- Email: [email protected]
Mapping study of an ultrasonic bath for the extraction of andrographolide from Andrographis paniculata using ultrasound
Industrial Crops and Products 66 (2015) 312–318
Contents lists available at ScienceDirect
Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Mapping study of an ultrasonic bath for the extraction of andrographolide from Andrographis paniculata using ultrasound Priyanka R. Rao, 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
Article history: Received 13 August 2014 Received in revised form 11 November 2014 Accepted 26 November 2014 Keywords: Andrographolide Ultrasound assisted extraction Ultrasound bath Mapping
a b s t r a c t In the present work, extraction of andrographolide from Andrographis paniculata L. was carried out by mapping an ultrasound bath to identify the active and passive zones within it. This helps in accurately placing the extraction vessel in the bath for obtaining a maximum yield. Various parameters of extraction like choice of solvent, time of ultrasound exposure, solute to solvent ratio, temperature, position and depth of extraction vessel, the frequency used, and input power were investigated. Aluminum foil weight loss test was also performed at different positions and its results were compared with the results obtained for extraction at same positions. Results revealed a similar trend in variation for both the investigations. Highest yield of andrographolide obtained was 27.97 mg/g, at position 11, at a depth of 2.54 cm, in only 10 min of ultrasound irradiation, using 1:40 solute to solvent ratio, at a frequency of 22 kHz, utilizing 134 W of power with 50% ethanol as the solvent. Calorimetric studies were also carried out within the reaction vessel at these positions, to determine the actual power being utilized for extraction. Power dissipated at these locations differed from each other and maximum power was found to be dissipated at position 11. This study implies the significance of the above mentioned parameters in mapping of an ultrasound bath which can further be used for extraction of biomolecules from natural products. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Although medicinal plants have been widely used since the ancient times, the identity of compounds that showed the desired therapeutic effect remained unknown until a few decades back. Development in the field of medicinal chemistry and pharmacology led to their structural elucidation and further helped in understanding their biological activities in the human body. The chemists synthesized these compounds thus reducing the cost of their production (Ji et al., 2009). However, there are reports of adverse effects caused by synthesized drugs, which has revived the interest in the use of natural compounds (Csoka and Szyf, 2009). Andrograhis paniculata is a popular Ayurvedic and Chinese household remedy for the common cold, digestive issues, upper respiratory tract infections, and flu. Andrographolide is a bitter compound extracted from the leaves and stem of an herbaceous plant called A. paniculata (Caceres et al., 1997). It is a major diterpene lactone showing
Abbreviations: AP, andrographolide; UAE, ultrasound assisted extraction; MAE, microwave assisted extraction; SCFE, supercritical fluid extraction; ASE, assisted solvent extraction. ∗ Corresponding author. Tel.: +91 22 33612020; fax: +91 22 33612010/2020. E-mail address: [email protected] (V.K. Rathod). http://dx.doi.org/10.1016/j.indcrop.2014.11.046 0926-6690/© 2014 Elsevier B.V. All rights reserved.
multifarious activities like anti-pyretic, anti-inflammatory, antioxidant, anticancer, and hepatoprotective (Dhiman et al., 2012). It inhibits the in vitro proliferation of different tumor cell lines, representing various types of cancers. Andrographolide shows direct anticancer activity on cancer cells by cell-cycle arrest at G0/G1 phase through induction of cell-cycle inhibitory protein p27 and decreased expression of cyclin-dependent kinase 4 (CDK4). The immunostimulatory activity of andrographolide is seen by an increased proliferation of lymphocytes and production of interleukin-2 (Rajagopal et al., 2003). Recently water soluble andrographolide sulfonate, exerting anti-sepsis action in mice has been synthesized (Guo et al., 2012). Traditional extraction techniques have the drawbacks of longer extraction time, higher extraction temperature, increased solvent consumption, and mass transfer resistance. Novel extraction techniques like ultrasound assisted extraction (UAE), microwave assisted extraction (MAE), supercritical fluid extraction (SCFE), and accelerated solvent extraction (ASE) overcome these pitfalls (Shah Megha and Rohit Minal, 2013). Several reports suggest the advantages of these modern extraction techniques over conventional techniques to extract bioactives from natural sources. A comparative study of superheated water extraction (SWE) with conventional volatile isolation methods like hydrodistillation and soxhlet extraction on Buniumpersicum Boiss shows SWE to be a quicker and selective method with respect
P.R. Rao, V.K. Rathod / Industrial Crops and Products 66 (2015) 312–318
to the valuable oxygenated components (Mortazavi et al., 2010). MAE is popularly used as an advantageous method for the extraction of natural products. Report by Rafiee et al. indicates that MAE gives a higher extraction yield as compared to the conventional maceration method, for extraction of phenolic compounds from olive leaves (Rafiee et al., 2011). MAE is also reported to reduce the extraction time and solvent consumption in extraction of cottonseed oil from whole cottonseed (Taghvaei et al., 2014). It causes plant cells to burst due to localized internal superheating followed by leaching out of the biomolecules. The cell bursting phenomenon facilitates the entry of extracting solvent to solubilize out the target compound, thus leading to faster and more efficient extraction (Vasu et al., 2010). MAE has certain limitations that it cannot be used for heat sensitive materials and involves high investment costs. Most of these methods have some similarities with respect to solvent volume, extraction time, and extraction efficiency. SFE is one of the most promising extraction processes. It provides higher selectivity, shorter extraction times and does not use toxic organic solvents (Kaur et al., 2012). However, SCFE and ASE require greater financial investment and the presence of water in samples can cause hindrances in both techniques. UAE has been previously used for extracting biomolecules from a number of natural products. The basis of UAE is a well-known phenomenon called cavitation. As ultrasound waves pass through liquid medium it generates small vacuum filled cavitation bubbles within it. With an increase in the number of expansion and compression cycles within the medium, these bubbles implode abruptly. There is an increase in local temperature and pressure which facilitates greater solvent circulation, deeper solvent penetration within the cellular material and enhanced mass transfer, thus enhancing the extraction rate (Charpe and Rathod, 2012). Apart from considerable reduction in extraction time, the major benefits of UAE are that it increases the yield, requires a simple and easy to operate apparatus which helps in easy extraction of thermolabile compounds, without any degradation (Wang et al., 2013). Indirect sonication using an ultrasound bath with one or more transducers fitted at the bottom, is extensively used to extract bioactives from natural compounds (Tao et al., 2014). It is essential to know the active and passive zones in the bath to accurately place the extraction vessel in it which helps in obtaining a maximum extraction yield. The yield of ultrasound extraction depends on the location of transducers in the bath, power input, frequency of ultrasound, position of the extraction vessel, and its geometry (Santos and Lodeiro, 2009). The present report, concentrates on the optimization of the above mentioned parameters, which effect the extraction of andrographolide from A. paniculata. One of the methods of quantifying cavitational activity distribution is by investigating the secondary effects generated by cavitation. Secondary effects indicate the effects generated after bubble collapse (e.g., aluminum foil test, iodine dosimetry, electrochemical methods, etc.) (Sutkar and Gogate, 2009). So, to investigate the physical effects of cavitation in the bath at various positions, a comparison of aluminum foil weight loss test with that of andrographolide extraction is performed. To the best of our knowledge there are no reports available on mapping of an ultrasonic bath for extraction of andrographolide from A. paniculata. Furthermore, parameters like the choice of solvent, ultrasound irradiation time, solute to solvent ratio and the effect of temperature on extraction of andrographolide are also examined.
2. Materials and methods 2.1. Material Powdered leaves of A. paniculata was purchased from a local store in Mumbai. An average powder size of 0.6–1.1 mm was used.
313
The powder was sun dried and checked for its moisture content. The moisture content was found to be approximately, 7%. The standard for andrographolide (Aldrich 365,645–100 mg, 98% HPLC grade) was procured from Sigma–Aldrich. Acetonitrile and acidified water were used as the mobile phase for HPLC. Acetonitrile was of HPLC grade and ethanol used as the solvent was of AR grade. These solvents were purchased from high media, Mumbai, India for the experiments. Distilled water used as one of the mobile phases, was obtained from Millipore Milli Q 50HPLC grade. 2.2. Analysis of andrographolide Separation of andrographolide from its extract was done using HPLC (Agilent 1260 infinity high performance auto sampler) equipped with C18 column. Mobile phase used for HPLC analysis comprised of acetonitrile and acidified water in the ratio 60:40 v/v. Isocratic elution was used to perform HPLC analysis where the flow rate was set at 1.0 mL/min throughout the run with an injection volume of 5 L. The peak for andrographolide was detected at 223 nm with a retention time of 7.2 min. 3. Experimental work 3.1. Soxhlet extraction Soxhlet extraction of A. paniculata was carried out for 4 h using aqueous ethanol as the solvent. The dried sample was packed in a sample tube, above the extraction solvent. On heating, the solvent evaporated and cooled into a liquid in a condenser after which it re-entered the sample tube. This continued for several hours until the extraction was complete. Andrographolide content was then estimated using HPLC. 3.2. Ultrasound assisted extraction of A. paniculata Ultrasound assisted extraction of A. paniculata was carried out by indirect sonication in a dual frequency operated ultrasound bath operating at 25 and 40 kHz (model no. 6.5l 250H/DTC/DF). A rectangular tank having internal dimensions 300 × 150 × 150 mm, fitted with five transducers at the bottom, arranged in a zigzag manner was used. Fig. 1 shows a schematic representation of the top view of ultrasound bath. The rated power of the bath was 222 W. All experiments were performed thrice and their average values with standard deviations are reported. Ultrasound assisted extraction was carried out in a cylindrical flat bottom glass vessel of a specific height and diameter, at various positions and heights to obtain maximum yield (Kulkarni and Rathod, 2014). Extraction was carried out for 10 min at a temperature of 27 ± 4 ◦ C. Effect of parameters like choice of solvent, ultrasound exposure time, temperature, solute to solvent ratio, position of the vessel in the bath, depth of the vessel, frequency of sound waves, and power input, on the extraction yield of andrographolide was investigated. A number of solvents including water, various concentrations of ethanol, methanol, and isopropyl alcohol were screened for extraction. Solid to solvent ratio of 1:40 was maintained throughout the study. The variation in cavitation activity at various positions in the bath was then confirmed by aluminum foil test. A piece of accurately weighed aluminum foil was taken in the glass vessel and sonicated in similar conditions like A. paniculata, at same positions. The weight loss of aluminum foil in the vessel at these positions was compared with the extraction yield of andrographolide at identical positions (Pugin, 1987). Also, power dissipation studies at these positions, to estimate the amount of power actually being utilized for extracting andrographolide, were done in detail. Calorimetric studies were performed for the same purpose with 3.5 liters of water in the bath.
314
P.R. Rao, V.K. Rathod / Industrial Crops and Products 66 (2015) 312–318
Fig. 1. A schematic representation of the top view of ultrasound bath.
4.1. Soxhlet extraction The extract was collected after every 1 h and analyzed for AP content using HPLC. The maximum yield of andrographolide obtained after 4 h of soxhlet extraction was 42.46 mg/g. This yield was considered to be a reference for comparison of yield of soxhlet extraction with that of UAE. 4.2. Effect of position of extraction vessel To examine the variation in cavitation activity at different locations in the bath, it is of prime importance to study the effect of vessel position in the bath. Use of multiple transducers reportedly gives more uniform and intense cavitational activity as compared to a single transducer irradiation (Shirsath et al., 2012). Hence a bath fitted with multiple transducers was used. The entire bath was divided in 15 uniform sections where the extraction vessel was placed, as shown in Fig. 1. With the use of flat bottom vessel, minimum loss of ultrasound wave is expected to occur. Therefore, a flat bottom vessel with 1.3 cm diameter and 17.5 cm height already optimized by Kulkarni and Rathod (2014a) was used. The vessel was covered with aluminum foil to avoid loss of solvent. It was then placed at a depth of 1 cm from the bottom of the ultrasound bath. Extraction was carried out for 10 min at 22 kHz and 40 kHz using 1:40 solute to solvent ratio with maximum power input of 212 W. After 10 min the extract was filtered and analyzed for andrographolide content by HPLC. The vessel was then placed at all the remaining positions, one at a time, under similar conditions and analyzed for andrographolide content. It was observed that the highest yield of andrographolide was obtained at position 11 followed by position 5 and 8 at both frequencies as shown in Fig. 2. The yield of AP obtained at position 11 at 22 kHz and 40 kHz was 22.10 mg/g and 17.91 mg/g, respectively. Positions 5, 8, and 11 have three transducers in their locality which produce a synergistic cavitation effect thus creating conducive conditions for higher yield in the vessel at these positions in comparison to others. Also, literature suggests that cavitation phenomenon is higher in the vertical plane of transducers than the horizontal or radial plane (Sravan Kumar et al., 2009). Thus from the collated results, it can be concluded that
the proximity of extraction vessel in the vertical plane of the transducers is reflected by an increase in extraction yield. Based on our results position 11 was considered to be the most suitable position in the bath to perform extraction. 4.3. Aluminum foil weight loss test Aluminum foil weight loss test was done to evaluate the distribution of cavitation effect throughout the bath. At low frequencies the mechanical effect of ultrasound is predominant (Viet et al., 2014). Thus, 22 kHz frequency was selected for the experiment. A piece of aluminum foil was weighed and subjected to similar conditions of sonication as that for crude material, i.e., 22 kHz frequency, 212 W power,10 min, 50% ethanol, 1:40 solid to solvent ratio. The foil was again weighed after 10 min of ultrasound irradiation (Pugin, 1987). This study was performed at all 15 positions of the bath at a depth of 1 cm from the base of the bath. Results showed that a maximum foil weight loss was seen at position 11, followed by position 5, and 8, connoting that cavitation intensity was supreme at these positions. The result of this investigation was compared with that of the finding of position study experiment. Parallel trends in both the graphs were seen when plotted and compared. Fig. 3 illustrates the obtained results. The highest yield of andrographolide as well as a maximum foil weight loss, both was obtained at position 11, corroborating that cavitation intensity
25
Yield of AP (mg/g)
4. Results and discussion
20 15 10 5 0 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15
Position of vess el Fig. 2. Effect of position of extraction vessel in the ultrasound bath.
25
35
120
20
30
90
15
60
10
30
5
0
0 1
2
3
4
5
6 7 8 9 10 11 12 13 14 15 Position of vessel
Yield of AP in mg/g
150
Yield of AP (mg/g)
Alumihnum foil weight loss (mg)
P.R. Rao, V.K. Rathod / Industrial Crops and Products 66 (2015) 312–318
25 20 15 10 5 0 1
Yield of AP (mg/g)
315
2
3
4
5
6
7
8
9 10 11 12 13 14 15
Position of vess el
Aluminum foil weight loss (mg)
Fig. 3. Comparison of aluminum foil weight loss study and extraction yield of AP.
1.01 cm
2.54 cm
5.08 cm
Fig. 4. Effect of depth of extraction vessel on extraction yield.
was non uniform throughout the bath with a maximum intensity at position 11.
obtaining a maximum yield at 2.54 cm, which is closer to 3.4 cm, is justified.
4.4. Effect of frequency 4.6. Choice of solvent
4.5. Effect of depth of extraction vessel Depth at which the extracting vessel is placed in the bath plays a significant role in affecting the final yield. Increasing the distance between extracting vessel and transducers brings down the cavitation activity (Kanthale et al., 2003). Hence it is advantageous to establish an optimum height at which the vessel must be kept to obtain maximum extraction. Keeping the previously mentioned experimental conditions constant, the depth of the vessel from bottom of the bath was varied between 1.27 cm and 5.08 cm. Three different depths were selected (1.27 cm, 2.54 cm, and 5.08 cm) and extraction was carried out in a flat bottom glass vessel keeping it at all 15 positions. The results observed are illustrated in Fig. 4. Maximum yield of AP was obtained at a depth of 2.54 cm in all the vessels. The yields at position 11, 5, and 8 were comparatively higher. Ultrasound passes through water contained in the bath in form of waves. Each wave has its maxima and minima. Ultrasound irradiation at maxima is very powerful. For transducers operating at 22 kHz, the wavelength of ultrasound in water is equal to half the product of velocity of sound in water (1500 m/s) and the frequency at which the bath is operated (22 kHz). Thus the maxima of ultrasound wave are likely to be around 3.4 cm (De Castro and Capote, 2006). Hence
Choice of solvent is a critical parameter which affects the extraction yield. The solubility of a compound in a particular solvent increases with solvent polarity but reduces with an increase in solvent viscosity, surface tension and vapor pressure. Also, it becomes difficult to attain cavitation with an increase in surface tension and vapor pressure of the solvent (Gogate et al., 2003). Polar solvents assist in increasing the cell wall permeability causing an improved contact between the solvent and solid, thereby increasing the extraction yield (Jadhav et al., 2009). The solvents were selected on the basis of solubility of AP in them. Various solvents screened were methanol, different concentrations of ethanol (100%, 75%, 50%, and 25%), isopropyl alcohol and water. Fig. 5 shows the yield of andrographolide in different solvents. UAE in these solvents was carried out in a glass vessel for 10 min at 212 W using 1:40 solid to solvent ratio. Among the various solvents investigated, highest extraction yield was seen in methanol followed by 50% ethanol; whereas, water showed minimum yield. Andrographolide is sparingly soluble in water due to which a reduced yield is expected to be obtained. Methanol gave the highest yield of andrographolide because of its high polarity, less viscosity, and surface tension. Although methanol is not regarded as a safe solvent, extraction in methanol was performed to determine its AP extracting property (Hemwimol et al., 2006). Thus extraction was not carried out in methanol for further experiments inspite of obtaining a higher yield in it. Various concentrations of aqueous ethanol were also 25 20 Yield of AP (mg/g)
Ultrasound bath having a dual frequency, operating at 22 kHz and 40 kHz was used for extraction. Experiments were carried out at all 15 positions under previously mentioned conditions at both these frequencies. Results showed a higher yield (22.10 mg/g at position 11) at 22 kHz whereas a lower yield (17.91 mg/g at position 11) was seen at 40 kHz. The energy released by implosion of bubbles generated by ultrasound is sufficient to cause cell wall disruption and further release of its content into the solvent (Vilkhu et al., 2008). This indicates that UAE is a physical process. At high frequency the vapor pressure of solvent rises. The cavitation bubbles generated might thus be filled with the solvent which causes them to implode with lesser intensity. Also it is difficult for a liquid to generate voids at high frequency. This creates an unfavorable condition for cavitation to occur leading to the formation of fewer cavitation bubbles (Viet et al., 2014). All these reasons possibly attribute to an increased extraction yield at 22 kHz. Power dissipation was then calculated at both the frequencies. It was seen that 62.14 W of power was dissipated at 22 kHz and 44.46 W at 40 kHz which also supports a higher yield at 22 kHz than 40 kHz.
15 10 5 0
water
IPA
100% EtOH 75% EtOH 50% EtOH 25% EtOH Solvents
Fig. 5. Yield of andrographolide in various solvents.
316
P.R. Rao, V.K. Rathod / Industrial Crops and Products 66 (2015) 312–318
32
25
Yield of AP (mg/g)
Yield of AP ( mg/g)
20 15 10 5 0
0
4
8
12 Time (min)
16
20
24
30
28
26
30
screened to carry out extraction of AP. The highest extraction yield was obtained in 50% ethanol. This percentage of solvent mixture provides the most favorable polarity for maximum extraction of AP. Also the presence of water causes swelling of plant material thus increasing the solid-solvent contact. But the use of high amount of water (more than 60%) promotes the production of free radicals due to ultrasound induced dissociation of water. The presence of free radicals causes oxidative reactions to coexist with extraction which in turn reduces the extraction yield (Kanthale et al., 2003). This might be the reason for obtaining of comparatively lower yield of AP at water concentration of more than 50%. Andrographolide has a large hydrocarbon moiety along with some polar functions which makes it better soluble in ethanol (Wongkittipong et al., 2004). Thus further experiments were carried out in 50% ethanol. 4.7. Effect of ultrasound exposure time To avoid excessive use of ultrasound energy it is mandatory to determine the optimum time of extraction. UAE of A. paniculata was carried out for 4, 6, 8, 10, 14, 18, and 20 min at 212 W power in 50% ethanol as solvent. Fig. 6 depicts the effect of time on extraction yield of andrographolide. It was seen that until 10 min, yield of andrographolide increased rapidly after which there was no significant increase in yield up to 20 min. Almost all of andrographolide was extracted in the solvent within 10 min of ultrasound exposure. Hence 10 min was chosen as extraction time for further experiments. 4.8. Solute to solvent ratio The amount of solvent available for solute to leach out is an important factor affecting the extraction to prevent the immoderate use of solvent. The effect of solute to solvent ratio on extraction yield of andrographolide was investigated. The results are shown in Fig. 7. It was observed that the yield of andrographolide increased 35
Yield of AP (mg/g)
30 25 20 15 10 5 0
1:20
1:30
1:40
1:50
1:60
Solute to solvent ratio Fig. 7. Effect of solid to solvent ratio on extraction yield of andrographolide.
40
50
60
Temperature (° C)
Fig. 6. Effect of ultrasound time on extraction yield of andrographolide.
Fig. 8. Effect of temperature on extraction yield.
when the solid to solvent ratio was increased from 1:20 to 1:40 and then until 1:60 ratio no significant increase in the extraction yield was seen. When the amount of solvent increases a larger concentration gradient between solute and solvent exists, hence the mass transfer increases. Once there is sufficient amount of solvent available for extraction, further addition of solvent does not affect the extraction yield. More solid particles cause scattering of ultrasound waves thus reducing the ultrasound energy transferred into the vessel (Gogate, 2008). An increase in cavitation threshold is found to occur with more solid contaminants. This results in reduced ultrasound intensity and poor cavitation conditions. Hence optimum yield of AP was obtained at higher solid to solvent ratio (Kulkarni and Rathod, 2014b). As there was a marginal change in extraction yield after 1:40 ratio, this was taken as optimized ratio and used for all further experiments. 4.9. Effect of temperature Extraction temperature adds to the energy and cost consumption and hence must be optimized. Extraction was carried out at different bath temperatures of 30 ◦ C, 40 ◦ C, 50 ◦ C, and 60 ◦ C. Extraction yield of AP was found to be optimum at 40 ◦ C after which up to 60 ◦ C there was no significant improvement in yield as shown in Fig. 8. An increase in temperature increases the solubility of AP in the solvent, reduces the solvent viscosity and density resulting in an increase in mass transfer. As an outcome of these effects extraction yield increases (Charpe and Rathod, 2012). At lower temperature although fewer bubbles are formed, they implode with high intensity. Conversely at higher temperature more bubbles are formed which implode with lesser intensity resulting in reduced cavitation and lower extraction yield (Vetal et al., 2013). These might be the possible reasons for an optimum extraction yield at 40 ◦ C. 4.10. Effect of power input Cost per unit volume of the extraction process escalates with the use of large amount of power by ultrasound. Any equipment cannot work at 100% efficiency and loss of energy is bound to occur in the form of heat. Therefore, optimizing the power consumption for any process is crucial to make it cost effective and avoid wastage of energy. The ultrasound bath was operated at seven different powers ranging from 45 W to 212 W at 22 kHz. The experiments were carried out under previously mentioned conditions at the following powers: 45, 65, 88, 111, 134, 155, 185, and 212 W. The result obtained is illustrated in Fig. 9. It was observed that when power was increased from 45 W to 212 W, there was a marked increase in extraction yield from 17.96 mg/g to 28.73 mg/g. The significant rise in yield can be attributed to the fact that as the power was increased from 45 W to 200 W ultrasonic waves with larger ampli-
Yield of AP (mg/g)
P.R. Rao, V.K. Rathod / Industrial Crops and Products 66 (2015) 312–318
35 30 25 20 15 10 5 0
317
Table 1 Power dissipation at 15 positions of the ultrasound bath. Power dissipation in Watt
45
65
88
111
134
155
200
212
Power (W) Fig. 9. Effect of power input on extraction yield.
Position
65 W
88 W
111 W
134
155 W
200 W
212 W
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
18.27 21.93 22.84 21.09 24.67 20.1 19.19 23.76 22.84 20.1 24.67 19.19 18.27 21.09 21.93
21.93 25.58 27.22 25.58 28.33 22.84 21.93 29.29 27.22 23.76 29.29 22.84 21.09 24.67 26.5
27.41 33.81 34.72 32.9 36.55 31.07 30.15 35.64 35.64 31.98 36.55 31.07 29.29 31.98 33.81
34.72 39.29 40.21 39.29 42.03 36.55 35.64 42.95 41.12 37.46 42.95 36.55 35.64 38.8 40.21
39.29 43.86 44.78 43.86 47.5 41.12 40.21 46.6 45.69 42.03 47.5 41.12 38.8 42.95 44.78
48.43 53.05 54.83 53.05 56.66 50.26 49.35 56.66 54.83 51.17 56.66 50.26 48.43 52.09 53.91
52.09 56.66 57.57 56.66 61.23 53.91 53.05 59.4 58.07 54.83 62.14 53.91 52.09 55.74 57.57
Table 2 Ratio between yield of AP and power dissipation at 11th position.
Fig. 10. Plot of position of vessel vs ratio of yield of AP and power dissipation.
tude travel through the solvent thus creating more bubbles which collapse violently. The violent bubble collapse causes the molecules to mix better, thereby improving the mass transfer rate (Zhang et al., 2008). After 134 W of power input, until 212 W, there was a marginal difference in the rise of extraction yield. This might be because nearly all molecules of andrographolide would have been extracted in the solvent at 134 W. Power dissipation studies were performed calorimetrically, within the extraction vessel to calculate the actual power consumed for extraction. Power dissipated was found to be 24.67, 29.29, 36.55, 42.95, 47.5, 56.66, and 62.14 W for 65, 88, 111, 134, 155, 185, and 212 W, respectively. Although there was a noticeable increase in power dissipation after 134 W, the increase in extraction yield was marginal. The ratio of extraction yield and power dissipation at various powers, at the 11th position was also calculated. Table 2 illustrates the ratio between yield of AP and power dissipation at various powers, at 11th position. The ratios remained fairly similar over different powers and a maximum ratio was obtained at 65 W. But the extraction yield of AP at 65 W was only 19.54 mg/g as compared to 27.9 mg/g at 134 W. Therefore, considering the vast difference between the extraction yields, 134 W was taken to be the optimal power. As 134 W was found to be the optimum power, the ratio extraction yield and power dissipation at 134 W was calculated over all 15 positions. Fig. 10 shows ratio of yield of AP and power dissipation at 134 W, at 15 different positions. Maximum ratio was found at position 11 which supports earlier discussion of selection of 11th position. Furthermore, the power dissipated in the extraction vessel at all 15 positions, at various powers was also determined by calorimetric studies, based on temperature variation within the extraction vessel. Extraction vessel was immersed at a depth of 2.54 cm from base of the bath. Extraction was carried out for 18 min, using 100% duty cycle, with 3500 mL of water as the bulk of solvent in the bath, considering the bath to be an adiabatic system. Rise in temperature after 18 min was noted and the power dissipated was calculated. The result for power dissipation study is tabulated in Table 1. The result showed that power dissipation varied significantly at all 15 positions. This suggested that acoustic power utilized for extraction of AP at these positions was different. Highest power was dissipated at the 11th position for every power investigated thus confirming
Extraction yield Of AP (mg/g)
Power (Watt)
Power dissipation (Watt)
Yield of AP/power dissipation
17.98 19.54 21.32 23.84 27.91 28.37 28.52 28.77
45 65 88 111 134 155 200 212
21.55 24.67 29.29 36.55 42.95 47.5 56.66 62.14
0.78 0.79 0.73 0.65 0.65 0.59 0.5 0.46
the reason behind getting a highest yield at this position. Variation in the power dissipated within the reaction vessel evinced a variation in cavitation intensity in the bath. 5. Conclusion From the above performed experiments it can be concluded that parameters like choice of solvent, time of ultrasound exposure, solute to solvent ratio, position and depth of extraction vessel, frequency of sound waves and input power played a pivotal role in escalating the extraction yield. Extraction of andrographolide from A. paniculata L. was carried out using soxhlet and ultrasound assisted extraction. Soxhlet extraction gave a yield of 42.46 mg/g of andrographolide. Highest extraction yield of andrographolide of 27.97 mg/g at 22 kHz frequency and 134 W of power was obtained in 10 min of ultrasound exposure with 1:40 solid to solvent ratio, in 50% ethanol at position 11 and 2.54 cm depth from bottom of the bath. 65.87% extraction yield was obtained by UAE as compared to soxhlet extraction, in 10 min. Further power dissipation study showed that the power dissipated within the extraction vessel at different locations in the bath was different. Thus ultrasound intensity throughout the bath was found to be non-uniform and it is hence imperative to optimize all these parameters to obtain good extraction yield. References Caceres, D.D., Hancke, D.D., Burgos, R.A., 1997. Prevention of common colds with Andrographis paniculata dried extract: a pilot double blind trial. Phytomedicine 4, 101–104. Charpe, T.W., Rathod, V.K., 2012. Process intensification extraction of glycyrrhizic acid from licorice root using ultrasound: process intensification studies. Chem. Eng. Process. Process Intensif. 54, 37–41, http://dx.doi.org/10.1016/j.cep.2012.01.002. Csoka, A.B., Szyf, M., 2009. Epigenetic side-effects of common pharmaceuticals: A potential new field in medicine and pharmacology. Med. Hypotheses 73, 770–780, http://dx.doi.org/10.1016/j.mehy.2008.10.039.
318
P.R. Rao, V.K. Rathod / Industrial Crops and Products 66 (2015) 312–318
De Castro, M.D.L., Capote, F.P., 2006. Analytical Applications of Ultrasound. Elsevier. Dhiman, A., Goyal, J., Sharma, K., Nanda, A., Dhiman, S., 2012. Review on medicinal prespectives of Andrographis paniculata Nees. J. Pharm. Sci. Innov. 1, 1–4. Gogate, P.R., Marie, A., Pandit, A.B., 2003. Some aspects of the design of sonochemical reactors. Ultrason. Sonochem. 10, 325–330, http://dx.doi.org/10.1016/S1350-4177(03)103-2. Gogate, P.R., 2008. Cavitational reactors for process intensification of chemical processing applications: a critical review. Chem. Eng. Process. 47, 515–527, http://dx.doi.org/10.1016/j.cep.2007.09.014. Guo, W., Liu, W., Chen, G., Hong, S., Qian, C., Xie, N., Sun, Y., Xu, Q., 2012. International immunopharmacology water-soluble andrographolide sulfonate exerts anti-sepsis action in mice through down-regulating p38 MAPK, STAT3 and NF-B pathways. Int. Immunopharmacol. 14, 613–619, http://dx.doi.org/10.1016/j.intimp.2012.09.002. Hemwimol, S., Pavasant, P., Shotipruk, A., 2006. Ultrasound-assisted extraction of anthraquinones from roots of Morinda citrifolia. Ultrason. Sonochem 13, 543–548, http://dx.doi.org/10.1016/j.ultsonch.2005.09.009. Jadhav, D., Rekha, B.N., Gogate, P.R., Rathod, V.K., 2009. Extraction of vanillin from vanilla pods: a comparison study of conventional soxhlet and ultrasound assisted extraction. J. Food Eng. 93, 421–426, http://dx.doi.org/10.1016/j.jfoodeng.2009.02.007. Ji, H., Li, X., Zhang, H., 2009. EMBO Report194–200. Kanthale, P.M., Gogate, P.R., Pand it, A.B., Wilhelm, A.M., 2003. Mapping of an ultrasonic horn: link primary and secondary effects of ultrasound. Ultrason. Sonochem 10, 331–335, http://dx.doi.org/10.1016/S1350-4177(03)104-4. Kaur, K., Shivhare, U., Basu, S., Vijaya Raghavan, G.S., 2012. Kinetics of extraction of -carotene from tray dried carrots by using supercritical fluid extraction technique. Food Nutr. Sci. 3, 591–595, http://dx.doi.org/10.4236/fns.2012.35081. Kulkarni, V.M., Rathod, V.K., 2014a. Mapping of an ultrasonic bath for ultrasound assisted extraction of mangiferin from Mangifera indica leaves. Ultrason. Sonochem 21, 606–611, http://dx.doi.org/10.1016/j.ultsonch.2013.08.021. Kulkarni, V.M., Rathod, V.K., 2014b. Extraction of mangiferin from Mangifera indica leaves using three phase partitioning coupled with ultrasound. Ind. Crop. Prod., http://dx.doi.org/10.1016/j.indcrop.2013.10.032. Mortazavi, S.V., Eikani, M.H., Mirzaei, H., Jafari, M., Golmohammad, F., 2010. Extraction of essential oils from Bunium persicum Boiss. using superheated water. Food Bioprod. Process. 88, 222–226. Pugin, B., 1987. Qualitative characterization of ultrasound reactors for heterogeneous sonochemistry. Sonochemistry 25, 8–11. Rafie, Z., Jafari, S.M., Alami, M., Khomeiri, M., 2011. Microwave assisted extraction of phenolic compounds from olive leaves; a comparison with maceration. J. Anim. Plant Sci. 21 (4), 738–745. Rajagopal, S., Kumar, R.A., Deevi, D.S., Satyanarayana, C., Rajagopalan, R., 2003. Andrographolide: a potential cancer therapeutic agent isolated from Andrographis paniculata. J. Exp. Therapeut. Oncol. 3, 147–158. Santos, H.M., Lodeiro, C., 2009. The pgffffower of ultrasound, in: ultrasound. Chem. Anal. Appl., 1–16.
Shah Megha, V., Rohit Minal, C., 2013. Novel techniques for isolation and extraction of phyto-constituents from herbal plants. Am. J. Phytomedicine Clin. Ther. 3, 338–350. Shirsath, S.R., Sonawane, S.H., Gogate, P.R., 2012. Intensification of extraction of natural products using ultrasonic irradiations – a review of current status. Chem. Eng. Process. Process Intensif. 53, 10–23, http://dx.doi.org/10.1016/j.cep.2012.01.003. Sravan Kumar, S., Balasubrahmanyam, A., Shyam Sundar, P., Gogate, P.R., Deshpande, V.D., Shukla, S.R., Pandit, A.B., 2009. Characterization of sonochemical reactor for physicochemical transformations. Ind. Eng. Chem. Res. 48, 9402–9407. Sutkar, V.S., Gogate, P.R., 2009. Design aspects of sonochemical reactors: techniques for understanding cavitational activity distribution and effect of operating parameters. Chem. Eng. J. 155, 26–36, http://dx.doi.org/10.1016/j.cej.2009.07.021. Taghvaei, M., Jafari, S.M., Assadpoor, E., Nowrouzieh, S., Alishah, O., 2014. Optimization of microwave-assisted extraction of cottonseed oil and evaluation of its oxidative stability and physicochemical properties. Food Chem. 160, 90–97, http://dx.doi.org/10.1016/j.foodchem.2014.03.064. Tao, C., Wu, D., Zhang, Q., Sun, D., 2014. Ultrasound-assisted extraction of phenolics from wine lees: modeling, optimization and stability of extracts during storage. Ultrasonics. Sonochem. 21, 706–715, http://dx.doi.org/10.1016/j.ultsonch.2013.09.005. Vasu, S., Palaniyappan, V., Badami, S., 2010. A novel microwave assisted extraction for isolation of andrographolide from Andrographis paniculata and its in vitro antioxidant activity. Nat. Prod. Res. 24 (16), 1560–1567, http://dx.doi.org/10.1080/14786419.2010.495071. Vetal, M.D., Lade, V.G., Rathod, V.K., 2013. Extraction of ursolic acid from Ocimum sanctum by ultrasound: process intensification and kinetic studies. Chem. Eng. Process. Process Intensif. 69, 24–30, http://dx.doi.org/10.1016/j.cep.2013.01.011. Viet, K., Tran, B., Kimura, T., Kondo, T., Koda, S., 2014. Quantification of frequency dependence of mechanical effects induced by ultrasound. Ultrason. Sonochem. 21, 716–721, http://dx.doi.org/10.1016/j.ultsonch.2013.08.018. Vilkhu, K., Mawson, R., Simons, L., Bates, D., 2008. Applications and opportunities for ultrasound assisted extraction in the food industry – a review. Innov. Food Sci. Emerg. Technol. 9, 161–169, http://dx.doi.org/10.1016/j.ifset.2007.04.014. Wang, J., Zhao, Y., Tian, Y., Yan, C., Guo, C., 2013. Ultrasound-assisted extraction of total phenolic compounds from Inula helenium. Sci. World J., 1–5. Wongkittipong, R., Prat, L., Damronglerd, S., Gourdon, C., 2004. Solid–liquid extraction of andrographolide from plants – experimental study, kinetic reaction and model. Sep. Purif. Technol. 40, 147–154, http://dx.doi.org/10.1016/j.seppur.2004.02.002. Zhang, Z., Wang, L., Li, D., Jiao, S., Dong, X., Mao, Z., 2008. Ultrasound-assisted extraction of oil from flaxseed. Sep. Purif. Technol. 62, 192–198, http://dx.doi.org/10.1016/j.seppur.2008.01.014.
Contents lists available at ScienceDirect
Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Mapping study of an ultrasonic bath for the extraction of andrographolide from Andrographis paniculata using ultrasound Priyanka R. Rao, 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
Article history: Received 13 August 2014 Received in revised form 11 November 2014 Accepted 26 November 2014 Keywords: Andrographolide Ultrasound assisted extraction Ultrasound bath Mapping
a b s t r a c t In the present work, extraction of andrographolide from Andrographis paniculata L. was carried out by mapping an ultrasound bath to identify the active and passive zones within it. This helps in accurately placing the extraction vessel in the bath for obtaining a maximum yield. Various parameters of extraction like choice of solvent, time of ultrasound exposure, solute to solvent ratio, temperature, position and depth of extraction vessel, the frequency used, and input power were investigated. Aluminum foil weight loss test was also performed at different positions and its results were compared with the results obtained for extraction at same positions. Results revealed a similar trend in variation for both the investigations. Highest yield of andrographolide obtained was 27.97 mg/g, at position 11, at a depth of 2.54 cm, in only 10 min of ultrasound irradiation, using 1:40 solute to solvent ratio, at a frequency of 22 kHz, utilizing 134 W of power with 50% ethanol as the solvent. Calorimetric studies were also carried out within the reaction vessel at these positions, to determine the actual power being utilized for extraction. Power dissipated at these locations differed from each other and maximum power was found to be dissipated at position 11. This study implies the significance of the above mentioned parameters in mapping of an ultrasound bath which can further be used for extraction of biomolecules from natural products. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Although medicinal plants have been widely used since the ancient times, the identity of compounds that showed the desired therapeutic effect remained unknown until a few decades back. Development in the field of medicinal chemistry and pharmacology led to their structural elucidation and further helped in understanding their biological activities in the human body. The chemists synthesized these compounds thus reducing the cost of their production (Ji et al., 2009). However, there are reports of adverse effects caused by synthesized drugs, which has revived the interest in the use of natural compounds (Csoka and Szyf, 2009). Andrograhis paniculata is a popular Ayurvedic and Chinese household remedy for the common cold, digestive issues, upper respiratory tract infections, and flu. Andrographolide is a bitter compound extracted from the leaves and stem of an herbaceous plant called A. paniculata (Caceres et al., 1997). It is a major diterpene lactone showing
Abbreviations: AP, andrographolide; UAE, ultrasound assisted extraction; MAE, microwave assisted extraction; SCFE, supercritical fluid extraction; ASE, assisted solvent extraction. ∗ Corresponding author. Tel.: +91 22 33612020; fax: +91 22 33612010/2020. E-mail address: [email protected] (V.K. Rathod). http://dx.doi.org/10.1016/j.indcrop.2014.11.046 0926-6690/© 2014 Elsevier B.V. All rights reserved.
multifarious activities like anti-pyretic, anti-inflammatory, antioxidant, anticancer, and hepatoprotective (Dhiman et al., 2012). It inhibits the in vitro proliferation of different tumor cell lines, representing various types of cancers. Andrographolide shows direct anticancer activity on cancer cells by cell-cycle arrest at G0/G1 phase through induction of cell-cycle inhibitory protein p27 and decreased expression of cyclin-dependent kinase 4 (CDK4). The immunostimulatory activity of andrographolide is seen by an increased proliferation of lymphocytes and production of interleukin-2 (Rajagopal et al., 2003). Recently water soluble andrographolide sulfonate, exerting anti-sepsis action in mice has been synthesized (Guo et al., 2012). Traditional extraction techniques have the drawbacks of longer extraction time, higher extraction temperature, increased solvent consumption, and mass transfer resistance. Novel extraction techniques like ultrasound assisted extraction (UAE), microwave assisted extraction (MAE), supercritical fluid extraction (SCFE), and accelerated solvent extraction (ASE) overcome these pitfalls (Shah Megha and Rohit Minal, 2013). Several reports suggest the advantages of these modern extraction techniques over conventional techniques to extract bioactives from natural sources. A comparative study of superheated water extraction (SWE) with conventional volatile isolation methods like hydrodistillation and soxhlet extraction on Buniumpersicum Boiss shows SWE to be a quicker and selective method with respect
P.R. Rao, V.K. Rathod / Industrial Crops and Products 66 (2015) 312–318
to the valuable oxygenated components (Mortazavi et al., 2010). MAE is popularly used as an advantageous method for the extraction of natural products. Report by Rafiee et al. indicates that MAE gives a higher extraction yield as compared to the conventional maceration method, for extraction of phenolic compounds from olive leaves (Rafiee et al., 2011). MAE is also reported to reduce the extraction time and solvent consumption in extraction of cottonseed oil from whole cottonseed (Taghvaei et al., 2014). It causes plant cells to burst due to localized internal superheating followed by leaching out of the biomolecules. The cell bursting phenomenon facilitates the entry of extracting solvent to solubilize out the target compound, thus leading to faster and more efficient extraction (Vasu et al., 2010). MAE has certain limitations that it cannot be used for heat sensitive materials and involves high investment costs. Most of these methods have some similarities with respect to solvent volume, extraction time, and extraction efficiency. SFE is one of the most promising extraction processes. It provides higher selectivity, shorter extraction times and does not use toxic organic solvents (Kaur et al., 2012). However, SCFE and ASE require greater financial investment and the presence of water in samples can cause hindrances in both techniques. UAE has been previously used for extracting biomolecules from a number of natural products. The basis of UAE is a well-known phenomenon called cavitation. As ultrasound waves pass through liquid medium it generates small vacuum filled cavitation bubbles within it. With an increase in the number of expansion and compression cycles within the medium, these bubbles implode abruptly. There is an increase in local temperature and pressure which facilitates greater solvent circulation, deeper solvent penetration within the cellular material and enhanced mass transfer, thus enhancing the extraction rate (Charpe and Rathod, 2012). Apart from considerable reduction in extraction time, the major benefits of UAE are that it increases the yield, requires a simple and easy to operate apparatus which helps in easy extraction of thermolabile compounds, without any degradation (Wang et al., 2013). Indirect sonication using an ultrasound bath with one or more transducers fitted at the bottom, is extensively used to extract bioactives from natural compounds (Tao et al., 2014). It is essential to know the active and passive zones in the bath to accurately place the extraction vessel in it which helps in obtaining a maximum extraction yield. The yield of ultrasound extraction depends on the location of transducers in the bath, power input, frequency of ultrasound, position of the extraction vessel, and its geometry (Santos and Lodeiro, 2009). The present report, concentrates on the optimization of the above mentioned parameters, which effect the extraction of andrographolide from A. paniculata. One of the methods of quantifying cavitational activity distribution is by investigating the secondary effects generated by cavitation. Secondary effects indicate the effects generated after bubble collapse (e.g., aluminum foil test, iodine dosimetry, electrochemical methods, etc.) (Sutkar and Gogate, 2009). So, to investigate the physical effects of cavitation in the bath at various positions, a comparison of aluminum foil weight loss test with that of andrographolide extraction is performed. To the best of our knowledge there are no reports available on mapping of an ultrasonic bath for extraction of andrographolide from A. paniculata. Furthermore, parameters like the choice of solvent, ultrasound irradiation time, solute to solvent ratio and the effect of temperature on extraction of andrographolide are also examined.
2. Materials and methods 2.1. Material Powdered leaves of A. paniculata was purchased from a local store in Mumbai. An average powder size of 0.6–1.1 mm was used.
313
The powder was sun dried and checked for its moisture content. The moisture content was found to be approximately, 7%. The standard for andrographolide (Aldrich 365,645–100 mg, 98% HPLC grade) was procured from Sigma–Aldrich. Acetonitrile and acidified water were used as the mobile phase for HPLC. Acetonitrile was of HPLC grade and ethanol used as the solvent was of AR grade. These solvents were purchased from high media, Mumbai, India for the experiments. Distilled water used as one of the mobile phases, was obtained from Millipore Milli Q 50HPLC grade. 2.2. Analysis of andrographolide Separation of andrographolide from its extract was done using HPLC (Agilent 1260 infinity high performance auto sampler) equipped with C18 column. Mobile phase used for HPLC analysis comprised of acetonitrile and acidified water in the ratio 60:40 v/v. Isocratic elution was used to perform HPLC analysis where the flow rate was set at 1.0 mL/min throughout the run with an injection volume of 5 L. The peak for andrographolide was detected at 223 nm with a retention time of 7.2 min. 3. Experimental work 3.1. Soxhlet extraction Soxhlet extraction of A. paniculata was carried out for 4 h using aqueous ethanol as the solvent. The dried sample was packed in a sample tube, above the extraction solvent. On heating, the solvent evaporated and cooled into a liquid in a condenser after which it re-entered the sample tube. This continued for several hours until the extraction was complete. Andrographolide content was then estimated using HPLC. 3.2. Ultrasound assisted extraction of A. paniculata Ultrasound assisted extraction of A. paniculata was carried out by indirect sonication in a dual frequency operated ultrasound bath operating at 25 and 40 kHz (model no. 6.5l 250H/DTC/DF). A rectangular tank having internal dimensions 300 × 150 × 150 mm, fitted with five transducers at the bottom, arranged in a zigzag manner was used. Fig. 1 shows a schematic representation of the top view of ultrasound bath. The rated power of the bath was 222 W. All experiments were performed thrice and their average values with standard deviations are reported. Ultrasound assisted extraction was carried out in a cylindrical flat bottom glass vessel of a specific height and diameter, at various positions and heights to obtain maximum yield (Kulkarni and Rathod, 2014). Extraction was carried out for 10 min at a temperature of 27 ± 4 ◦ C. Effect of parameters like choice of solvent, ultrasound exposure time, temperature, solute to solvent ratio, position of the vessel in the bath, depth of the vessel, frequency of sound waves, and power input, on the extraction yield of andrographolide was investigated. A number of solvents including water, various concentrations of ethanol, methanol, and isopropyl alcohol were screened for extraction. Solid to solvent ratio of 1:40 was maintained throughout the study. The variation in cavitation activity at various positions in the bath was then confirmed by aluminum foil test. A piece of accurately weighed aluminum foil was taken in the glass vessel and sonicated in similar conditions like A. paniculata, at same positions. The weight loss of aluminum foil in the vessel at these positions was compared with the extraction yield of andrographolide at identical positions (Pugin, 1987). Also, power dissipation studies at these positions, to estimate the amount of power actually being utilized for extracting andrographolide, were done in detail. Calorimetric studies were performed for the same purpose with 3.5 liters of water in the bath.
314
P.R. Rao, V.K. Rathod / Industrial Crops and Products 66 (2015) 312–318
Fig. 1. A schematic representation of the top view of ultrasound bath.
4.1. Soxhlet extraction The extract was collected after every 1 h and analyzed for AP content using HPLC. The maximum yield of andrographolide obtained after 4 h of soxhlet extraction was 42.46 mg/g. This yield was considered to be a reference for comparison of yield of soxhlet extraction with that of UAE. 4.2. Effect of position of extraction vessel To examine the variation in cavitation activity at different locations in the bath, it is of prime importance to study the effect of vessel position in the bath. Use of multiple transducers reportedly gives more uniform and intense cavitational activity as compared to a single transducer irradiation (Shirsath et al., 2012). Hence a bath fitted with multiple transducers was used. The entire bath was divided in 15 uniform sections where the extraction vessel was placed, as shown in Fig. 1. With the use of flat bottom vessel, minimum loss of ultrasound wave is expected to occur. Therefore, a flat bottom vessel with 1.3 cm diameter and 17.5 cm height already optimized by Kulkarni and Rathod (2014a) was used. The vessel was covered with aluminum foil to avoid loss of solvent. It was then placed at a depth of 1 cm from the bottom of the ultrasound bath. Extraction was carried out for 10 min at 22 kHz and 40 kHz using 1:40 solute to solvent ratio with maximum power input of 212 W. After 10 min the extract was filtered and analyzed for andrographolide content by HPLC. The vessel was then placed at all the remaining positions, one at a time, under similar conditions and analyzed for andrographolide content. It was observed that the highest yield of andrographolide was obtained at position 11 followed by position 5 and 8 at both frequencies as shown in Fig. 2. The yield of AP obtained at position 11 at 22 kHz and 40 kHz was 22.10 mg/g and 17.91 mg/g, respectively. Positions 5, 8, and 11 have three transducers in their locality which produce a synergistic cavitation effect thus creating conducive conditions for higher yield in the vessel at these positions in comparison to others. Also, literature suggests that cavitation phenomenon is higher in the vertical plane of transducers than the horizontal or radial plane (Sravan Kumar et al., 2009). Thus from the collated results, it can be concluded that
the proximity of extraction vessel in the vertical plane of the transducers is reflected by an increase in extraction yield. Based on our results position 11 was considered to be the most suitable position in the bath to perform extraction. 4.3. Aluminum foil weight loss test Aluminum foil weight loss test was done to evaluate the distribution of cavitation effect throughout the bath. At low frequencies the mechanical effect of ultrasound is predominant (Viet et al., 2014). Thus, 22 kHz frequency was selected for the experiment. A piece of aluminum foil was weighed and subjected to similar conditions of sonication as that for crude material, i.e., 22 kHz frequency, 212 W power,10 min, 50% ethanol, 1:40 solid to solvent ratio. The foil was again weighed after 10 min of ultrasound irradiation (Pugin, 1987). This study was performed at all 15 positions of the bath at a depth of 1 cm from the base of the bath. Results showed that a maximum foil weight loss was seen at position 11, followed by position 5, and 8, connoting that cavitation intensity was supreme at these positions. The result of this investigation was compared with that of the finding of position study experiment. Parallel trends in both the graphs were seen when plotted and compared. Fig. 3 illustrates the obtained results. The highest yield of andrographolide as well as a maximum foil weight loss, both was obtained at position 11, corroborating that cavitation intensity
25
Yield of AP (mg/g)
4. Results and discussion
20 15 10 5 0 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15
Position of vess el Fig. 2. Effect of position of extraction vessel in the ultrasound bath.
25
35
120
20
30
90
15
60
10
30
5
0
0 1
2
3
4
5
6 7 8 9 10 11 12 13 14 15 Position of vessel
Yield of AP in mg/g
150
Yield of AP (mg/g)
Alumihnum foil weight loss (mg)
P.R. Rao, V.K. Rathod / Industrial Crops and Products 66 (2015) 312–318
25 20 15 10 5 0 1
Yield of AP (mg/g)
315
2
3
4
5
6
7
8
9 10 11 12 13 14 15
Position of vess el
Aluminum foil weight loss (mg)
Fig. 3. Comparison of aluminum foil weight loss study and extraction yield of AP.
1.01 cm
2.54 cm
5.08 cm
Fig. 4. Effect of depth of extraction vessel on extraction yield.
was non uniform throughout the bath with a maximum intensity at position 11.
obtaining a maximum yield at 2.54 cm, which is closer to 3.4 cm, is justified.
4.4. Effect of frequency 4.6. Choice of solvent
4.5. Effect of depth of extraction vessel Depth at which the extracting vessel is placed in the bath plays a significant role in affecting the final yield. Increasing the distance between extracting vessel and transducers brings down the cavitation activity (Kanthale et al., 2003). Hence it is advantageous to establish an optimum height at which the vessel must be kept to obtain maximum extraction. Keeping the previously mentioned experimental conditions constant, the depth of the vessel from bottom of the bath was varied between 1.27 cm and 5.08 cm. Three different depths were selected (1.27 cm, 2.54 cm, and 5.08 cm) and extraction was carried out in a flat bottom glass vessel keeping it at all 15 positions. The results observed are illustrated in Fig. 4. Maximum yield of AP was obtained at a depth of 2.54 cm in all the vessels. The yields at position 11, 5, and 8 were comparatively higher. Ultrasound passes through water contained in the bath in form of waves. Each wave has its maxima and minima. Ultrasound irradiation at maxima is very powerful. For transducers operating at 22 kHz, the wavelength of ultrasound in water is equal to half the product of velocity of sound in water (1500 m/s) and the frequency at which the bath is operated (22 kHz). Thus the maxima of ultrasound wave are likely to be around 3.4 cm (De Castro and Capote, 2006). Hence
Choice of solvent is a critical parameter which affects the extraction yield. The solubility of a compound in a particular solvent increases with solvent polarity but reduces with an increase in solvent viscosity, surface tension and vapor pressure. Also, it becomes difficult to attain cavitation with an increase in surface tension and vapor pressure of the solvent (Gogate et al., 2003). Polar solvents assist in increasing the cell wall permeability causing an improved contact between the solvent and solid, thereby increasing the extraction yield (Jadhav et al., 2009). The solvents were selected on the basis of solubility of AP in them. Various solvents screened were methanol, different concentrations of ethanol (100%, 75%, 50%, and 25%), isopropyl alcohol and water. Fig. 5 shows the yield of andrographolide in different solvents. UAE in these solvents was carried out in a glass vessel for 10 min at 212 W using 1:40 solid to solvent ratio. Among the various solvents investigated, highest extraction yield was seen in methanol followed by 50% ethanol; whereas, water showed minimum yield. Andrographolide is sparingly soluble in water due to which a reduced yield is expected to be obtained. Methanol gave the highest yield of andrographolide because of its high polarity, less viscosity, and surface tension. Although methanol is not regarded as a safe solvent, extraction in methanol was performed to determine its AP extracting property (Hemwimol et al., 2006). Thus extraction was not carried out in methanol for further experiments inspite of obtaining a higher yield in it. Various concentrations of aqueous ethanol were also 25 20 Yield of AP (mg/g)
Ultrasound bath having a dual frequency, operating at 22 kHz and 40 kHz was used for extraction. Experiments were carried out at all 15 positions under previously mentioned conditions at both these frequencies. Results showed a higher yield (22.10 mg/g at position 11) at 22 kHz whereas a lower yield (17.91 mg/g at position 11) was seen at 40 kHz. The energy released by implosion of bubbles generated by ultrasound is sufficient to cause cell wall disruption and further release of its content into the solvent (Vilkhu et al., 2008). This indicates that UAE is a physical process. At high frequency the vapor pressure of solvent rises. The cavitation bubbles generated might thus be filled with the solvent which causes them to implode with lesser intensity. Also it is difficult for a liquid to generate voids at high frequency. This creates an unfavorable condition for cavitation to occur leading to the formation of fewer cavitation bubbles (Viet et al., 2014). All these reasons possibly attribute to an increased extraction yield at 22 kHz. Power dissipation was then calculated at both the frequencies. It was seen that 62.14 W of power was dissipated at 22 kHz and 44.46 W at 40 kHz which also supports a higher yield at 22 kHz than 40 kHz.
15 10 5 0
water
IPA
100% EtOH 75% EtOH 50% EtOH 25% EtOH Solvents
Fig. 5. Yield of andrographolide in various solvents.
316
P.R. Rao, V.K. Rathod / Industrial Crops and Products 66 (2015) 312–318
32
25
Yield of AP (mg/g)
Yield of AP ( mg/g)
20 15 10 5 0
0
4
8
12 Time (min)
16
20
24
30
28
26
30
screened to carry out extraction of AP. The highest extraction yield was obtained in 50% ethanol. This percentage of solvent mixture provides the most favorable polarity for maximum extraction of AP. Also the presence of water causes swelling of plant material thus increasing the solid-solvent contact. But the use of high amount of water (more than 60%) promotes the production of free radicals due to ultrasound induced dissociation of water. The presence of free radicals causes oxidative reactions to coexist with extraction which in turn reduces the extraction yield (Kanthale et al., 2003). This might be the reason for obtaining of comparatively lower yield of AP at water concentration of more than 50%. Andrographolide has a large hydrocarbon moiety along with some polar functions which makes it better soluble in ethanol (Wongkittipong et al., 2004). Thus further experiments were carried out in 50% ethanol. 4.7. Effect of ultrasound exposure time To avoid excessive use of ultrasound energy it is mandatory to determine the optimum time of extraction. UAE of A. paniculata was carried out for 4, 6, 8, 10, 14, 18, and 20 min at 212 W power in 50% ethanol as solvent. Fig. 6 depicts the effect of time on extraction yield of andrographolide. It was seen that until 10 min, yield of andrographolide increased rapidly after which there was no significant increase in yield up to 20 min. Almost all of andrographolide was extracted in the solvent within 10 min of ultrasound exposure. Hence 10 min was chosen as extraction time for further experiments. 4.8. Solute to solvent ratio The amount of solvent available for solute to leach out is an important factor affecting the extraction to prevent the immoderate use of solvent. The effect of solute to solvent ratio on extraction yield of andrographolide was investigated. The results are shown in Fig. 7. It was observed that the yield of andrographolide increased 35
Yield of AP (mg/g)
30 25 20 15 10 5 0
1:20
1:30
1:40
1:50
1:60
Solute to solvent ratio Fig. 7. Effect of solid to solvent ratio on extraction yield of andrographolide.
40
50
60
Temperature (° C)
Fig. 6. Effect of ultrasound time on extraction yield of andrographolide.
Fig. 8. Effect of temperature on extraction yield.
when the solid to solvent ratio was increased from 1:20 to 1:40 and then until 1:60 ratio no significant increase in the extraction yield was seen. When the amount of solvent increases a larger concentration gradient between solute and solvent exists, hence the mass transfer increases. Once there is sufficient amount of solvent available for extraction, further addition of solvent does not affect the extraction yield. More solid particles cause scattering of ultrasound waves thus reducing the ultrasound energy transferred into the vessel (Gogate, 2008). An increase in cavitation threshold is found to occur with more solid contaminants. This results in reduced ultrasound intensity and poor cavitation conditions. Hence optimum yield of AP was obtained at higher solid to solvent ratio (Kulkarni and Rathod, 2014b). As there was a marginal change in extraction yield after 1:40 ratio, this was taken as optimized ratio and used for all further experiments. 4.9. Effect of temperature Extraction temperature adds to the energy and cost consumption and hence must be optimized. Extraction was carried out at different bath temperatures of 30 ◦ C, 40 ◦ C, 50 ◦ C, and 60 ◦ C. Extraction yield of AP was found to be optimum at 40 ◦ C after which up to 60 ◦ C there was no significant improvement in yield as shown in Fig. 8. An increase in temperature increases the solubility of AP in the solvent, reduces the solvent viscosity and density resulting in an increase in mass transfer. As an outcome of these effects extraction yield increases (Charpe and Rathod, 2012). At lower temperature although fewer bubbles are formed, they implode with high intensity. Conversely at higher temperature more bubbles are formed which implode with lesser intensity resulting in reduced cavitation and lower extraction yield (Vetal et al., 2013). These might be the possible reasons for an optimum extraction yield at 40 ◦ C. 4.10. Effect of power input Cost per unit volume of the extraction process escalates with the use of large amount of power by ultrasound. Any equipment cannot work at 100% efficiency and loss of energy is bound to occur in the form of heat. Therefore, optimizing the power consumption for any process is crucial to make it cost effective and avoid wastage of energy. The ultrasound bath was operated at seven different powers ranging from 45 W to 212 W at 22 kHz. The experiments were carried out under previously mentioned conditions at the following powers: 45, 65, 88, 111, 134, 155, 185, and 212 W. The result obtained is illustrated in Fig. 9. It was observed that when power was increased from 45 W to 212 W, there was a marked increase in extraction yield from 17.96 mg/g to 28.73 mg/g. The significant rise in yield can be attributed to the fact that as the power was increased from 45 W to 200 W ultrasonic waves with larger ampli-
Yield of AP (mg/g)
P.R. Rao, V.K. Rathod / Industrial Crops and Products 66 (2015) 312–318
35 30 25 20 15 10 5 0
317
Table 1 Power dissipation at 15 positions of the ultrasound bath. Power dissipation in Watt
45
65
88
111
134
155
200
212
Power (W) Fig. 9. Effect of power input on extraction yield.
Position
65 W
88 W
111 W
134
155 W
200 W
212 W
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
18.27 21.93 22.84 21.09 24.67 20.1 19.19 23.76 22.84 20.1 24.67 19.19 18.27 21.09 21.93
21.93 25.58 27.22 25.58 28.33 22.84 21.93 29.29 27.22 23.76 29.29 22.84 21.09 24.67 26.5
27.41 33.81 34.72 32.9 36.55 31.07 30.15 35.64 35.64 31.98 36.55 31.07 29.29 31.98 33.81
34.72 39.29 40.21 39.29 42.03 36.55 35.64 42.95 41.12 37.46 42.95 36.55 35.64 38.8 40.21
39.29 43.86 44.78 43.86 47.5 41.12 40.21 46.6 45.69 42.03 47.5 41.12 38.8 42.95 44.78
48.43 53.05 54.83 53.05 56.66 50.26 49.35 56.66 54.83 51.17 56.66 50.26 48.43 52.09 53.91
52.09 56.66 57.57 56.66 61.23 53.91 53.05 59.4 58.07 54.83 62.14 53.91 52.09 55.74 57.57
Table 2 Ratio between yield of AP and power dissipation at 11th position.
Fig. 10. Plot of position of vessel vs ratio of yield of AP and power dissipation.
tude travel through the solvent thus creating more bubbles which collapse violently. The violent bubble collapse causes the molecules to mix better, thereby improving the mass transfer rate (Zhang et al., 2008). After 134 W of power input, until 212 W, there was a marginal difference in the rise of extraction yield. This might be because nearly all molecules of andrographolide would have been extracted in the solvent at 134 W. Power dissipation studies were performed calorimetrically, within the extraction vessel to calculate the actual power consumed for extraction. Power dissipated was found to be 24.67, 29.29, 36.55, 42.95, 47.5, 56.66, and 62.14 W for 65, 88, 111, 134, 155, 185, and 212 W, respectively. Although there was a noticeable increase in power dissipation after 134 W, the increase in extraction yield was marginal. The ratio of extraction yield and power dissipation at various powers, at the 11th position was also calculated. Table 2 illustrates the ratio between yield of AP and power dissipation at various powers, at 11th position. The ratios remained fairly similar over different powers and a maximum ratio was obtained at 65 W. But the extraction yield of AP at 65 W was only 19.54 mg/g as compared to 27.9 mg/g at 134 W. Therefore, considering the vast difference between the extraction yields, 134 W was taken to be the optimal power. As 134 W was found to be the optimum power, the ratio extraction yield and power dissipation at 134 W was calculated over all 15 positions. Fig. 10 shows ratio of yield of AP and power dissipation at 134 W, at 15 different positions. Maximum ratio was found at position 11 which supports earlier discussion of selection of 11th position. Furthermore, the power dissipated in the extraction vessel at all 15 positions, at various powers was also determined by calorimetric studies, based on temperature variation within the extraction vessel. Extraction vessel was immersed at a depth of 2.54 cm from base of the bath. Extraction was carried out for 18 min, using 100% duty cycle, with 3500 mL of water as the bulk of solvent in the bath, considering the bath to be an adiabatic system. Rise in temperature after 18 min was noted and the power dissipated was calculated. The result for power dissipation study is tabulated in Table 1. The result showed that power dissipation varied significantly at all 15 positions. This suggested that acoustic power utilized for extraction of AP at these positions was different. Highest power was dissipated at the 11th position for every power investigated thus confirming
Extraction yield Of AP (mg/g)
Power (Watt)
Power dissipation (Watt)
Yield of AP/power dissipation
17.98 19.54 21.32 23.84 27.91 28.37 28.52 28.77
45 65 88 111 134 155 200 212
21.55 24.67 29.29 36.55 42.95 47.5 56.66 62.14
0.78 0.79 0.73 0.65 0.65 0.59 0.5 0.46
the reason behind getting a highest yield at this position. Variation in the power dissipated within the reaction vessel evinced a variation in cavitation intensity in the bath. 5. Conclusion From the above performed experiments it can be concluded that parameters like choice of solvent, time of ultrasound exposure, solute to solvent ratio, position and depth of extraction vessel, frequency of sound waves and input power played a pivotal role in escalating the extraction yield. Extraction of andrographolide from A. paniculata L. was carried out using soxhlet and ultrasound assisted extraction. Soxhlet extraction gave a yield of 42.46 mg/g of andrographolide. Highest extraction yield of andrographolide of 27.97 mg/g at 22 kHz frequency and 134 W of power was obtained in 10 min of ultrasound exposure with 1:40 solid to solvent ratio, in 50% ethanol at position 11 and 2.54 cm depth from bottom of the bath. 65.87% extraction yield was obtained by UAE as compared to soxhlet extraction, in 10 min. Further power dissipation study showed that the power dissipated within the extraction vessel at different locations in the bath was different. Thus ultrasound intensity throughout the bath was found to be non-uniform and it is hence imperative to optimize all these parameters to obtain good extraction yield. References Caceres, D.D., Hancke, D.D., Burgos, R.A., 1997. Prevention of common colds with Andrographis paniculata dried extract: a pilot double blind trial. Phytomedicine 4, 101–104. Charpe, T.W., Rathod, V.K., 2012. Process intensification extraction of glycyrrhizic acid from licorice root using ultrasound: process intensification studies. Chem. Eng. Process. Process Intensif. 54, 37–41, http://dx.doi.org/10.1016/j.cep.2012.01.002. Csoka, A.B., Szyf, M., 2009. Epigenetic side-effects of common pharmaceuticals: A potential new field in medicine and pharmacology. Med. Hypotheses 73, 770–780, http://dx.doi.org/10.1016/j.mehy.2008.10.039.
318
P.R. Rao, V.K. Rathod / Industrial Crops and Products 66 (2015) 312–318
De Castro, M.D.L., Capote, F.P., 2006. Analytical Applications of Ultrasound. Elsevier. Dhiman, A., Goyal, J., Sharma, K., Nanda, A., Dhiman, S., 2012. Review on medicinal prespectives of Andrographis paniculata Nees. J. Pharm. Sci. Innov. 1, 1–4. Gogate, P.R., Marie, A., Pandit, A.B., 2003. Some aspects of the design of sonochemical reactors. Ultrason. Sonochem. 10, 325–330, http://dx.doi.org/10.1016/S1350-4177(03)103-2. Gogate, P.R., 2008. Cavitational reactors for process intensification of chemical processing applications: a critical review. Chem. Eng. Process. 47, 515–527, http://dx.doi.org/10.1016/j.cep.2007.09.014. Guo, W., Liu, W., Chen, G., Hong, S., Qian, C., Xie, N., Sun, Y., Xu, Q., 2012. International immunopharmacology water-soluble andrographolide sulfonate exerts anti-sepsis action in mice through down-regulating p38 MAPK, STAT3 and NF-B pathways. Int. Immunopharmacol. 14, 613–619, http://dx.doi.org/10.1016/j.intimp.2012.09.002. Hemwimol, S., Pavasant, P., Shotipruk, A., 2006. Ultrasound-assisted extraction of anthraquinones from roots of Morinda citrifolia. Ultrason. Sonochem 13, 543–548, http://dx.doi.org/10.1016/j.ultsonch.2005.09.009. Jadhav, D., Rekha, B.N., Gogate, P.R., Rathod, V.K., 2009. Extraction of vanillin from vanilla pods: a comparison study of conventional soxhlet and ultrasound assisted extraction. J. Food Eng. 93, 421–426, http://dx.doi.org/10.1016/j.jfoodeng.2009.02.007. Ji, H., Li, X., Zhang, H., 2009. EMBO Report194–200. Kanthale, P.M., Gogate, P.R., Pand it, A.B., Wilhelm, A.M., 2003. Mapping of an ultrasonic horn: link primary and secondary effects of ultrasound. Ultrason. Sonochem 10, 331–335, http://dx.doi.org/10.1016/S1350-4177(03)104-4. Kaur, K., Shivhare, U., Basu, S., Vijaya Raghavan, G.S., 2012. Kinetics of extraction of -carotene from tray dried carrots by using supercritical fluid extraction technique. Food Nutr. Sci. 3, 591–595, http://dx.doi.org/10.4236/fns.2012.35081. Kulkarni, V.M., Rathod, V.K., 2014a. Mapping of an ultrasonic bath for ultrasound assisted extraction of mangiferin from Mangifera indica leaves. Ultrason. Sonochem 21, 606–611, http://dx.doi.org/10.1016/j.ultsonch.2013.08.021. Kulkarni, V.M., Rathod, V.K., 2014b. Extraction of mangiferin from Mangifera indica leaves using three phase partitioning coupled with ultrasound. Ind. Crop. Prod., http://dx.doi.org/10.1016/j.indcrop.2013.10.032. Mortazavi, S.V., Eikani, M.H., Mirzaei, H., Jafari, M., Golmohammad, F., 2010. Extraction of essential oils from Bunium persicum Boiss. using superheated water. Food Bioprod. Process. 88, 222–226. Pugin, B., 1987. Qualitative characterization of ultrasound reactors for heterogeneous sonochemistry. Sonochemistry 25, 8–11. Rafie, Z., Jafari, S.M., Alami, M., Khomeiri, M., 2011. Microwave assisted extraction of phenolic compounds from olive leaves; a comparison with maceration. J. Anim. Plant Sci. 21 (4), 738–745. Rajagopal, S., Kumar, R.A., Deevi, D.S., Satyanarayana, C., Rajagopalan, R., 2003. Andrographolide: a potential cancer therapeutic agent isolated from Andrographis paniculata. J. Exp. Therapeut. Oncol. 3, 147–158. Santos, H.M., Lodeiro, C., 2009. The pgffffower of ultrasound, in: ultrasound. Chem. Anal. Appl., 1–16.
Shah Megha, V., Rohit Minal, C., 2013. Novel techniques for isolation and extraction of phyto-constituents from herbal plants. Am. J. Phytomedicine Clin. Ther. 3, 338–350. Shirsath, S.R., Sonawane, S.H., Gogate, P.R., 2012. Intensification of extraction of natural products using ultrasonic irradiations – a review of current status. Chem. Eng. Process. Process Intensif. 53, 10–23, http://dx.doi.org/10.1016/j.cep.2012.01.003. Sravan Kumar, S., Balasubrahmanyam, A., Shyam Sundar, P., Gogate, P.R., Deshpande, V.D., Shukla, S.R., Pandit, A.B., 2009. Characterization of sonochemical reactor for physicochemical transformations. Ind. Eng. Chem. Res. 48, 9402–9407. Sutkar, V.S., Gogate, P.R., 2009. Design aspects of sonochemical reactors: techniques for understanding cavitational activity distribution and effect of operating parameters. Chem. Eng. J. 155, 26–36, http://dx.doi.org/10.1016/j.cej.2009.07.021. Taghvaei, M., Jafari, S.M., Assadpoor, E., Nowrouzieh, S., Alishah, O., 2014. Optimization of microwave-assisted extraction of cottonseed oil and evaluation of its oxidative stability and physicochemical properties. Food Chem. 160, 90–97, http://dx.doi.org/10.1016/j.foodchem.2014.03.064. Tao, C., Wu, D., Zhang, Q., Sun, D., 2014. Ultrasound-assisted extraction of phenolics from wine lees: modeling, optimization and stability of extracts during storage. Ultrasonics. Sonochem. 21, 706–715, http://dx.doi.org/10.1016/j.ultsonch.2013.09.005. Vasu, S., Palaniyappan, V., Badami, S., 2010. A novel microwave assisted extraction for isolation of andrographolide from Andrographis paniculata and its in vitro antioxidant activity. Nat. Prod. Res. 24 (16), 1560–1567, http://dx.doi.org/10.1080/14786419.2010.495071. Vetal, M.D., Lade, V.G., Rathod, V.K., 2013. Extraction of ursolic acid from Ocimum sanctum by ultrasound: process intensification and kinetic studies. Chem. Eng. Process. Process Intensif. 69, 24–30, http://dx.doi.org/10.1016/j.cep.2013.01.011. Viet, K., Tran, B., Kimura, T., Kondo, T., Koda, S., 2014. Quantification of frequency dependence of mechanical effects induced by ultrasound. Ultrason. Sonochem. 21, 716–721, http://dx.doi.org/10.1016/j.ultsonch.2013.08.018. Vilkhu, K., Mawson, R., Simons, L., Bates, D., 2008. Applications and opportunities for ultrasound assisted extraction in the food industry – a review. Innov. Food Sci. Emerg. Technol. 9, 161–169, http://dx.doi.org/10.1016/j.ifset.2007.04.014. Wang, J., Zhao, Y., Tian, Y., Yan, C., Guo, C., 2013. Ultrasound-assisted extraction of total phenolic compounds from Inula helenium. Sci. World J., 1–5. Wongkittipong, R., Prat, L., Damronglerd, S., Gourdon, C., 2004. Solid–liquid extraction of andrographolide from plants – experimental study, kinetic reaction and model. Sep. Purif. Technol. 40, 147–154, http://dx.doi.org/10.1016/j.seppur.2004.02.002. Zhang, Z., Wang, L., Li, D., Jiao, S., Dong, X., Mao, Z., 2008. Ultrasound-assisted extraction of oil from flaxseed. Sep. Purif. Technol. 62, 192–198, http://dx.doi.org/10.1016/j.seppur.2008.01.014.