20 (2013) 1026–1032
Fig. 1. The structure of all-trans-lycopene.
successively through ultrasound method [17–20]. Compared to
conventional solvent extraction, UAE can increase extraction yield
and extraction efficiency, reduce solvent consumption, resulting in
saving extraction time and energy input with high reproducibility
[16]. In addition, the advantages of UAE, compared with supercrit-
ical fluid (CO2) extraction, are that UAE can accelerate mass trans-
fer by mechanical effects and simplify manipulation with cheaper
equipment.
The acoustic cavitation is the main mechanism for the high
extraction efficiency of ultrasonic treatment. The collapse of cav-
itation bubbles can produce bursts of energy and cause extraor-
dinarily high temperature and pressure, which can enhance the
penetration of solvent into the matrix and accelerate the chem-
ical reactions of targeted compounds dramatically. In recent
studies, researchers are more concerned with changes in the
structure and properties of bioactive compounds during the pro-
cess of ultrasound-assisted extraction. Tiwari et al. [21] observed
that high intensity ultrasound caused the anthocyanins decom-
position and color degradation of grape juice. Ying et al. [19]
found the physical structure of cell walls of extraction tissues
and the properties of polysaccharides were drastically changed
under UAE. However, there is little sonochemical research
available regarding the structure and bioactivity changes of
carotenoids such as lycopene. Likewise, the stability of all-
trans-lycopene under high intensity ultrasonic treatment is
rarely reported.
In the present work, the effects of different factors in UAE on the
extraction yield of all-trans-lycopene (Fig. 1) from red grapefruit
were investigated. In addition, the stability and isomerisation of
all-trans-lycopene under UAE were evaluated and compared to
CSE by HPLC-PAD analysis. The objectives of this study were to effi-
ciently enhance the productivity and selectivity of all-trans-lyco-
pene with less degradation in extraction process and provide
information to understand the mechanism of ultrasound-assisted
extraction of all-trans-lycopene.
2. Materials and methods
2.1. Materials
Fresh red grapefruits (Citrus paradise Macf.) cultivar ‘Star Ruby’
were supplied by Zhejiang academy of agricultural sciences, China,
in December, 2011. Over-ripe and damaged fruits were discarded.
The selected grapefruits were peeled and seeded by hands, and
were then finely milled into a homogeneous puree with an electric
tissue grinder. The viscosity and brix of the puree were 23.8 mPa s
and 10.5% respectively. The plant materials were stored at 20 °C
in refrigerator for usage. The HPLC grade solvents, including
methyl-tert-butyl-ether (MTBE), methanol and ethyl acetate were
purchased from Thermo Fisher Scientific (Leicestershire, UK).
Petroleum ether (60–90 °C), acetone, hexane, ethanol (95%),
dichloromethane and butylated hydroxytoluene (BHT) were of
analytical grade and purchased from Sinopharm chemical reagent
Co., Ltd. (Shanghai, China). The standard all-trans-lycopene (purity
P90% from tomato) was purchased from Sigma–Aldrich (St. Louis,
MO, USA).
1027
2.2. Ultrasound-assisted extraction of lycopene
Ultrasound-assisted extraction was performed in an ultrasonic
probe processor (Scientz-IID, Ningbo scientz biotechnology Co.,
Ningbo, China) with a maximal input power of 950 W and an oper-
ating frequency range of 20–25 kHz. According to Fish et al. [22]
and the research of our team (not published), a solvent mixture
consisting of petroleum ether, acetone and 95% ethanol (2:1:1, V/
V/V) with 2% (W/V) dichloromethane and 0.05% (W/T) butylated
hydroxytoluene (BHT) was employed for the complete extraction
of all-trans-lycopene. The red grapefruit sample (10.0 g) was
placed into an amber erlenmeyer flask (100 mL) containing proper
volumes solvent, and then directly treated at 20 kHz at different
UAE parameters (assigned according to the experimental design)
with a probe microtip (diameter 10 mm) immersed 1 cm into the
solution. Meanwhile the flask was put into a low temperature ther-
mostatic ethanol (T = 0 °C) or water (T > 0 °C) bath (DC-0506, Shun-
ma instrument Co., Ltd., Nanjing, China). Samples were stayed in
the ethanol or water bath that was flowed from the low tempera-
ture thermostat through a hose to maintain the extraction temper-
ature. After standing and layering the treated solution for 10 min,
the nonpolar supernatant layers were collected as extracts and
then filtered by 0.22 lm hydrophobic membranes for HPLC analy-
sis. As a control, the conventional solvent extraction was carried
out in a JR-AB water bath oscillator (Jerel electric appliance Co.,
Ltd., Jiangsu, China) at 180 rpm, and the following procedure of
sample treatment was similar to UAE. The experimental process
should be protected from light for less attenuation of lycopene dur-
ing the extraction.
2.3. Calculation of ultrasonic intensity
The generated ultrasonic intensity delivered from the probe tip
was determined by the formula below [23]:
P
I¼
pr 2
where P is the absolute ultrasonic power, and r is the radius of
the probe tip. The ultrasonic power levels were designed as 10%,
30%, 50%, 70%, and 90% of maximum input power (950 W) which
were 95, 285, 475, 665, and 855 W, respectively. The calculated
ultrasonic wave intensities were 121, 363, 605, 847, and 1089 W/
cm2, respectively.
2.4. Experimental design
To evaluate the effect of each factor on all-trans-lycopene yield
under ultrasound treatment, single factor experiment design was
adopted for analyzing the influence of five different variables in
this study. The effects of extraction time (15, 30, 45, 60, 75, and
90 min), extraction temperature (0, 10, 20, 30, 40, and 50 °C), sol-
vent/material ratio (1:1, 2:1, 3:1, 4:1, and 5:1 mL/g), ultrasonic
intensity (121, 363, 605, 847, and 1089 W/cm2) and ultrasound
duty cycle (0, 33.3%, 40%, 50%, 66.7%, and 100%) were investigated
separately on the basis of extraction efficiency. The prior ultrasonic
conditions of each single-variable treatment were set as follows:
1028 Y. Xu, S. Pan / Ultrasonics Sonochemistry 20 (2013) 1026–1032

an extraction temperature of 30 °C, a solvent/material ratio of

3:1 mL/g, an extraction time of 30 min, an ultrasonic intensity of
605 W/cm2, with an interval between pulses of 2 s. Whilst the
CSE was performed at the extraction temperature of 30 °C, sol-
vent/material ratio of 3:1 mL/g and extraction time of 30 min.

2.5. Purification of lycopene

For the purpose of evaluating the effect of ultrasonic treatment

on the stability of all-trans-lycopene, the red grapefruit extracts
were purified by silica column chromatography before HPLC-PAD
analysis. The chromatographic column (50  1 cm) was packed
with silica gel (100 g) which was activated under 110 °C for 2 h.
The extracts (5 mL) were poured into the column and then eluted
by petroleum ether and acetone with different proportions from
49:1 to 9:1 (V/V). The deepest red color was collected during the
elution and then concentrated by rotary evaporation. After dissolv-
ing in 2 mL mobile phases and filtering through a 0.22 lm mem-
brane filter, the purified lycopene was analyzed by HPLC-PAD. Fig. 2. Effect of time on the extraction yield of all-trans-lycopene under UAE and
CSE. Different letters on bars indicate significant differences (p < 0.05).

2.6. HPLC analysis of lycopene

The concentration of all-trans-lycopene and the identification of
lycopene isomers were conducted with HPLC-PAD analytical meth-
od. A Waters 2695 HPLC system (Waters Corp., MA, USA) equipped
with a Waters 2996 photodiode array detector (PAD) and a poly-
meric C30 reversed phase column (250  4.6 mm, i.d. 5 lm, YMC,
Inc. Wilmington, NC, USA) was employed for analysis of lycopene.
The compound eluted isocratically with mobile phase methyl-tert-
butyl-ether/methanol/ethyl acetate (40:50:10, v/v/v) at a flow rate
of 1 mL/min [13] and the detection spectrometric wavelength of
476 nm. The sample volume of 10 lL was injected and column
temperature was maintained at 35 °C.
Identification of all-trans-lycopene was verified by comparison
of retention time and UV–VIS spectrum with authentic standard,
in addition, the identity of other cis-isomers were confirmed by
absorption spectral characteristics data and Q ratios (the absor-
bance ratio of the cis peak to the main absorption peak) as de-
scribed in the literature [24]. Quantification of all-trans-lycopene
was carried out by external standard curve of pure lycopene stan-
dard, which was plotted with concentration from 0 to 250 lg/mL
at intervals of 25 lg/mL, and the concentraction of all-trans-lyco-
pene was expressed as microgram per gram of tissue (lg/g tissue).
The lowest detection limit (DL) and quantization limit (QL) were
0.74 lg/mL and 1.95 lg/mL that were calculated by a method of
Lee and Chen [24].
2.7. Statistical analysis
All treatments and HPLC analysis were conducted in triplicate
and the results were expressed as means ± SD. The data were eval-
uated by statistical analysis of variance (ANOVA) and Duncan’s
multiple range test (MRT) using SPSS software version 18.0 to com-
pare the effects of various factors under ultrasonic treatment. The
significant differences of the mean values were determined at
p < 0.05 levels.
3. Results and discussion
3.1. Effect of extraction time
The effect of extraction time on the yield of all-trans-lycopene
from red grapefruit are revealed in Fig. 2. The lycopene yield in-
creased gradually as extraction time increased from 15 to 60 min
(p < 0.05) and then did not continue to increase with time ranging
from 60 to 90 min (p > 0.05) after the yield reached maximal value
at 60 min in CSE. Whereas the optimum yield of lycopene was
achieved when extraction was performed for 30 min under UAE
and prolonged sonication time from 45 to 90 min reduced the lyco-
pene yield significantly (p < 0.05). In all the figures, values marked
with different letters under various extraction conditions stood for
the significant differences (p < 0.05) of extraction yield.
The results indicate that conventional method of extraction was
more time-consuming and the process of solid–liquid mass trans-
fer was more slowed down compared to UAE. After the diffusing of
all-trans-lycopene extracts arrived at extraction equilibrium at
60 min, the lycopene yield of CSE did not show noticeable change
and this phenomenon was probably due to the mild heating condi-
tion with a low temperature of 30 °C. Nevertheless, a decreasing
trend may occur with prolonged heating time because of the deg-
radation reaction of dissolved lycopene. With the advantage of
ultrasonic, UAE had a higher extraction efficiency that obtained
1.81 times of maximum yield when extraction time was 30 min
comparing to CSE.
According to the investigation, the cavitation effect of ultra-
sound, including macro-turbulence created by the implosion of
cavitation micro-bubbles and microjets generated by the cavita-
tion on the surface of plant matrix, facilitated the interior lycopene
release to extraction solvent. Additionally, most of all-trans-lyco-
pene in plant cells diffused at the early stage of extraction with a
sharp increase of extraction yield in the first 30 min. However, pro-
longed time for more than 30 min induced the degradation and
isomerisation of all-trans-lycopene and led to the decrease of lyco-
pene yield. The results are in agreement with Zhang and Liu [25],
who reported that 29.1 min of extraction time was optimum to ex-
tract lycopene with ultrasound from tomato paste while longer
time led to the oxidative decomposition of lycopene. This phenom-
enon might be due to the ultrasonic degradation. It is well known
that the chemical decomposition of some natural active constitu-
ents are likely happened on account of the negative effects of
long-time ultrasonic irradiation, resulting in a decrease of extrac-
tion yield [25]. In fact, the degradation of all-trans-lycopene was
due to the collapse of cavitation micro-bubbles in ultrasound treat-
ment. When the cavitation micro-bubbles collapse asymmetrically
onto the surfaces of target extracts, the generated microjets scour
the surfaces and cause damage to plant matrix in the solution [26].
The continuous collapse of cavitation bubbles could be responsible
for the decomposition of lycopene structure and the decline of its
stability. Moreover, it is also potential that the high reactive
 Y. Xu, S. Pan / Ultrasonics Sonochemistry 20 (2013) 1026–1032
hydroxyl radicals produced by cavitation effect in the matrix with
the existence of small quantities of water could cause the decom-
position or isomerisation of lycopene. As already discussed, ultra-
sonic time presented a significant impact on lycopene yield, but
additional extraction time was not necessary for obtaining more
lycopene from red grapefruit as prolonged time will degrade and
isomerize the extracted lycopene.
3.2. Effect of extraction temperature
The extraction efficiency of all-trans-lycopene was influenced
by various factors. Fig. 3 shows the extraction yield under CSE
was lowest at 0 °C and was not significant different as temperature
increased from 10 to 30 °C (p > 0.05), whereas it significantly in-
creased with rising temperature from 30 to 50 °C (p < 0.05). In
comparison, the yield of all-trans-lycopene had an obvious apogee
at 30 °C (p < 0.05) under UAE. Statistical analysis (ANOVA) stated
that there were significant differences on the extraction yield be-
tween CSE and UAE when extraction temperature ranged from 0
to 50 °C. The highest extraction yield of UAE was 1.89 times than
CSE at 30 °C and 1.64 times than the maximum yield of CSE at
50 °C.
The effect of extraction temperature on the yield under CSE is
probably due to the acceleration of molecular thermal motion
and the increase of solubility of all-trans-lycopene as temperature
raising from 30 to 50 °C. The variation of extraction yield under
UAE could be explained by the combined action of acoustic cavita-
tion and thermal effect, and these two effects were significantly
influenced by extraction temperature. Although the increase of
temperature may accelerate thermal activity which promoted the
solubility of extracts, higher temperature (40 °C) resulted in the
decrease of cavitation intensity. The physical characteristics of sol-
vent such as surface tension, vapour pressure and viscosity are the
major factors impacting cavitation intensity, and the vapour pres-
sure is the crucial factor among these properties [27]. The temper-
ature displays a positive effect on vapour pressure, therefore, high
temperature leads to the increase of vapour pressure of solvent
molecules within cavitation mrico-bubbles [28,29], causing the
damping of the collapse and the decrease of cavitation intensity.
Thus, temperature was a sensitive variable for ultrasonic extrac-
tion of all-trans-lycopene from red grapefruit, and the optimum
temperature was 30 °C which gave the highest extraction yield of
all-trans-lycopene when cavitation effect and thermal effect ar-
rived at an equilibrium state with the variation of temperature.
Fig. 3. Effect of temperature on the extraction yield of all-trans-lycopene under UAE
and CSE. Different letters on bars indicate significant differences (p < 0.05).
1029
Additionally, the total lycopene yield under UAE was remark-
ably enhanced by 64% compared with CSE, which was due to the
increase of mass transfer between the solvent and plant matrix
by cavitation during the ultrasound treatment of samples. The col-
lapse of cavitation bubbles in the liquid with the release of huge
amount of energy leads to better cell disruption through the gen-
eration of microjets directed towards the solid surface. The high
temperature and pressure released in the collapse create hotspots
that can dramatically accelerate the chemical reactivity into the li-
quid and can also break open the cell walls of grapefruit tissue to
facilitate the release of lycopene into the medium.
3.3. Effect of solvent/material ratio
As shown in Fig. 4, the extraction yield of all-trans-lycopene in-
creased rapidly (p < 0.05) with increasing solvent/material ratio
ranged from 1:1 to 3:1 (mL/g), while there was no significant
change (p > 0.05) on the yield of all-trans-lycopene as solvent/
material ratio continued to increase from 3:1 to 5:1 (mL/g) both
under UAE and CSE. The reason was that higher ratio of solvent
to material could cause greater concentration difference which
accelerated mass transfer and facilitated the diffusion of all-
trans-lycopene into the medium. However, after the mass transfer
process reached its maximum, futher increase of solvent to mate-
rial ratio prolonged the distance of diffusion from solvent to inte-
rior matrix and could hardly enhance any more lycopene yield.
This phenomenon was also observed by Ying et al. [19], who found
that a higher ratio of solvent to raw material did not lead to the
obvious increase of polysaccharides yield from mulberry leaves
by ultrasonic extraction.
According to the ANOVA statistical analysis, there were signifi-
cant differences between CSE and UAE (p < 0.05) in extraction yield
throughout the range of experimental solvent/material ratio. For
example, the yield of UAE was 2.06 times than CSE (p < 0.05) when
the solvent/material ratio was 3:1 mL/g. The high extraction effi-
ciency of UAE is mainly owing to the generation of cavitation by
ultrasonic irradiation in the liquid. Furthermore, there exists
microscopic mixing which is generated by cavitation, cavity oscil-
lations and acoustic streaming [30]. These effects can improve the
extracting capacity of solvent with promoting solvent penetration
into plant cell walls to extract the targeted compounds, and disrupt
the cellular materials to facilitate the release of all-trans-lycopene
from tissues into the extraction medium. Therefore, the results
indicate that ultrasound had a distinct advantage of increasing
Fig. 4. Effect of solvent/material ratio on the extraction yield of all-trans-lycopene
under UAE and CSE. Different letters on bars indicate significant differences
(p < 0.05).
1030 Y. Xu, S. Pan / Ultrasonics Sonochemistry 20 (2013) 1026–1032
extraction yield compared with CSE at different levels of solvent/
material ratio.
3.4. Effect of ultrasonic intensity
Fig. 5 shows the effect of ultrasonic intensity on the extraction
yield of all-trans-lycopene in UAE. A positive effect on the lycopene
yield was observed with the increase of ultrasonic intensity rang-
ing from 0 to 605 W/cm2. In this range of variation, the extraction
yield of lycopene and the intensity of ultrasound was in a logarith-
mic function relationship (R2 = 0.994, p < 0.05) through nonlinear
fitting calculation. However, the yield of lycopene then decreased
significantly when ultrasonic intensity increased from 605 to
1089 W/cm2 (p < 0.05). This phenomenon indicated that ultrasonic
intensity was critical in extracting all-tans-lycopene. An obvious
increasing trend of lycopene yield with stronger ultrasonic inten-
sity transmitted to the medium can be explained that greater num-
ber of cavitation micro-bubbles was created into the liquid and the
bubbles collapsed more drastically as ultrasonic intensity increas-
ing from 0 to 605 W/cm2, which facilitated the disruption of tissue
cell walls and accelerated the diffusion of lycopene into the med-
ium. When the acoustic intensity was greater than 605 W/cm2,
these cavitation bubbles could grow too big to collapse or collapse
gently which resulted in the reduction of cavitation effect. Like-
wise, the excessive bubbles produced by higher acoustic intensity
may hamper the transmission or cause the scattering of ultrasonic
waves [31]. Moreover, the thermal effect also presented a negative
influence on the yield of lycopene that the heat produced by ultra-
sound may not completely dissipate in a short period of time and
the absorption of heat could lead to the decomposition of all-
trans-lycopene as ultrasound intensity increasing from 605 to
1089 W/cm2.
3.5. Effect of ultrasound duty cycle
The effect of sonication in pulse mode with different duty cycle
compared to continuous mode (100% duty cycle) was studied and
is shown in Fig. 6. As can be seen extraction yield enhanced signif-
icantly with the increase of duty cycle from 0 to 66.7% (p < 0.05)
and then reached a maximum. When the duty cycle increased to
100% as continuous ultrasound, the lycopene yield decreased sig-
nificantly (p < 0.05) and even less than the yield at 50% duty cycle
(p < 0.05). The results indicate that the utilization of pulsed ultra-
sound with proper pulse intervals can be more efficient than con-
Fig. 5. Effect of ultrasonic intensity on the extraction yield of all-trans-lycopene
under UAE. Different letters on the line indicate significant differences (p < 0.05).
Fig. 6. Effect of duty cycle on the extraction yield of all-trans-lycopene under UAE.
Different letters on the line indicate significant differences (p < 0.05).
tinuous sonication and reduce the electrical energy consumption,
which are in agreement with previous findings. Kobus [32] found
that pulsed ultrasound effectively accelerated the extraction of
bioactive components from dried roots of valerian. Kadkhodaee
and Hemmati-Kakhki [33] found that sonication at 50% duty cycle
beneficially impacted the extraction process and increased the
extraction yield of active compounds from saffron in comparison
of continuous sonication.
The reason for these observations may be that the formation of
degassing bubbles under continuous-wave ultrasound spatially re-
stricted the effective region of sonochemical reaction. Because of
volumetric oscillation, the clusters of degassing bubbles can pre-
vent the propagation of ultrasound by absorbing and scattering
the sound waves, which result in the violent expansion and con-
traction of spatial region for these bubbles and thus limit the sono-
chemical reaction. In virtue of the inactive period of ultrasound,
the pulse modulation of the ultrasound suppresses the generation
of degassing bubbles and this can favor the clarification of the cav-
itation zone and the enhancement of sonochemical reactions.
Moreover, the existence of residual cavitation nuclei during the
inactive period of pulsed ultrasound is more conducive to the for-
mation of cavitation bubbles for the next active ultrasonic period
[34,35]. To clear the effect of duty cycle on extraction yield, further
work is under study.
3.6. HPLC analysis of lycopene extractions
The reverse-phase chromatographic analysis of purified lyco-
pene which were extracted with CSE and UAE under fixed condi-
tions as mentioned in Section 2.4. are illustrated by Figs. 7a and
b, respectively. The chromatographic, spectral characteristics data
and Q ratios of all-trans-lycopene and cis-isomers obtained by
HPLC-PAD are summarized in Tables 1a and b for CSE and UAE,
separately. As seen in the above chromatography patterns, there
were different degrees of all-trans-lycopene degradation via isom-
erisation occurred in the course of different extraction processes. In
Fig. 7a, peak 4 was positively identified as all-trans-lycopene based
on the retention time and absorption spectrum with reference
standard. Peaks 1, 2 and 3 were tentatively identified as 15-cis-,
13-cis- and 9-cis-lycopene respectively, because a hypsochromic
shift of 6, 6 and 6 nm occurred when compared to the all-trans-
compound and the Q ratios were 0.60, 0.56 and 0.14 respectively,
which were similar to that reported in the literature [36]. Fig. 7b
shows the HPLC chromatogram of lycopene extracted by UAE.
 Y. Xu, S. Pan / Ultrasonics Sonochemistry 20 (2013) 1026–1032 1031

Likewise, peak 6 was positively identified as all-trans-lycopene

Fig. 7a. HPLC chromatogram of lycopene extracted by CSE. Chromatographic
conditions described in text. See Table 1a for peak identification.
Fig. 7b. HPLC chromatogram of lycopene extracted by UAE. Chromatographic
conditions described in text. See Table 1b for peak identification.
with the standard. Peak 1 and 2 were tentatively identified as 9,
130 -di-cis- and 9,13-di-cis-lycopene based on a stronger hypso-
chromic shift of 16 and 12 nm and Q-ratios (Table 1b) [36]. It has
been suggested that the di-cis-lycopene could be shifted to a short-
er wavelength than mono-cis-lycopene with a larger magnitude
[24]. Peak 3, 4 and 5 were tentatively identified as 15-cis-, 13-cis-
and 9-cis-lycopene, respectively, because of a cis peak at 360 nm
with a hypsochromic shift of 6 nm and the Q ratios were similar
to the references by other authors [24,36].
From the above analysis, it points out that during the extraction
process lycopene underwent geometrical isomerisation with the
presence of heat or ultrasonic, resulting in the formation of cis-iso-
mers. Results of lycopene geometrical isomerisation during the
processes of CSE and UAE are reported in Table 2. Due to the ther-
mal effect or acoustic cavitation effect, all-trans-lycopene was
gradually transformed into a range of different cis-isomers. It can
be seen from Table 2 that the mainly formed isomer was 13-cis-
lycopene both in CSE and UAE with the proportion of 6.91% and
9.99% of total lycopene, respectively. Compared to CSE, there was
a larger amount of all-trans-lycopene converted into cis-isomers
in UAE with the retention proportion of 87.1%, whereas the all-
trans-lycopene preserved in CSE was 91.5% of total lycopene. Addi-
tionally, more variety of cis-lycopene isomers was observed after
ultrasound treatment in the represent of 9,130 -di-cis- and 9,13-
di-cis-lycopene. This phenomenon can be attributed to the combi-
nation of caviation effect and thermal effect in UAE that the contin-
uous collapse of cavitation bubbles and excess heat provided
appreciable activation energy for lycopene isomerisation which re-
sulted in the decline of the stability of all-trans-lycopene.
Heating process of CSE and UAE can provide thermal energy
which induced the isomerization of all-trans-lyocpene to the
unstable cis-isomers. Furthermore, the conversion of all-trans-lyco-
pene into cis-isomers can be facilitated through cavitation effect in
UAE. High reactive molecules, such as hydroxyl radicals, and the
hotspots released from the collapse of cavitation bubbles can dra-
matically trigger or accelerate the chemical reactivity to promote
the chemical degradation with more formation of unstable cis-iso-
mers. The extraction condition of CSE was relatively mild than UAE,
so that less cis-isomers were obtained in CSE as a result of thermal
effect alone. In the present study, we only analyzed the isomeriza-
tion products of lycopene and further work should be progressed
to systematically evaluate the stability of lycopene and reveal the

Table 1a
Identification and chromatographic data for isomers of lycopene in red grapefruit (Citrus paradise Macf.) after conventional solvent extraction.

Peak no. Retention time (min) Compound k (nm) found k (nm) reported [36] Q-ratio found
Found Reported [36]
1 10.133 15-cis-lycopene 360 444 466 498 362 446 470 500 0.60 0.61
2 11.392 13-cis-lycopene 360 439 466 496 362 440 470 508 0.56 0.55
3 16.291 9-cis-lycopene 360 442 466 498 362 446 470 500 0.14 0.12
4 27.250 all-trans-lycopene 446 472 503 452 476 506

Table 1b
Identification and chromatographic data for isomers of lycopene in red grapefruit (Citrus paradise Macf.) after ultrasound-assisted extraction.

Peak no. Retention time (min) Compound k (nm) found k (nm) reported [36] Q-ratio found
Found Reported [36]
0
1 5.725 9,13 -di-cis-lycopene 356 445 456 490 368 458 488 0.16 0.2
2 8.133 9,13-di-cis-lycopene 361 434 460 491 344 440 464 494 0.1 0.1
3 10.983 15-cis-lycopene 360 445 466 501 362 446 470 500 0.72 0.61
4 12.266 13-cis-lycopene 360 440 466 496 362 440 470 508 0.61 0.55
5 17.291 9-cis-lycopene 360 439 466 498 362 446 470 500 0.14 0.12
6 28.841 all-trans-lycopene 446 472 503 452 476 506
1032 Y. Xu, S. Pan / Ultrasonics Sonochemistry 20 (2013) 1026–1032
Table 2
Comparsion of lycopene isomers extracted from red grapefruit (Citrus paradise Macf.) with CSE and UAE by HPLC analysis.
Extraction technique Lycopene isomer contents (% of purified extract)
All-trans 9-cis
CSE 91.5 ± 0.48 0.42 ± 0.06
UAE 87.1 ± 0.38 1.14 ± 0.11
degradation mechanism of lycopene in the process of ultrasound-
assisted extraction.
4. Conclusions
The results obtained in this study indicate that each factor of
ultrasonic treatment significantly influenced the extraction yield
of all-trans-lycopene from red grapefruit under ultrasound-as-
sisted extraction. The extraction efficiency was shown to be a func-
tion of time, temperature, solvent/material ratio, ultrasonic
intensity and duty cycle. In comparison with CSE, ultrasound
incredibly reduced the processing time and largely enhanced the
extraction yield. The relatively low temperature under UAE had a
positive effect on lycopene yield and could limit the degradation
of all-trans-lycopene during extraction. An optimal solvent/mate-
rial ratio could be adopted for moderating solvent consumption.
Proper ultrasonic intensity was necessary to optimize the extrac-
tion yield. Pulsed ultrasound at certain intervals could be more
efficient than continuous sonication with the advantage of saving
electrical energy. Although these findings demonstrate that the
use of ultrasonic offers considerable advantages on lycopene yield,
low extraction efficiency and degradation of all-trans-lycopene
could occur if conditions of UAE are not optimized. Isomerisation
reaction of lycopene was observed during the process of ultra-
sound-assisted extraction and the isomers were analyzed and
identified by means of HPLC-PAD. Therefore, more attention
should be invested in the application of ultrasound technique for
lycopene extraction. The present study provides very useful infor-
mation for optimizing ultrasonic extraction conditions of lycopene
from red grapefruit according to the various factors studied. A new
method to make isomers of lycopene was also discovered.
Acknowledgment
This research was supported by ‘948’ project of Chinese Minis-
try of Agriculture (2006-Z25) and the key scientific and technolog-
ical projects of Hubei province (2007AA204A02).
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