Ultrasonics Sonochemistry 21 (2014) 606–611

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

Mapping of an ultrasonic bath for ultrasound assisted extraction

of mangiferin from Mangifera indica leaves
Vrushali M. Kulkarni, Virendra K. Rathod ⇑
Chemical Engineering Department, Institute of Chemical Technology, Matunga, Mumbai 400019, India

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

Article history: The present work deals with the mapping of an ultrasonic bath for the maximum extraction of mangiferin
Received 17 April 2013 from Mangifera indica leaves. I3 liberation experiments (chemical transformations) and extraction
Received in revised form 28 August 2013 (physical transformations) were carried out at different locations in an ultrasonic bath and compared.
Accepted 29 August 2013
The experimental findings indicated a similar trend in variation in an ultrasonic bath by both these
Available online 7 September 2013
methods. Various parameters such as position and depth of vessel in an ultrasonic bath, diameter and
shape of a vessel, frequency and input power which affect the extraction yield have been studied in detail.
Keywords:
Maximum yield of mangiferin obtained was approximately 31 mg/g at optimized parameters: distance of
Mapping
Ultrasonic bath
2.54 cm above the bottom of the bath, 7 cm diameter of vessel, flat bottom vessel, 6.35 cm liquid height,
Extraction 122 W input power and 25 kHz frequency. The present work indicates that the position and depth of ves-
Natural products sel in an ultrasonic bath, diameter and shape of a vessel, frequency and input power have significant
Mangiferin effect on the extraction yield. This work can be used as a base for all ultrasonic baths to obtain maximum
efficiency for ultrasound assisted extraction.
Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Conventional extraction techniques like soxhlet extraction

In recent years, there has been tremendous increase in the use
of natural products due its applications in the pharmaceutical,
nutraceutical, cosmetic and other sectors. Sources of these natural
drugs could be plant, microorganisms or animals. Although micro-
organisms are the preferred sources, plant sources are similarly
important as some compounds can be obtained from plants. Mang-
iferin, chemically a xanthonoid, is one of the important natural
drugs, which has wide applications in pharmaceutical and other
related industries [1]. It shows antioxidant, antitumor and antiviral
properties [2–6]. Owing to complex structure of mangiferin, it is
very difficult to synthesize it chemically and extracting mangiferin
from natural sources is the best possible alternative for its produc-
tion. Mangiferin is present in various natural sources such as
Mangifera indica, Swertia anguistifolia Ham and Anemarrhena
asphodeloides rhizomes. Among these sources, M. indica is selected
because despite it being widely available in India, it is less
exploited. Reports are available on its medicinal uses [7–10]. Gen-
erally, mango leaves are regarded as sacred and people use it for
religious purposes. Therefore, in present work, there has been an
attempt of extracting mangiferin from M. indica using conventional
and advance extraction techniques.
⇑ Corresponding author. Tel.: +91 22 33612020; fax: +91 22 33611020.
E-mail address: vk.rathod@ictmumbai.edu.in (V.K. Rathod).
require longer time and heating. Advance extraction techniques
such as ultrasonic assisted extraction, supercritical fluids or micro-
waves assisted extraction increase the productivity by influencing
factors like the kinetics through the partial mass transfer rates, the
interfacial area or the driving force [11–12].
Application of ultrasound for the extraction of high-cost
materials has potential as it is simple, efficient and economical
alternative when compared to other extraction processes
[13–16]. One of the major advantages of using ultrasound assisted
extraction process is that it requires very less time for extraction as
compared to the conventional techniques [17]. Ultrasound assisted
extraction of natural products is usually performed in an ultrasonic
bath having transducers, which are located at the base of ultrasonic
bath. The task of the transducers is to convert energy into ultra-
sound. The function of generator is to transform the electrical en-
ergy from the input power source into a suitable form for
energizing the transducers at the desired frequencies [18]. Taking
into account the role of transducers, generators and sound waves;
the present work deals with the mapping i.e., effect of the position
of transducers, variations in input power and frequency of sound
waves on extraction of mangiferin from M. indica. Furthermore,
the shape of the reaction vessel is essential as energy is reflected
when the ultrasonic wave impinges against any solid surface. Thus,
the effect of geometry of the vessel in ultrasound bath should also
be studied to minimize attenuation of the ultrasonic wave. Much of
the work has been carried out on optimization of extraction

1350-4177/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ultsonch.2013.08.021
 V.M. Kulkarni, V.K. Rathod / Ultrasonics Sonochemistry 21 (2014) 606–611 607

process parameters using ultrasound assisted extraction. However, 150

Distance from the corner (mm)

very few reports are available on mapping of ultrasonic bath for 55
30
125 45 30
the extraction of natural products. In this paper, we explain the 1 4 7 10 13
mapping of ultrasonic bath in detail for the extraction of a natural 100 18
material. Previously similar experiments were performed with the 40
75 2 5 8 11 14
natural dye [19]. However, we have additionally studied the effect
of frequency, input power, height and geometry of the vessel. It is 50
observed that mapping helps in relating the primary effects (local 3 6 9 12 15
25 70
pressure field) of ultrasound activity with the observed secondary 120
55
110

effects (such as chemical reaction) [20]. In UAE, geometric 0

parameters are also essential for design of an efficient and cost 0 50 100 150 200 250 300
effective process. This paper describes the mapping of ultrasonic Distance from the corner (mm)

bath along with the parameters affecting the ultrasound assisted Transducers Test Tubes
extraction.
300 mm

2. Materials and methods

2.1. Material
Green (mature) leaves of M. indica were obtained from the Insti-
tute of Chemical Technology garden at Matunga, Mumbai, India.
Leaves were cleaned and sun dried for 48 h. The dried leaves were
then powdered and stored in an air tight container at a cool place.
Moisture content of the powder was approximately 9%. Mangiferin
(Sigma M3547-100 mg P98% HPLC grade), procured from Sigma–
Aldrich was used as the reference standard. All solvents were of
analytical grade, purchased from Hi Media Ltd., Mumbai, India.
Distilled water used as a solvent was obtained from Millipore Milli
Q 50 HPLC grade. Methanol and acidified water (0.1% acetic acid)
were used as mobile phase. Extracts of 5 ll were prepared from
each sample and injected into HPLC. The peak areas at 254 nm
wavelength were then measured.
2.2. Analysis of mangiferin
The quantification of mangiferin was carried out using HPLC
(Agilent 1260 infinity high performance auto sampler) equipped
with C18 column. The analysis was performed by isocratic elution
with a flow rate of 1 ml/min. Mobile phase used was methanol and
acidified water in 30:70 v/v ratio.
3. Experiment
3.1. Soxhlet extraction
150 mm
76.2
50.8 mm
25.4 mm
10.16 mm
Fig. 1. Schematic representation of arrangement of test tubes in ultrasonic bath.
leaves was studied. Experiments were performed for 4 min
keeping the temperature of solvent inside the glass tube at
29 ± 2 °C. All experiments were performed three times and average
values have been reported with standard deviations. The findings
were further validated for all parameters by using Weissler reac-
tion or decomposition of KI. When aqueous solution of potassium
iodide (KI) is exposed to ultrasound, decomposition of KI takes
place forming I3 which can be analyzed using UV Spectrophotom-
eter in the range of 352–354 nm wavelengths [21]. The reaction is
typically used as a measure to check the variation in cavitation
activity [21–23]. In order to study the energy efficiency of the
ultrasonic bath, calorimetric studies were performed. Amount of
energy dissipated in the bulk of liquid was calculated by measuring
the rise in temperature of a fixed quantity of water (3000 ml) in an
ultrasonic bath for a given time. Power dissipation was calculated
as per Sutkar et al. [21].

Soxhlet extraction was used as reference method for compari-

son with the ultrasound assisted extraction. Soxhlet extraction
was performed with 0.5 g of M. indica dry adult leaves powder with
200 ml distilled water as a solvent contained in a 200 ml round
bottom flask. After 5 h of extraction, the concentration of mangif-
erin was determined by HPLC analysis.
3.2. Ultrasound assisted extraction
Ultrasound assisted extraction was carried out in an ultrasonic
bath (Model 6.5l200 H, Dakshin, India) with a working frequency of
25 and 40 kHz and a maximum input power of up to 210 W. The
ultrasonic bath consisted of a rectangular tank of internal dimen-
sions 300  150  150 mm having four transducers at the bottom
of the bath. Fig. 1 shows schematic representation of ultrasonic
bath used for mapping. All the experiments were carried out in
glass tubes of known dimension placed in an ultrasonic bath at
known height and position. The influence of number of parameters
like position of vessel, frequency of sound waves, input power, and
geometry of vessel on ultrasound assisted extraction of M. indica
4. Results and discussions
4.1. Soxhlet extraction
After 5 h, mangiferin yield of 57 mg/g in adult leaves was
obtained from soxhlet extraction. Yield of extraction from this
conventional method of extraction was further compared with
the ultrasound assisted extraction.
4.2. Effect of position of vessel
As transducers are located at the bottom of the ultrasonic bath,
one of the main parameters influencing ultrasound assisted extrac-
tion is the position of vessel in the ultrasound bath. To check the
variations in the ultrasonic intensity distribution, the ultrasonic
bath was segregated into five segments and each segment was fur-
ther sub-divided into three. Thus there were 15 different positions
as numbered in Fig. 1. A flat bottom glass test tube having 1.3 cm
diameter and 17.5 cm height was placed at one of the locations
608 V.M. Kulkarni, V.K. Rathod / Ultrasonics Sonochemistry 21 (2014) 606–611
(shown in Fig. 1) in an ultrasonic bath. The test tube was placed in
such a way that the bottom of the test tube was at a height 1 cm
from the bottom of the bath as shown in Fig 1. Solute to solvent ra-
tio was maintained 1:40, input power was 204 W, frequency used
was 25 and 40 kHz separately and extraction was carried out for
4 min. After 4 min, extract from the test tube was centrifuged
and analysis was done using HPLC. Similarly, the test tube was
placed in all the other locations keeping the height of liquid con-
stant and the experiment was carried out. Results obtained are
shown in Fig. 2. It was observed that the test tube placed at
positions 5 and 11 resulted in a higher yield as compared to other
positions. We have thus selected 5th position in further experi-
ments to optimize other parameters. The yield of mangiferin was
obtained to be approximately 26 mg/g and 23 mg/g at 25 and
40 kHz, respectively at 5th position.
The findings were further validated by using Weissler reaction
or decomposition of KI. Decomposition of KI was carried out at
15 different locations, one location at a time, as shown in Fig. 1.
The concentration of I3 was measured at 352 nm as mentioned
earlier. Results obtained are shown in Fig. 3. When Weissler reac-
tion was used, the concentration of I3 was obtained to be approx-
imately 6 and 5 ppm at 25 and 40 kHz, respectively at 5th position.
The experimental findings by using Weissler reaction were in
agreement with those observed in the extraction process.
Thus the effect of variation in cavitation in a bath on chemical
transformations and physical transformations or extraction indi-
cated a similar trend. Concentration of I3 was higher at 5th,
11th and 8th positions similar to the results obtained for extraction
process. Similar findings were reported by Kumar et al. [19] where
they compared the extraction of dye (physical extraction) with the
KI degradation. The KI oxidation occurs due to OH radical gener-
ated by sonolytic dissociation of water (sonochemical transforma-
28
26
Yield of mangiferin (mg/g)
24
22
20
18
16
14
12
10
1 2
Fig. 2. Effect of position of extraction vessel at 25 and 40 kHz.
6
− (ppm)
5
Concentration of
0
1 2
Fig. 3. Validation of effect of position of extraction vessel using Weissler reaction at 25 and 40 kHz.
tion). The extraction of biomolecule occurs due to the propagation
of the shock wave by the cavity collapse. Even though the cause of
the Iodine dosimetry and extraction are different, the same princi-
pal, i.e., cavitation phenomenon, ultimately governs them. Hence
both are expected to show a similar trend. Inside the ultrasonic
bath, the 5th, 11th and 8th positions were the only positions with
two transducers in the vicinity and thus the highest three yields
were obtained in these positions. Amongst these positions, the dis-
tance from the nearest transducer is the least at 5th position and
thus has a higher yield than the other two positions. Hence, it is
concluded that positions closer to the location above the transduc-
ers have a higher intensity of ultrasound waves, which can result in
better extraction. In studies related to ultrasound assisted extrac-
tion using probe sonicator, it has been reported that ultrasound
intensity is higher in axial direction (vertical plane) as compared
to radial direction (horizontal plane) [19]. It has been reported that
standing waves result in higher cavitation at specific locations in
axial direction. The results obtained in the ultrasonic bath show
a similar trend as seen in the studies related to probe sonicator.
Thus we conclude that even though amplitude in the ultrasonic
bath and probe sonicator may differ, the basic principle that ultra-
sound intensity is higher in axial direction as compared to radial
direction with respect to transducers will hold. The position study
indicated that the intensity of the ultrasound in an ultrasound as-
sisted extraction is non-uniform being higher in the region above
the transducers. Hence the position of vessel is optimized for
obtaining best possible results.
4.3. Effect of frequency
The extraction was carried out at two different frequencies 40
and 25 kHz at 204 W. From Fig. 2, it has been found that the
3 4 5 6 7 8 9 10 11 12 13 14 15
Position of vessel in ultrasound bath
3 4 5 6 7 8 9 10 11 12 13 14 15
Position of vessel in ultrasound bath
 V.M. Kulkarni, V.K. Rathod / Ultrasonics Sonochemistry 21 (2014) 606–611
extraction was higher at 25 kHz. Yield of mangiferin was obtained
to be around 26 and 22 mg/g at 25 and 40 kHz, respectively. It has
been stated in literature that the cavitation yield decreases as fre-
quency increases because the cavitation bubbles tend to be smaller
and less energetic, thereby reducing the yield [24]. Previous stud-
ies have reported similar trend in their research where low fre-
quency was found to be preferable [25–27]. The scattering and
attenuation of sound waves is less at lower frequencies and also
cavitation is easily possible at lower frequency as compared to
higher frequency. This could be the possible reason for higher
extraction yield at lower frequency [25]. Additionally, power dissi-
pation was calculated and found to be 53 and 47.6 W for 25 and
40 kHz, respectively. Since the power dissipated for 25 kHz is high-
er than that in 40 kHz, higher yield was obtained at 25 kHz. Thus
further experiments were performed at 25 kHz as the yield ob-
tained is higher at 25 kHz.
4.4. Effect of depth of extraction vessel
The effect of the depth of vessel (inside the ultrasonic bath) on
extraction yield was essential to obtain most favorable position of
the vessel where the intensity of ultrasound waves would be high-
er. Experiments were performed for four different depths i.e.,
1.016, 2.54, 5.08 and 7.62 cm as shown in Fig. 1. Each experiment
was performed by keeping other experimental conditions constant
as previously stated and results obtained are shown in Fig. 4. It is
observed that the extraction yield was optimum at 2.54 cm depth
for all the tubes. Experiments were performed in the range of
25–30 °C. Within this range, the velocity of sound is approximately
equal to 1500 m/s. Since velocity is a product of frequency and
wavelength, at 25 kHz frequency the wavelength would be equal
to 6 cm. Hence the maximum ultrasound intensity in ultrasound
bath would be half the wavelength which will be around 3 cm
above the transducers [27]. As per the results, the extraction yield
was highest at 2.54 cm (which is close to 3 cm). Similarly, the
extraction yield was higher for 5th position supporting our earlier
results of position optimization. Hence for further studies, 5th
position and 2.54 cm depth were selected.
4.5. Effect of diameter and shape of extraction vessel
One of the essential variables which affect ultrasound assisted
extraction is the geometry of the vessel in which extraction is
being carried out. To check the effect of the dimensions of the ves-
sel on ultrasound assisted extraction process, the diameter of the
test tubes were varied. Vessels with different diameters selected
were 1.3, 1.5, 2.5, 3, 3.5, 5 and 7 cm while all other experimental
conditions were kept constant. Results obtained are shown in
Fig. 5. Further, the effect of bottom shape of test tube in ultrasound
30
Yield of mangiferin (mg/g)
28
26
24
22
20
18
16
14
12
10
1 2 3 4 5 6 7 8 9 10 11 12 13
Position of vessel in ultrasound bath
1.016 cm 2.54 cm 5.08 cm 7.62 cm
Fig. 4. Effect of depth of extraction vessel.
609
31
Yield of mangiferin (mg/g)
29
27
25
1.3 1.5 2.5 3 3.5 5 7
Diameter of (cm)
Fig. 5. Effect of diameter of extraction vessel.
bath was also tested. Flat bottom test tube was replaced by round
bottom test tube and results obtained are shown in Fig. 6. It was
observed that as the diameter increases the ultrasound assisted
extraction also increases up to some limit and then it remains con-
stant. In a vessel with diameter 1.3 cm, yield of mangiferin was ob-
tained to be approximately 26 mg/g, while in vessels with 3.5, 5
and 7 cm, yield was approximately 31 mg/g. Increase in yield with
the increase in diameter can be attributed to the fact that as diam-
eter increases, surface area for ultrasound exposure increases. Also,
as the diameter of the vessel increases, liquid height decreases.
One of the reasons of getting higher yield in 7 cm diameter vessel
could be attributed to the fact that that corresponding liquid height
in 7 cm tube is nearer to the half wavelength (3 cm) compared to
the liquid height of other vessels [28]. Hence it is necessary to opti-
mize the liquid height in vessel. Yield of mangiferin was obtained
to be approximately 28 mg/g in flat bottom vessel while it was
approximately 26 mg/g in round bottom vessel. This is attributed
to the fact that the transfer of energy is much more effective when
sound impinges on flat surface than when it hits underside of
spherical container where some energy is reflected away [27].
For further studies 7 cm flat bottom vessel was used taking into ac-
count its larger surface area for ultrasound assisted extraction.
4.6. Effect of variation in liquid height
Keeping the position, diameter, depth, frequency, input power,
solute to solvent ratio and extraction time constant, liquid height
was varied within the vessel of diameter 7 and 3.5 cm to obtain
the optimum liquid volume. Liquid heights were maintained at
3.81, 6.35 and 8.89 cm, respectively. Yield of mangiferin after
extraction in two different vessels at various liquid heights is
shown in Fig. 7. Extraction was higher at 3.81 cm compared to
6.35 and 8.89 cm in both the vessels. Power density was calculated
at heights 3.81, 6.35 and 8.89 cm. It was observed that power den-
sity decreases with increase in liquid height. Power density at 3.81,
30
Yield of mangiferin (mg/g)
28
26
24
14 15
22
Flat bottom test tube Round bottom test tube
Fig. 6. Effect of shape of extraction vessel.
610
29
Yield of mangiferin (mg/g)
27
25
23
21
19
17
15
3.81 6.35
Variation of liquid height in
7 cm tube 3.5 cm tube
Fig. 7. Effect of variation in liquid height (cm) in extraction vessel.
6.35 and 8.89 for 3.5 cm diameter tube was 5.5, 4.5 and 3.2 W/cm3,
respectively while for 7 cm diameter tube was 1.39, 0.81, and
0.58 W/cm3, respectively. Thus the decrease in extraction yield
with the increase in the liquid height could be because there is a
decrease in power density when reaction volume increases. Studies
have also stated that with large volumes, ultrasound efficiency de-
creases due to scatter of ultrasonic energy in bulk solution [27].
Also, low sonication volume allows inducing more collisions
among particles and thereby increasing the total area exposed to
solvent extraction [18]. One of the possible reasons could be that
liquid height corresponding to 3.81 cm and 6.35 cm is closer to half
wavelength than that corresponding to 8.89 cm. Hence increased
cavitation is obtained at those heights [28].
4.7. Effect of input power
One of the major parameters affecting the cost of the process,
especially at industrial scale is input power. Hence, it is necessary
to optimize input power and check its effect on ultrasound assisted
extraction. Input power selected was 42, 83, 92, 103, 110, 122, 163
and 204 W, respectively from 20–100% i.e., from 42 to 204 W,
while other parameters were kept constant. Results obtained are
shown in Fig. 8. Studies have stated that high input power results
in more disintegration of cells and gives higher mass transfer
which results in more yields [29]. In the present work, it was ob-
served that initially till 60% input power or 122 W, the extraction
of mangiferin was nearly constant. While from 122 W a sudden in-
crease in the yield of mangiferin was observed and it remained
constant up to 100% input power. Increase in the yield with the in-
crease in input power was observed because of the larger ampli-
tude of ultrasound traveling through the solvent. Thus higher the
power input, the more violently the cavity collapse, and thus high-
er extraction can be attained. At 122 W, the energy released was
optimum to extract all the mangiferin as the yield was constant
28
Yield of mangiferin (mg/g)
26
24
22
20
42 83 92 103 110
Input power (W)
Fig. 8. Effect of input power on yield of mangiferin.
V.M. Kulkarni, V.K. Rathod / Ultrasonics Sonochemistry 21 (2014) 606–611
after 122 W. This could be the possible reason for the increase in
the yield at 122 W [30]. Thereafter to estimate the actual input
power being used, power dissipation was calculated based on calo-
rimetric study. It was observed that power dissipated in a vessel
was 12.7, 21.2, 24.6, 27.4, 32.5, 35.3, 44.5 and 53.4 W, respectively.
Thus as the input power increased above 122 W, there was an in-
crease in the power dissipation but the extraction yield remained
the same. Hence, it is beneficial to optimize the input power before
carrying out at larger scale as it will help us to conserve power and
thus will contribute in making the process more economical.
8.89
vessel (cm)
5. Conclusion
In the present work, mapping of ultrasonic bath and optimiza-
tion of parameters affecting ultrasound assisted extraction of
mangiferin from M. indica leaves were successfully carried out.
Parameters such as position, depth and geometry of extraction ves-
sel, variation of liquid height in extraction vessel, effect of fre-
quency and power were studied. Maximum yield of mangiferin
was obtained approximately 31 mg/g at optimized parameters
such as 5th position, distance of 2.54 cm above the bottom of the
bath, 7 cm diameter of vessel, flat bottom vessel, 6.35 cm liquid
height, 122 W input power and 25 kHz frequency. From the com-
parison of ultrasound assisted extraction with soxhlet extraction,
it was observed that approximately 54% yield was obtained from
ultrasound assisted extraction in only 4 min. Hence the study of
mapping of ultrasonic bath and parameters like power and fre-
quency will help in avoiding unnecessary consumption of power,
which in turn will help to make the process more cost effective.
Therefore, mapping of the ultrasonic bath will be helpful for ultra-
sound assisted extraction techniques.
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