Ultrasonic Extraction of Active Compounds from Saffron
R. Kadkhodaee, A. Hemmati-Kakhki
Department of Food Technology
Khorasan Research Centre for Technology Development
Mashad
Iran
Keywords: crocins, picrocrocine, pulsed sonication, safranal, ultrasound
Abstract
The active compounds of saffron were extracted using high power
ultrasound at a constant frequency of 30 kHz. The effect of acoustic intensity,
time and mode of sonication on the extraction yield of three major constituents
of saffron was investigated at 20 °C. The efficiency of the process was compared
with that of cold water extraction method proposed by ISO. The results showed
that ultrasound largely improved the extraction rate and incredibly reduced the
process time. The extraction yield increased with the increase of time and
amplitude of sonication. It was also found that the use of pulsed ultrasound with
short pulse intervals was more efficient than continuous sonication.
INTRODUCTION
Saffron is the dried stigmas of the flowers of Crocus sativa Linnaeus, which is
cultivated in many countries including Iran, Spain, Greece, India, and china; with the
former producing greater than 90 % of the world’s total annual production (Sampathu
et al., 1984; Negbie, 1999). It is the most expensive spice in the world increasingly
gaining interest among the customers for the delicate flavour, bitter taste and
attractive yellow colour it impacts on food. The main constituents responsible for
these characteristics are crocins, picrocrocin and safranal. Crocins are water soluble
derivatives of crocetin, glucosyl esters of 8,8’-diapocarotene-8,8’-dioic acid,
representing the yellow pigments of saffron (Sampathu et al., 1984). Picrocrocin, 4(β-D-glucopyranosyloxy)-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde, is the
glucoside responsible for the bitter taste of saffron from which safranal is derived
(Zarghami and Heinz, 1971). Safranal, 2,6,6-trimethyl-1,3-cyclohexadiene-1carboxaldehyde, is a monoterpene considered to be the major component constituting
saffron volatile components (Zarghami and Heinz, 1971; Tarantilis et al., 1994).
The quality of saffron is determined by its coloring strength, aroma and taste.
These quality attributes are highly dependent on soil, climate, rainfall, time of harvest,
and post harvest operations of saffron. Numerous methods have been proposed for
quality determination of saffron involving preparation of saffron extract followed by
measurement the quantity of crocins, picrocrocins, and safranal (Basker and Negbi,
1985; Sujata et al., 1992; Orfanou and Tsimidou, 1996; ISO, 2003; Kanakis et al.,
2004). Preparation of extract has a crucial impact on the accuracy of the results. It has
been reported that the type of solvent, time and method of extraction not only
considerably affect the diffusion rate of the components across the cell wall but also
their stability (Himeno and Sano, 1987; Sujata et al., 1992; Tarantilis et al., 1994;
Orfanou and Tsimidou, 1996). Crocins have been shown to undergo degradation
during prolonged extraction time in aqueous media of high water activity. It has also
been demonstrated that the use of alcohol or water-alcohol results in higher extraction
rate compared to water (Orfanou and Tsimidou, 1996). Accordingly, the methods of
extraction are being steadily revised and modified techniques proposed.
The use of high power ultrasound in the extraction of functional and bioactive
components of plant materials has been reported by many researchers (Salisova et al.,
1997; Vinatoru et al., 1997; Melecchi et al., 2002; Bruni et al., 2002; Albu et al.,
2004; Schinor et al., 2004; Wang and Weller, 2006). It has been shown that
ultrasound largely improves the extraction rate by disrupting plant cells and hence
increasing the diffusion of the cell contents across the cell wall. The beneficial effects
of sound waves on extraction are attributed to the formation and asymmetrical
collapse of microcavities in the vicinity of cell walls leading to the generation of
microjets rupturing the cells. It is also thought that the pulsation of bubbles causes
acoustic streaming which improves mass transfer rate by preventing the solvent layer
surrounding the plant tissue from getting saturated and hence enhancement of
convection.
The research reported here is aimed at the ultrasound-assisted extraction of
active principles from saffron. Observations and comparisons will be made with
reference to the methodology proposed by the International Standards Organization
(ISO 3632-2:2003).
MATERIALS AND METHODS
Commercial samples of saffron stigmas were purchased from local market.
The samples were ground with a pestle and mortar and passed through a 0.5 mm
mesh. The prepared sample was stored in a hermetic bag at 4 °C temperature until
use.
Preparation of Extracts
1. Cold Water Extraction Using Ultrasound. 0.25 g of sample was extracted with
20 ml of distilled water in a flat-bottomed glass tube using direct sonication, Dr.
Hielscher ultrasonic processor, Germany (Model UP 50H), at a frequency of 30 kHz
for 1, 3, 5 and 10 min. A titanium sonotrode of 3 mm in diameter was used in all
experiments with the tip placed 1.5 cm below the surface of liquid. The temperature
was kept constant at 20 °C throughout sonication by immersing the glass tube in a
refrigerated water bath. Continuous and pulsed sonication at 0.2 and 0.5 duty cycles
and 20, 60 and 100 % of the available amplitude were used. The above sonication
times were corrected with respect to the duty cycle used when working on pulse
mode. After sonication the sample was immediately transferred into a 500 ml
volumetric flask and made to the volume with distilled water. 20 ml of this suspension
was then diluted to 200 ml followed by filtration through cellulose acetate membrane
(pore size: 0.45 µm). The absorbance readings of the filtrate were measured on a UVVis recording spectrophotometer, Shimadzu, Japan (Model UV-160A) at 257, 330
and 440 nm representing  max for picrocrocine, safranal, and crocins, respectively.
Distilled water was used as blank. The results were expressed as E 1%max :
A max  10000
E1%max 
m 100  H 
where A is the absorbance at λmax, m the mass of saffron sample (g), and H is the mass
fraction of moisture and volatile content of the sample. H was determined to be 6.5 %
for the samples used in this study.
2. Cold Water Extraction without Ultrasound. 0.25 g of sample was extracted with
500 ml of distilled water for 1 hr according to ISO 3632-2: 2003. The suspension was
stirred using a magnetic stirrer during extraction and the temperature kept constant at
20 °C by immersing the flask in a refrigerated water bath. A 20 ml aliquot of this
solution was transferred to a 200 ml volumetric flask, diluted to the mark with
distilled water and then filtered as explained above. Absorbance readings were taken
at the same wavelengths given in section 1.
3. Statistical Analysis. Triplicate samples were used. A completely randomized
factorial design was performed to determine the significance of difference between
means by calculating Fisher static (F) at a 5 % confidence interval. Comparison
among means of the two extraction methods was carried out by Duncan’s Multiple
Range Test. Microsoft Excel and SAS 8.2 (SAS Institute In., Cary, USA) were used
for data analysis.
RESULTS AND DISCUSSION
The results clearly indicated that increasing sonication time increased the
yield, with up to 15 % enhancement for safranal towards the end of extraction period.
The rate of extraction was high during the first 5 minutes and then decreased
thereafter. An explanation for this is that there is a large concentration gradient for the
solutes in the plant cells and extraction solvent at the initial stage of extraction
resulting in higher extraction rates. Additionally, the constituents located in the
surface layers of the particles are more readily accessible than those in the deeper
regions which hardly diffuse out and hence slow down the rate of extraction.
Comparison of the results obtained in the presence of ultrasound with those of
1%
ISO procedure revealed that continuous sonication resulted in greater E 440
values at
1%
1%
all times and amplitudes tested, whereas E330
and E 257
gained higher values only at
longer sonication times or higher amplitudes (Table 1). Pulsed sonication led to
various extraction rates clearly indicating that using low duty cycle did not have
positive effect on extraction yield, although sonication at the maximum amplitude for
10 min resulted in E 1% values comparable to those of cold water extraction (Table 2).
On the other hand, as the data given in Table 3 show, sonication at high burst cycle
beneficially impacted the process of extraction causing larger amounts of the plant
constituents to diffuse out and hence E1%max values much greater than those of
continuous sonication. This is depicted in Figures 1a, b and c illustrating changes in
E1%
max values in the course of extraction at the amplitude of 100 %. These
observations indicate that a pulse is the more efficient, the shorter the interval
between pulses. In a pulsed ultrasound field the bubbles volume concentration in the
path of the acoustic wave is lower than that of a continuous field which improves the
acoustic transparency of the cavitation field. This is due to the fact that in the pulsed
field the time between two pulses acts as a rest time during which the small bubbles
and the unstable cavities generated by bubble collapses, depending on their sizes,
dissolve away or float out of the cavitation zone and the initial conditions of the liquid
are restored (Atchley et al., 1988; Johri et al., 1988; Francescutto et al., 1999). This
reduces the bubble-bubble interactions and the probability of losing the spherical
shape of the bubble at the early stage of collapse. Therefore, the bubble collapses are
more efficient and hence higher amount of heat and pressure is generated in the core
of bubble upon implosion. Another reason for the improvement of sonochemical
reactions in a pulsed field can be the disintegration of the stable agglomerations of
cavitation bubbles or prevention of their formation. The bubbles inside the cluster are
shielded from the ultrasound field by the bubbles at the outside part of the cluster and
act strongly on each other. Both theses factors diminish the efficiency of the bubble
collapse. The pulse modulation of the ultrasound prevents the clustering and this can
favor the clarification of the cavitation zone and thus maximization of the process
yield as reported by some authors (Flynn and Curch, 1988a, b; Wan et al., 1996).
The effect of acoustic amplitude on extraction of crocins, safranal and
picrocrocine is shown in Figures 2-4. As can be seen increasing amplitude
increases E1%max , with up to 11, 13, and 12 % enhancement for picrocrocine, safranal,
and crocins, respectively, when pulsed sonication at 100 % amplitude and 0.5 duty
cycle was used. The increased yield suggests that either additional cells have been
ruptured or extraction from the inner parts of the matrix has been accelerated. These
can be attributed to the cavitational effects, which caused the intensification of mass
transfer and thus closed interaction between the solvent and the plant tissues. It is
demonstrated that at increasing amplitudes, cavitational bubble collapse is more
violent since the resonant bubble size is proportional to the amplitude of ultrasounic
wave (Suslick et al., 1987; Suslick and Price, 1999, Li et al., 2004). Bubble collapse
in the vicinity of plant membranes may cause strong shear forces to be exerted that
can cause microfractures to be formed in plant tissues (Vinatoru et al., 1997;
Vinatoru, 2001). Additionally, increasing amplitude may cause microcavities to be
formed inside the cells that could contribute to disruption of the walls from the inside
(Vinatoru et al., 1997).
CONCLUSIONS
These results obtained in this study propose ultrasound as a potent alternative
for efficient extraction of active components from saffron. In comparison to cold
water extraction ultrasound improves the efficiency and reduces the processing time.
The rate of extraction was shown to be a function of time, amplitude and mode of
sonication. The increase of time and amplitude had a positive effect on the extraction
yield. Pulsed sonication at short intervals was found to be more efficient than
continuous sonication. Although these findings demonstrate that the use of ultrasound
produces greater extraction yields and thus can be considered in the future revision of
the ISO specification standard, further experiments are yet required before it is
introduced for industrial applications.
ACKNOWLEGEMENTS
Financial support for this project from Khorasan Science and technology Park
is gratefully acknowledged. The authors also thank Ms. Sara Sobhani and Ms. Zahra
Adabi for their great assistance in the experimentation and analysis.
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Table 1. Comparison of E1%max values obtained using continuous
sonication and ISO procedure
1%
1%
1%
Amplitude Sonication
E 257
E330
E 440
(%)
time (min)
20
1
84.2±0.8 35.9±0.8 233.3±0.7
3
85.3±1.1 37.1±0.6 234.3±3.3
5
87.4±2.6 37.4±0.1 235.9±2.7
10
88.1±4.6 39.0±1.2 237.5±3.0
60
1
3
5
10
85.3±1.1
87.7±1.9
88.6±1.0
90.1±1.7
37.0±0.5
37.2±0.1
37.9±0.8
39.4±0.6
233.9±4.3
236.6±3.3
237.0±2.3
238.3±3.9
100
1
3
5
10
87.4±0.6
89.0±1.5
89.3±2.1
91.8±0.7
37.0±0.5
37.8±0.4
38.6±3.4
39.4±0.6
234.0±3.3
236.6±3.1
237.6±1.1
239.3±3.3
89.0±1.6
38.6±1.1
226.9±2.6
ISO
Table 2. Comparison of E1%max values obtained
sonication at 0.2 cycle and ISO procedure
1%
1%
Amplitude Sonication
E 257
E330
(%)
time (min)
20
1
80.7±0.8 33.1±0.7
3
85.0±0.4 35.4±0.05
5
85.5±1.1 36.4±0.7
10
86.4±1.9 36.5±0.8
using pulsed
1%
E 440
194.3±3.9
210.0±0.5
215.1±2.5
220.2±6.6
60
1
3
5
10
82.9±3.8
86.3±1.2
86.8±1.2
89.3±1.2
34.4±2.3
36.2±0.3
37.0±0.3
38.8±1.6
204.0±16
210.4±0.9
216.3±4.8
222.6±0.8
100
1
3
5
10
84.7±1.6
86.8±2.4
87.6±0.5
90.0±1.9
35.1±1.3
36.5±1.8
37.2±0.6
38.9±0.7
204.8±5.4
212.0±2.9
218.5±0.5
226.9±1.7
89.0±1.6
38.6±1.1
226.9±2.6
ISO
Table 3. Comparison of E1%max values obtained
sonication at 0.5 cycle and ISO procedure
1%
1%
Amplitude Sonication
E 257
E330
(%)
time (min)
20
1
88.5±1.8 33.2±0.9
3
89.6±0.4 33.7±0.05
5
91.8±0.7 39.1±1.1
10
95.0±1.5 39.1±1.2
using pulsed
1%
E 440
224.2±7.9
228.5±1.5
232.0±2.2
242.8±4.8
60
1
3
5
10
89.2±1.3
90.0±0.5
95.2±2.1
95.3±1.6
37.7±0.5
38.7±0.3
39.70.2
40.5±1.2
226.2±5.6
233.8±2.0
239.5±1.4
243.3±1.6
100
1
3
5
10
89.6±1.4
90.5±0.05
95.4±2.6
96.1±1.8
38.7±0.8
39.3±1.1
40.5±1.5
41.1±1.0
227.9±6.2
234.4±0.3
245.4±3.6
246.1±5.4
89.0±1.6
38.6±1.1
226.9±2.6
ISO
250
42
(a)
(b)
40
230
E330
E440
240
220
38
36
210
34
200
0
2
4
6
8
10
0
12
2
4
6
8
10
12
T ime (min)
T ime (min)
98
(c)
E257
94
90
86
82
0
2
4
6
8
10
12
T ime (min)
Fig. 1. Changes in E1%max as a function of time during sonication at 100 % amplitude.
(a) crocins, (b) safranal, and (c) picrocrocine. ♦, continuous sonication; ▲, pulsed
sonication at 0.2 cycle; ■, pulsed sonication at 0.5 cycle.
240
230
(b)
(a)
220
E440
E440
238
236
234
210
200
232
190
0
20
40
60
80
120
100
0
20
40
Amplitude (%)
60
80
100
120
Amplitude (%)
250
(c)
E440
240
230
220
0
20
40
60
80
100
120
Amplitude (%)
Fig. 2. Influence of acoustic amplitude on the extraction yield of crocins. (a)
continuous sonication, (b), pulsed sonication at 0.2 cycle, (c) pulsed sonication at
0.5 cycle. ■, 1min; ▲, 3 min; ♦, 5 min; ×, 10 min.
40
40
(a)
(b)
38
E330
E330
38
36
36
34
34
32
0
20
40
60
80
100
120
0
20
40
Amplitude (%)
60
80
100
120
Amplitude (%)
42
(c)
E330
38
34
30
0
20
40
60
80
100
120
Amplitude (%)
Fig. 3. Influence of acoustic amplitude on the extraction yield of safranal. (a)
continuous sonication, (b), pulsed sonication at 0.2 cycle, (c) pulsed sonication at
0.5 cycle. ■, 1min; ▲, 3 min; ♦, 5 min; ×, 10 min.
92
94
(b)
(a)
90
E257
E257
88
84
86
82
80
0
20
40
60
80
100
120
0
20
40
Amplitude (%)
60
80
100
120
Amplitude (%)
98
(c)
E257
94
90
86
0
20
40
60
80
100
120
Amplitude (%)
Fig. 4. Effect of acoustic amplitude on the extraction yield of picrocrocine. (a)
continuous sonication, (b), pulsed sonication at 0.2 cycle, (c) pulsed sonication at
0.5 cycle. ■, 1min; ▲, 3 min; ♦, 5 min; ×, 10 min.