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Ultrasonically enhanced extraction of luteolin and apigenin from the leaves of Perilla frutescens (L.) Britt. using liquid carbon dioxide and ethanol
Ultrasonics Sonochemistry 29 (2016) 19–26
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Ultrasonically enhanced extraction of luteolin and apigenin from the leaves of Perilla frutescens (L.) Britt. using liquid carbon dioxide and ethanol Hirofumi Kawamura a,b, Kenji Mishima a,⇑, Tanjina Sharmin a, Shota Ito a, Ryo Kawakami a, Takafumi Kato a, Makoto Misumi c, Tadashi Suetsugu c, Hideaki Orii d, Hiroyuki Kawano e, Keiichi Irie e, Kazunori Sano e, Kenichi Mishima e, Takunori Harada f, Salim Mustofa g, Fauziyah Hasanah h, Yusraini Dian Inayati Siregar h, Hilyatuz Zahroh h, Lily Surayya Eka Putri h, Agus Salim h a
Department of Chemical Engineering, Faculty of Engineering, Fukuoka University, 8-19-1, Nanakuma Jonan-ku, Fukuoka 814-0180, Japan Department of Seasoning and Foods Division, San-Ei Gen F.F.I., Inc., 1-1-11, Sanwa-cho, Toyonaka, Osaka 561-8588, Japan c Department of Electronics Engineering and Computer Science, Faculty of Engineering, Fukuoka University, 8-19-1, Nanakuma Jonan-ku, Fukuoka 814-0180, Japan d Department of Electrical Engineering, Faculty of Engineering, Fukuoka University, 8-19-1, Nanakuma Jonan-ku, Fukuoka 814-0180, Japan e Department of Neuropharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1, Nanakuma Jonan-ku, Fukuoka 814-0180, Japan f Department of Applied Chemistry, Faculty of Engineering, Oita University, 700 Dannoharu, Oita-shi 870-1192, Japan g Research Center for Technology of Nuclear Industrial Material, Indonesia Nuclear Energy Agency, Gedung 42, Kawasan PUSPIPTEK Serpong, Tangerang Selatan, Banten 15419, Indonesia h Faculty of Science and Technology, Syarif Hidayatullah State Islamic University (UIN) Jakarta, JL.Ir.H.Juanda Ciputat, Tangerang, Indonesia b
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Article history: Received 25 June 2015 Received in revised form 24 August 2015 Accepted 24 August 2015 Available online 24 August 2015 Keywords: Liquid carbon dioxide extraction Luteolin Apigenin Ultrasonic irradiation
a b s t r a c t The present study reports on the ultrasonic enhancement of the liquid carbon dioxide (CO2) extraction of luteolin and apigenin from the leaves of Perilla frutescens (L.) Britt., to which ethanol is added as a cosolvent. The purpose of this research is also to investigate the effects of the particle size, temperature, pressure, irradiation power, irradiation time, and ethanol content in the liquid CO2 solution on the extraction yield using single-factor experiments. We qualitatively and quantitatively analyzed the yields in the extract using HPLC (high-performance liquid chromatography). The liquid CO2 mixed with ethanol was used at temperatures of 5, 20 and 25 °C with extraction pressures from 8 to 14 MPa. The yields of luteolin and apigenin in the extraction were clearly enhanced by the ultrasound irradiation, but the selectivity of the extract was not changed. The yields of luteolin and apigenin in the extract were also significantly improved by adjusting the operating temperature, the irradiation time, and the ethanol content in the liquid CO2 solution, but no change in the selectivity of the extract was observed. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Perilla frutescens (L.) Britt. Var. crispa (Thunb.) is a common edible plant of the mint family, grown primarily in such Asian countries as China, India, and Japan [1,2]. It was introduced into Japan as Japanese basil (Shiso) from China in the 8th–9th century and is now grown extensively. P. frutescens leaves are frequently used as a fresh vegetable in Asian cooking as a garnish and to process pickles. However, it is most often used as an herbal medicine to treat a wide variety of ailments such as asthma, coprostasis and sitotoxism [2,3]. P. frutescens leaves are not only used in medicine
⇑ Corresponding author. E-mail address: [email protected] (K. Mishima). http://dx.doi.org/10.1016/j.ultsonch.2015.08.016 1350-4177/Ó 2015 Elsevier B.V. All rights reserved.
or food but also in skin creams and soap preparations. Previous investigations have reported P. frutescens to be one of the richest sources of natural polyphenols that exhibit significant pharmacological activities such as anti-allergy, anti-inflammatory [4–6] and anti-oxidant [3] properties. Therefore, worldwide interest in the consumption and use of P. frutescens leaves and products has dramatically and increased. Among the natural polyphenols of low molecular weight, luteolin (30 ,40 ,5,7-tetrahydroxyflavone) and apigenin (40 ,5,7,-trihydroxyflavone) are reportedly the main flavones that exhibit those pharmacological effects [3]. However, as these compounds are usually present in low concentrations, a great deal of research needs to be conducted to develop more effective and selective extraction methods for the recovery of these compounds from the raw materials.
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Various elution and separation methods have been developed for the separation of luteolin and apigenin from plant materials, such as solvent, Soxhlet, or heat reflux extraction [7,8]. Although these methods are effective, they can easily lead to the degradation of heat-sensitive compounds and leave traces of toxic solvent in the solute. Alternatives to conventional solvent extraction, such as supercritical carbon dioxide (supercritical CO2) extraction, have provoked considerable interest in many fields [9–17]. In our previous paper [10,11,17], the possibility of the extraction and separation of flavonoids using SC-CO2 was demonstrated. Although carbon dioxide (CO2) is nonflammable, nontoxic, inexpensive and environmentally benign with an easily accessible critical condition, the extraction of flavones from leaves using SC-CO2 has been little reported on an industrial scale. The reason why SC-CO2 is not used is the excessive pressure sensitivity of the solvent power of SC-CO2 and the low solubility of flavones in SC-CO2. Generally, the pressure sensitivity of the solvent power of the SC-CO2 comes from the change in density of the solvent. However, when the gas phase and the liquid phase coexist in an extraction vessel, the density change in the liquid CO2 is very small with a change in the operating pressure. In the liquid CO2 extraction process, the excessive sensitivity of the solvent power to changes in the operating pressure can thereby be avoided. Therefore, we used liquid CO2 instead of SC-CO2 as the extraction solvent and added ethanol as a cosolvent [14]. Recently, supercritical fluid technology enhanced by ultrasonic treatment has been proposed to produce a greater yield at lower temperatures together with a shorter extraction time for several applications, allowing the extraction of temperature-sensitive components [18]. This can be simply achieved by the induction of an ultrasonic transducer into the extraction unit. The ultrasound-assisted extraction can be used with any solvent, but a literature survey [19] has shown that ultrasound more efficiently enhances solvent extractions involving neat or aqueous ethanol than when applied to any other classical method. The optimization process is also taken into consideration, as various parameters significantly affect the success of the ultrasound-enhanced extraction process. The optimum condition can be obtained by investigating multiple parameters such as particle size, temperature, pressure, ultrasonic power and irradiation time, and cosolvent concentration. This condition was considered achieved when a high yield was obtained. In the present study, we describe the possibility of the ultrasonic-assisted enhancement of the liquid CO2 extraction of luteolin and apigenin from P. frutescens leaves, where liquid CO2 is used as an extraction solvent, and ethanol is added as a cosolvent. We also determined the effect of various parameters (particle size, temperature, pressure, ultrasonic power and irradiation time, and cosolvent (ethanol) concentration) on the extraction yield using the single-factor method. 2. Materials and methods 2.1. Raw materials and reagents P. frutescens leaves were purchased from local markets in Fukuoka, dried at room temperature, and ground into a fine powder by using a freezer mill 6750 (SPEX CentriPrep Co. Ltd., New Jersey, USA) and a mortar and pestle. The particles were ground by using the mortar and pestle and passed through sieves with openings of 0.18 mm and 0.8 mm to ensure their homogeneity. Afterwards, the mean particle diameter (Dpm) was determined by a laser diffraction particle size analyzer (SALD-2000, Shimadzu Co. Ltd., Kyoto, Japan). Finally, powder particles with an average diameter of 0.015 mm (obtained using a freezer mill), assigned as size 1; an average diameter of 0.12 mm, assigned as size 2
(obtained by using a mortar and pestle); and an average diameter of 0.234 mm, assigned as size 3 (obtained by using a mortar and pestle), were separated for the experiments. Samples were stored in a cold and dark place because of their photosensitivity. Luteolin, apigenin, methanol, ethanol and acetic acid were purchased from Wako Co., Ltd., Osaka, Japan. Analytical grade methanol and ethanol were used as solvents for the conventional liquid extraction and the HPLC analysis. High-purity CO2 (more than 99%, Fukuoka Sanso Co., Ltd., Fukuoka, Japan) was used as received. 2.2. Equipment and procedure A schematic diagram of the experimental apparatus used for the liquid CO2 extractions with and without ultrasonic irradiation is shown in Fig. 1. The extraction cell was approximately 150 cm3 in volume (34 mm i.d. 165 mm long, Toyo Koatsu Co., Ltd., HIroshima, Japan). A titanium ultrasound horn is installed inside the upper part of the extraction cell and driven by electrical signals from an ultrasonic processor (VC-750, Sonic and Materials, Inc., Suffolk, UK). Liquid CO2 was pumped from a gas cylinder at a constant flow rate (2 cm3/min) through the dryer and cooling unit into the extraction cell by an HPLC pump (SCF-get, JASCO, Tokyo, Japan) up to the desired pressure. The cooling unit is used to cool the pump-head, which keeps the CO2 liquid. The back pressure regulator (880-81, JASCO), located at the outlet of the cell, is adjusted to maintain the desired pressure within an accuracy of ±0.3 MPa and monitored by a digital pressure gauge (Shinwa Electronics Co., Tokyo, Japan; model DD-501; accuracy ±0.3%). Prior to the pressurization, 0.3 g of a P. frutescens leaf sample was loaded directly into the high-pressure cell, and ultrasound was delivered to the pressurized fluid. The ultrasonic processor produced ultrasonic waves at a frequency of 20 kHz with a maximum power capability of 750 W. During the ultrasonic irradiation, waves of constant amplitude were delivered from the ultrasonic processor by automatically adjusting the power supply. The maximum amplitude was 61 lm, and the amplitude control was set at 100%. The extraction experiments were performed at a constant amplitude of 15.3 lm (the amplitude control was set at 25%). To prevent a rapid increase in the system temperature from the ultrasonic irradiation, the ultrasonic irradiation was performed intermittently (on time: 5 s; off time: 2 s), and the ultrasonic horn was cooled using a cooling jacket. The high-pressure cell is immersed in a water bath to monitor and control the temperature to an accuracy of ±0.1 °C. Ethanol was injected through a co-solvent feeder. The solvent composition xEtOH was defined by the mole fraction of ethanol in the solution of CO2 and ethanol and was controlled by adjusting the amount of ethanol. After the pressure and temperature of the high-pressure cell reached the desired values, the ultrasonic irradiation was performed using the ultrasonic processor. And in case of without ultrasonic irradiation, the high-pressure cell was set on a magnetic stirrer. After each experiment, the cell was slowly depressurized to atmospheric pressure. The extract was collected from the high-pressure cell through an expansion valve, tubes, and U-tube cold traps maintained at atmospheric pressure. The amount of CO2 used was measured by a gas flow meter. 2.2.1. Experimental design In our study, the liquid CO2 extraction of luteolin and apigenin from P. frutescens leaves was performed with and without ultrasound. A time-dependent assay was performed under atmospheric pressure to determine the enhancing effect of the ultrasonic irradiation on the yield. The extraction process was essentially affected by the solute solubility, which was sensitive to parameters such as the particle size, temperature, pressure, ultrasound irradiation time and power, and cosolvent (ethanol) concentration. Therefore,
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Fig. 1. Schematic diagram of the experimental apparatus for liquid CO2 extraction using ultrasonic irradiation.
the effects of particle size (sizes 1, 2 and 3), temperature (5, 20 and 25 °C), liquid CO2 pressure (8, 10 and 14 MPa), ultrasonic irradiation time (75, 125, 200 and 250 s) and power (25, 30 and 40% of nominal power), and mole fraction of ethanol in liquid CO2 (xEtOH = 0, 0.06, 0.131, 0.262, 0.393 and 1) on the extraction yield were investigated. 0.3 g of P. frutescens leaf sample was used in all experiments. The optimum condition was considered achieved when the highest yield was obtained. One parameter was changed at a time, while the other parameters were kept constant under the condition in which the highest yield achieved. Some of the analyses was carried out in triplicate. The compositions of the collected samples were analyzed using HPLC, and a scanning electron microscope (SEM, JEOL JSM6060, Tokyo, Japan) was used to image. The sample particles were stuck on a specimen holder and examined by a scanning electron microscope in a vacuum at an accelerating voltage of 10 kV. 2.2.2. HPLC Analysis Qualitative and quantitative information was obtained by HPLC. We modified the HPLC profiles that Cristea et al. originally developed [20] to more accurately separate the components in the extraction composite. This HPLC system consisted of a Tosoh LC-8010 system equipped with a UV detector. The detection wavelength was set at 343 nm. The separation of the extract was achieved using a TSK-GEL (Tosoh Co., Tokyo, Japan) ODS-80Ts column (150 4.6 mm) set to 40 °C. The injection volume was 100 lL. Two mobile phases were employed during the separations, (A) HPLC-grade methanol and (B) 1% acetic acid in water. The methanol gradient profile is shown in Table 1. The flow rate of the solvent was set at 1.0 mL/min. Table 1 Time course of methanol gradient for HPLC analysis. Time [min]
0
25
40
50
60
65
75
Methanol [vol%]
25
40
60
90
90
5
5
3. Results and discussion 3.1. Enhancing effect of ultrasound on extraction yield The enhancing effect of ultrasound irradiation on the yield of the P. frutescens leaves was determined under atmospheric pressure. Methanol and ethanol were used as the solvents, and the temperature was controlled at 25 °C. The total time of ultrasonic irradiation was 125 s. The experimental extraction curves for the yields of luteolin and apigenin with and without ultrasound are presented in Fig. 2a and b, respectively. It is shown that using either methanol or ethanol, the yields of luteolin and apigenin gradually increased over time until 60 min, at which point saturation occurred, indicating that the optimum time for extraction is approximately 60 min. Although the extract yield was greater with methanol, it is apparent that for both methanol or ethanol extractions, the total yield with ultrasound was higher than that without ultrasound at each identical time. The yields of luteolin with and without ultrasound were found to be 68.76 lg/g and 34.38 lg/g with methanol and 44.85 lg/g and 14.95 lg/g with ethanol, respectively. The yields of apigenin with and without ultrasound were found to be 71.11 lg/g and 61.80 lg/g with methanol and 48.14 lg/g and 25.36 lg/g with ethanol, respectively. These results imply that ultrasonic irradiation clearly improves the extraction yield of luteolin and apigenin. Based on this result, experiments on ultrasound-enhanced liquid CO2 extractions were conducted with different operating parameters to further the investigation. 3.2. Effect of extraction parameters on yield 3.2.1. Effect of particle size Ground P. frutescens leaves with sizes 1, 2, and 3 (Dpm of 0.015, 0.12 and 0.234 mm, respectively) were used to determine the effect of particle size on the extraction yield. Extractions were performed at 10 MPa and 25 °C. The total time of ultrasonic irradiation was 125 s. Fig. 3 shows the yields of luteolin and apigenin
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Fig. 2. Effect of ultrasound on extraction yields. The rates of extraction of luteolin (a) and apigenin (b) from leaves of P. frutescens powder using methanol or ethanol as the solvent at 25 °C, with and without ultrasound under atmospheric pressure. The total time of ultrasonic irradiation was 125 s. Symbols N and 4 show the mass extracted with and without ultrasound using ethanol, whereas j and h show the mass extracted with and without ultrasound using methanol.
extracted by liquid CO2 with and without ultrasound after 60 min. Both yields slightly decreased when the particle size changed from 0.015 to 0.2 mm, but there is no significant difference in the extraction yields among the different sizes. On the other hand, Fig. 3 shows a clear increase in the yield when ultrasound was applied. Generally, the extraction yields increase with a decrease in the particle size because of the shorter diffusion paths and the expanded contact area [21]. We considered that the grinding process increases the surface area, may disrupt the cell walls, reducing mass transfer resistance, and facilitates solvent access, consequently immediately increasing the transfer rate upon exposure to ultrasonic irradiation. Therefore, it is reasonable to use the reduced size particles for the rest of the experiments. 3.2.2. Effects of extraction temperature and pressure The effect of temperature on the total amounts of luteolin and apigenin extracted using liquid CO2 extraction with ethanol (xEtOH = 0.131) was studied with and without ultrasonic irradiation. For the calculation of the liquid CO2 density at various temperatures and pressures, we used the Peng–Robinson–Stryjek–Vera (PRSV) equations of state [22]. The pressure was set at 10 MPa, and the extraction was performed for 60 min. The total time of ultrasonic irradiation was 125 s. The temperature of the air bath was set to 5, 20 and 25 °C. As changing the temperature significantly affects the density of liquid CO2, changing the mole fraction ratio of ethanol to liquid CO2 (xEtOH) in the cell, additional ethanol was added to the extraction cell as a cosolvent before the extraction to balance the mole fraction ratio. Fig. 4a shows the effect of temperature on the yields of luteolin and apigenin. In the case of luteolin, the extraction yields gradually increased with the increasing temperature, and they were significantly further enhanced with ultrasound (44.85 lg/g and 48.14 lg/g, respectively at 25 °C), whereas the yields obtained for apigenin at 5 °C and 20 °C were clearly lower, and significant extraction was only achieved
at 25 °C and further enhanced by ultrasound. The explanation for this lies in the fact that, under the extraction conditions (temperature = 25 °C, pressure = 10 MPa), gas and liquid phases coexist in the extraction vessel, which was near the critical temperature of CO2, such as 32 °C. In this state, changing the temperature significantly affects the density of liquid CO2, which also changes the mole fraction ratio of ethanol to liquid CO2 (xEtOH) in the cell. We should take account of the high density gas–phase, which exist over liquid phase of CO2, thus a compromise between temperature and density must be achieved that may result into obtain the highest concentration at 25 °C. This result also implies that the extraction yields of luteolin or apigenin, are very much temperature specific. In addition, the effects of operating temperature on the extraction yield are similar to the results of other liquid CO2 extractions [13,16,17,22,23]. However, the tendency for elution at the extraction temperature negligibly differed between luteolin and apigenin. Moreover, in the presence of ultrasound, the yields of luteolin and apigenin at 5 °C and 20 °C were notably higher than those at 25 °C without ultrasound when the other extraction parameters were identical. This implies that the same yields of luteolin and apigenin could be obtained at a lower extraction temperature by applying ultrasound to the traditional liquid CO2 extraction. These results imply that ultrasonic irradiation improves the yields of the extractions. Furthermore, we examined the effect of the extraction pressure on the yields of luteolin and apigenin with and without ultrasonic irradiation using the liquid CO2 extraction with ethanol (xEtOH = 0.131) at 25 °C. The pressure was maintained at 8, 10 and 14 MPa. The temperature was set at 25 °C, as the highest yield was obtained at this temperature and also to avoid the unstable phases of gas and liquid that exist near the critical temperature of CO2, 32 °C. The total time of ultrasonic irradiation was 125 s. The densities of liquid CO2 at 8, 10, and 14 MPa at 25 °C are 766, 812, and 871 kg/m3, respectively [23]. As shown in Fig. 4b, the
Fig. 3. Effect of particle size on the extraction yields of luteolin (a) and apigenin (b) from the leaves of P. frutescens powder obtained by liquid CO2 extraction with ethanol (xEtOH = 0.131) at 10 MPa with ( ) and without ( ) ultrasonic irradiation.
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Fig. 4. Effects of temperature (a) and pressure (b) on the extraction yields of luteolin and apigenin from leaves of P. frutescens powder obtained by liquid CO2 extraction with ethanol (xEtOH = 0.131) at 10 MPa with and without ultrasonic irradiation. Symbols N and 4 show the mass extracted with and without ultrasound for luteolin, whereas j and h show the mass extracted with and without ultrasound for apigenin.
highest yields of luteolin and apigenin in ethanol were obtained at 10 MPa, and they were further successfully enhanced by ultrasonic irradiation. This shows that the eluent obtained at these pressures gave similar concentrations, implying that the yields of luteolin and apigenin were insensitive to the operating pressure of liquid CO2, and the extraction rate was clearly enhanced by ultrasonic irradiation. Although the yields of luteolin and apigenin were expected to be higher at 14 MPa, which gave a higher density of CO2 than at 10 MPa, the yields of luteolin and apigenin at 14 MPa were just slightly lower than that at 10 MPa. It is considered that the amount of ethanol, as the cosolvent, decreases in the liquid phase of CO2 because the ethanol distributed more to the vaporphase at 14 MPa. The results of the other liquid CO2 extractions are also similar for the effects of the operating pressure on the extraction yield to [13,16,17,22,23]. Under these extraction conditions (temperature = 25 °C, pressure = 8, 10, and 14 MPa), gas and liquid phases coexist in the extraction vessel. The change in the density of liquid CO2 is very small with a change in the operating pressure, so the solvent power of liquid CO2 is considered to be almost constant under these conditions. 3.2.3. Effect of ultrasonic power and irradiation time The effect of the ultrasonic power was studied using liquid CO2 extraction with ethanol (xEtOH = 0.131) at 25 °C and 10 MPa. The powers of ultrasound used were 0%, 10%, 25%, and 40% of the nominal power at a frequency of 20 kHz. The total time of ultrasonic irradiation was 125 s. As shown in Fig. 5a, the luteolin and apigenin yields first increased slightly with the change of ultrasonic power up to 40%. However, the luteolin and apigenin yields increased significantly when the ultrasonic irradiation time changed. To further examine the efficacy of ultrasound, different ultrasonic irradiation times were investigated - 0, 75, 125, 200 and 250 s – at 25% of the nominal power at a frequency of 20 kHz. The yields of luteolin and apigenin increased with an increase in the total time of
ultrasonic irradiation (Fig. 5), almost saturating at 200 s. Therefore, it is considered that the ultrasonic irradiation can improve the extraction yield, and 125 s of ultrasonic irradiation time gives a sufficient extraction effect. It is not easy to control the temperature when ultrasonic irradiation is applied using an ultrasonic horn. However, the temperature change inside the extractor is negligible. This may be because of the use of two strategies to control the temperature: the use of dedicated vessels that are specially designed to dissipate warming and the use of the pulse mode of ultrasonic application. Therefore, the temperature can be controlled inside the extractor. 3.2.4. Effect of mole fraction of cosolvent To elucidate the effect of changing the mole fraction (xEtOH) of ethanol on the yields of luteolin and apigenin, we performed the extraction using ethanol and liquid CO2 alone and in combination; xEtOH = 0, 1 denotes pure CO2 and ethanol, respectively, as shown in Fig. 6. The pressure was set at 10 MPa, the temperature was set at 25 °C, and the extraction was performed for 60 min. The total time of the ultrasonic irradiation was 125 s. To change the mole fraction of ethanol in the mixtures of liquid CO2 and ethanol over the range of 0 to 1, we used three experimental schemes: xEtOH = 0 uses only liquid CO2, and xEtOH = 1 is the traditional extraction with ethanol without liquid CO2. For other values of xEtOH, the extraction was performed with mixtures of liquid CO2 and ethanol with ethanol mole fractions set at 0.131, 0.26 and 0.39 to reveal the effects of the mole fraction of ethanol more precisely. The maximum yields of luteolin and apigenin, as shown in Fig. 6a and b, were obtained at xEtOH = 0.131, and the values were further enhanced by ultrasound. However, the extraction yield declined at higher ethanol concentrations (>0.131). This result implies that lower or higher mole fractions of ethanol reduce the yields of luteolin and apigenin under our extraction conditions. This result is consistent with the results of our previous CO2 extraction of Resveratrol [24]. One possible explanation is that mixtures of liquid CO2 with a low mole
Fig. 5. Effects of power (a) and irradiation time (b) of ultrasound on the yields of luteolin and apigenin from the leaves of P. frutescens powder using liquid CO2 extraction with ethanol (xEtOH = 0.131) at 25 °C and 10 MPa. Symbol N shows the mass extracted for luteolin, whereas j shows the mass extracted for apigenin.
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Fig. 6. Effect of the mole fraction of ethanol in the liquid CO2 and ethanol solvent on the yields of luteolin (a) and apigenin (b) extracted from the leaves of P. frutescens at 25 °C and 10 MPa with and without ultrasound. Symbols N and 4 show the mass extracted with and without ultrasound for luteolin, whereas j and h show the mass extracted with and without ultrasound for apigenin.
fraction of ethanol may facilitate the swelling of the leaf powder, which increases the surface area of solvent-sample contact [25]. 3.3. Extraction mechanism Many experiments have been conducted to optimize extraction conditions, but little work has been performed to elucidate the mechanism of ultrasonic extraction. In this paper, ultrasonic irradiation enhanced the extraction yield by approximately two times for both luteolin and apigenin compared to the extraction yield under mechanically stirred condition. Ultrasound may give a disturbance of the mixture of solid and liquid by a couple of routes, cavitation or vibration of the horn. We initially assumed that the most probable mechanism for the ultrasonic enhancement of extraction was through cavitation. Usually, ultrasound produces cavitation that generates bubbles, and the collapse of cavitation bubbles produces high-speed jets that drive the release of the cell contents [26,27]. Therefore, the production of cavitation bubbles near the cell walls is expected to wash out the disrupted cell contents together with enabling good penetration of the solvent into the cells through the ultrasonic jet. Ultrasonic cavitation also facilitates the collision of the particles, resulting in an exchange of energy and enhancing the mass transfer [28]. We also considered the intensification of mass transfer through cell disruption by milling the material before extraction. Even theoretically, the mechanism for the ultrasonic enhancement of extraction was more likely corresponding with cavitation than only vibration, it is difficult to prove. However, an aluminum foil test is the easiest method to apply in the laboratory to check whether ultrasonic irradiation generates enough cavitation or not. As a result of cavitation, it is known to cause the aluminum foils perforation. For this experiment, a series of aluminum foil sheets were placed in the most intense zones of sonication inside
the cell. A preliminary investigation was made to check the stability of the aluminum foils against the effects of pressurizing and depressurizing the cell with liquid CO2. After this experiment, no damage was observed on the aluminum foils (Fig. 7a). However, after exposure under specific conditions (20 kHz sonication frequency and 25% sonication amplitude) and defined length of time (125 s), round holes were examined on the foil (Fig. 7b). It is known that, the maximum perforations occur at the maximum intensity. In this experiment, the holes on the foil proves that the ultrasonic cavitation is working well. The pattern of foil damage indicates the distribution of ultrasonic energy within the liquid while the severity of damage and deformation is indicating the intensity of the ultrasonic cavitation field. To further clarify these arguments, we conducted additional observations by scanning electron microscopy (SEM) of the surface of the ground P. frutescens leaf particles with and without ultrasound. Direct evidence of the ultrasound effects on extraction yield can be observed. Images at 1000 magnification of the surface of the ground P. frutescens leaf particles (particle size 1) before and after extraction with and without ultrasonic irradiation are given in Fig. 8 a, b and c, respectively. In Fig. 8a, destructed and broken cell contents are observed on the surface of the untreated leaf particle sample surface. There was a small change observed in the sample without ultrasonic irradiation (Fig. 8b), which might be caused by the dissolution and denaturation of some of the cell walls from the continuous stirring. However, after the ultrasound-assisted extraction, the broken cell contents of the sample particle surface were completely washed out, and the texture was shrunken (Fig. 8c). This phenomenon can be explained by the collapse of cavitation bubbles near the cell walls, which may result in the production of an ultrasound jet, is expected to wash out the disrupted cell contents. On the other hand, this ultrasound jet can also facilitate better penetration of solvents into the cells by
Fig. 7. Aluminum foil test. (a) after pressurizing and de-pressurising the cell with liquid CO2 (b) after sonicated at 20 kHz sonication frequency and 25% sonication amplitude; Sonication time used for 125 s.
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Fig. 8. SEM images (1000, 10.0 kV) of untreated raw milled sample of leaves of P. frutescens (a) and of milled sample after liquid CO2 extraction without ultrasound (b) and with ultrasound (c).
Fig. 9. Comparison of a-mangostin yields of luteolin (a) and apigenin (b) from leaves of P. frutescen powder at 25 °C and 10 MPa with (j) and without ultrasound (h).
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driving the solvent towards the walls acting as a solvent micropump. This forces solvent into the cell dissolving the components which may cause the texture shrunken. Therefore, it is reasonable to infer that the effectiveness of the cleaning process is related to the intensity of the ultrasonic irradiation and resulting implosions of cavitation bubbles, which facilitates the mass transfer of luteolin and apigenin and improves the extraction efficiency. 3.4. Comparison of mass of extract in yield A comparison of the mass of extracts in the yields of luteolin and apigenin with and without ultrasound with ethanol and methanol and with mixtures of CO2 and ethanol (mole fraction of ethanol, xEtOH = 0.131) at 25 °C and 10 MPa is shown in Fig. 9a and b, respectively. Extractions were performed for 60 min in each case. This figure clearly shows that the highest yields of luteolin and apigenin in each condition are enhanced by ultrasonic irradiation than the extraction yield under mechanically stirred condition. Therefore, these comparisons are important to see the clear effect of ultrasound irradiation on the extraction yields. However, the highest improvement by ultrasonic irradiation were obtained with the mixtures of liquid CO2 and ethanol (mole fraction of ethanol, xEtOH = 0.131). 4. Conclusions Luteolin and apigenin were successfully extracted from the leaves of P. frutescens using a ultrasound-assisted liquid extraction method. Carbon dioxide and ethanol were used as the liquid solvent and cosolvent, respectively. Ultrasonic irradiation enhanced the extraction yields by approximately two times for both luteolin and apigenin. The yields of luteolin and apigenin in the liquid CO2 extraction were significantly improved by adjusting the operating temperature and pressure (25 °C, 10 MPa), irradiation time (total time = 125 s, amplitude = 15.3 lm, frequency = 20 kHz) and mole fraction of ethanol in the liquid CO2 solution (xEtOH = 0.131). Acknowledgment This work was partially supported by a Grant-in-Aid for Scientific Research (Grant Nos. 26420770 and 23560913). References [1] Y. Heci, Valuable ingredients from herb perilla: a mini review, Innov. Food Technol. 29–30 (2001) 32–33. [2] M. Asif, A. Kumar, Nutritional and functional characterization of Perilla frutescens seed oil and evaluation of its effect on gastrointestinal motility, Malaysian J. Pharm. Sci. 8 (2010) 1–15. [3] L. Meng, Y.F. Lozano, E.M. Gaydou, B. Li, Antioxidant activities of polyphenols extracted from Perilla frutescens Varieties, Molecules 14 (2009) 133–140. [4] H. Ueda, C. Yamazaki, M. Yamazaki, Luteolin as an anti-inflammatory and antiallergic constituent of Perilla frutescens, Biol. Pharm. Bull. 25 (2002) 1197– 1202. [5] T. Makino, Y. Furata, H. Wakushima, H. Fujii, K. Saito, Y. Kano, Anti-allergic effect of Perilla frutescens and its active constituents, Phytother. Res. 17 (2003) 240–243.
[6] C. Sanbongi, H. Takano, N. Osakabe, N. Sasa, M. Natsume, R. Yanagisawa, K.-I. Inoue, K. Sadakane, T. Ichinose, T. Yoshikawa, Effect of rosmarinic acid on allergic rhinitis in rats, Clin. Exp. Allergy 34 (2004) 971–977. [7] S.W. Park, C.S. Cho, N.H. Ryu, J.H. Kim, J.S. Kim, Luteolin extracted from Platycodon grandiflorum protects retinal pigment epithelial cells from oxidative stress-induced caspase-3 dependent apoptosis, Biomed. Prev. Nutr. 2 (2012) 77–80. [8] M. Bimakr, R.A. Rahman, F.S. Taip, A. Ganjoloo, L.M. Salleh, J. Salamat, A. Hamid, I.S.M. Zaidul, Comparison of different extraction methods for the extraction of major bioactive flavonoid compounds from spearmint (Mentha spicata L.) leaves, Food Bioprod. Process. 89 (2011) 67–72. [9] K. Mishima, T. Tokuyasu, K. Matsuyama, N. Komorita, T. Enjoji, M. Nagatani, Solubility of polymer in the mixtures containing supercritical carbon dioxide and antisolvent, Fluid Phase Equilib. 144 (1998) 299–305. [10] K. Mishima, S. Yamauchi, M. Ito, M. Ezawa, D. Tanabe, Measurement of solubilities of flavone and 3-hydroxyflavone in supercritical carbon dioxide by supercritical fluid chromatography, Solvent Extr. Res. Dev., Jpn. 6 (1999) 176–181. [11] K. Mishima, K. Matsuyama, M. Baba, T. Hirabaru, S. Yamauchi, K. Takahashi, N. Yamasaki, Solubilities of undecanolide and pentadecanolacton in supercritical carbon dioxide, J. Chem. Eng. Data 46 (2001) 69–72. [12] S. Maeda, K. Mishima, K. Matsuyama, M. Baba, T. Hirabaru, Solubilities of azobenzene, p-hydroxyazobenzene and p-dimethylazobenzene in supercritical carbon dioxide, J. Chem. Eng. Data 46 (2001) 647–650. [13] K. Mishima, K. Matsuyama, M. Baba, M. Ohuchi, T. Hakushi, Measurement of the solubilities of undecanolide and pentadecanolacton in supercritical carbon dioxide by supercritical fluid chromatography, Solvent Extr. Res. Dev., Jpn. 8 (2001) 215–222. [14] K. Mishima, K. Matsuyama, H. Ishikawa, K. Hayashi, S. Maeda, Measurement and correlation of solubilities of azo dyes and anthraquinone in supercritical carbon dioxide, Fluid Phase Equilib. 194 (2002) 895–904. [15] K. Mishima, K. Matsuyama, M. Baba, M. Chidori, Enzymatic dipeptide synthesis by surfactant-coated a-chymotrypsin complexes in supercritical carbon dioxide, Biotechnol. Prog. 19 (2003) 281–284. [16] K. Mishima, Biodegradable particle formation for drug and gene delivery using supercritical fluid and dense gas, Adv. Drug Deliv. Rev. 60 (2008) 411–432. [17] K. Mishima, R. Kawakami, H. Yokota, T. Harada, T. Kato, K. Irie, K. Mishima, M. Fujiwara, K. Matsuyama, S. Mustofa, A. Salim, Extraction of xanthones from pericarps of Garcinia mangostana Linn. with supercritical carbon dioxide and ethanol, Solvent Extr. Res. Dev., Jpn. 20 (2013) 79–89. [18] E.C. Schinor, M.J. Salvador, I.C.C. Turatti, Comparison of classical and ultrasound–assisted extractions of steroids and triterpenoids from three Chresta spp., Ultrason. Sonochem. 11 (2004) 415–421. [19] P. Garcia-Salas, A. Morales-Soto, A. Segura-Carretero, A. Fernández-Gutiérrez, Phenoliccompound-extraction systems for fruit and vegetable samples, Molecules 15 (2010) 8813–8826. [20] D. Cristea, I. Bareau, G. Vilarem, Identification and quantitative HPLC analysis of the main flavonoids present in weld (Reseda luteola L.), Dyes Pigm. 57 (2003) 267–272. [21] M. Goto, B.C. Roy, T. Hirose, Shrinking-core leaching model for supercritical fluid extraction, J. Supercrit. Fluids 9 (1996) 128–133. [22] R. Stryjek, J.H. Vera, PRSV — an improved peng-Robinson equation of state with new mixing rules for strongly nonideal mixtures, Can. J. Chem. Eng. 64 (1986) 334–340. [23] JSME data book: Thermophysical Properties of Fluids. The Japan Society of Mechanical Engineers (1983). [24] K. Mishima, Y.D.I. Siregar, H. Kawamura, R. Kawakami, S. Ito, Y. Inoue, T. Hirota, T. Harada, T. Kato, M. Misumi, T. Suetsugu, K. Irie, K. Mishima, S. Mustofa, F. Hasanah, H.Z. Zaharo, L.S.E. Putri, A. Salim, Extraction of resveratrol from melinjo (Gnetum gnemon L.) seeds using mixtures of liquid carbon dioxide and ethanol, Solvent Extr. Res. Dev., Jpn. 22 (2015) 69–77. [25] J. Jian-Bing, L. Xiang-hong, C. Mei-qiang, X. Zhi-chao, Improvement of leaching process of Geniposide with ultrasound, Ultrason. Sonochem. 13 (2006) 455–462. [26] J.L. Luque-García, M.D. Luque de Castro, Ultrasound: a powerful tool for leaching, Trends Anal. Chem. 22 (2003) 41–47. [27] A. Patist, D. Bates, Ultrasonic innovations in the food industry: from the laboratory to commercial production, Innov. Food Sci. Emerg. Technol. 9 (2008) 147–154. [28] M.A. Rostagno, A. Villares, E. Guillamón, A. García-Lafuente, J.A. Martínez, Sample preparation for the analysis of isoflavones from soybeans and soy foods, J. Chromatogr. A 1216 (2009) 2–29.
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Ultrasonically enhanced extraction of luteolin and apigenin from the leaves of Perilla frutescens (L.) Britt. using liquid carbon dioxide and ethanol Hirofumi Kawamura a,b, Kenji Mishima a,⇑, Tanjina Sharmin a, Shota Ito a, Ryo Kawakami a, Takafumi Kato a, Makoto Misumi c, Tadashi Suetsugu c, Hideaki Orii d, Hiroyuki Kawano e, Keiichi Irie e, Kazunori Sano e, Kenichi Mishima e, Takunori Harada f, Salim Mustofa g, Fauziyah Hasanah h, Yusraini Dian Inayati Siregar h, Hilyatuz Zahroh h, Lily Surayya Eka Putri h, Agus Salim h a
Department of Chemical Engineering, Faculty of Engineering, Fukuoka University, 8-19-1, Nanakuma Jonan-ku, Fukuoka 814-0180, Japan Department of Seasoning and Foods Division, San-Ei Gen F.F.I., Inc., 1-1-11, Sanwa-cho, Toyonaka, Osaka 561-8588, Japan c Department of Electronics Engineering and Computer Science, Faculty of Engineering, Fukuoka University, 8-19-1, Nanakuma Jonan-ku, Fukuoka 814-0180, Japan d Department of Electrical Engineering, Faculty of Engineering, Fukuoka University, 8-19-1, Nanakuma Jonan-ku, Fukuoka 814-0180, Japan e Department of Neuropharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1, Nanakuma Jonan-ku, Fukuoka 814-0180, Japan f Department of Applied Chemistry, Faculty of Engineering, Oita University, 700 Dannoharu, Oita-shi 870-1192, Japan g Research Center for Technology of Nuclear Industrial Material, Indonesia Nuclear Energy Agency, Gedung 42, Kawasan PUSPIPTEK Serpong, Tangerang Selatan, Banten 15419, Indonesia h Faculty of Science and Technology, Syarif Hidayatullah State Islamic University (UIN) Jakarta, JL.Ir.H.Juanda Ciputat, Tangerang, Indonesia b
a r t i c l e
i n f o
Article history: Received 25 June 2015 Received in revised form 24 August 2015 Accepted 24 August 2015 Available online 24 August 2015 Keywords: Liquid carbon dioxide extraction Luteolin Apigenin Ultrasonic irradiation
a b s t r a c t The present study reports on the ultrasonic enhancement of the liquid carbon dioxide (CO2) extraction of luteolin and apigenin from the leaves of Perilla frutescens (L.) Britt., to which ethanol is added as a cosolvent. The purpose of this research is also to investigate the effects of the particle size, temperature, pressure, irradiation power, irradiation time, and ethanol content in the liquid CO2 solution on the extraction yield using single-factor experiments. We qualitatively and quantitatively analyzed the yields in the extract using HPLC (high-performance liquid chromatography). The liquid CO2 mixed with ethanol was used at temperatures of 5, 20 and 25 °C with extraction pressures from 8 to 14 MPa. The yields of luteolin and apigenin in the extraction were clearly enhanced by the ultrasound irradiation, but the selectivity of the extract was not changed. The yields of luteolin and apigenin in the extract were also significantly improved by adjusting the operating temperature, the irradiation time, and the ethanol content in the liquid CO2 solution, but no change in the selectivity of the extract was observed. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Perilla frutescens (L.) Britt. Var. crispa (Thunb.) is a common edible plant of the mint family, grown primarily in such Asian countries as China, India, and Japan [1,2]. It was introduced into Japan as Japanese basil (Shiso) from China in the 8th–9th century and is now grown extensively. P. frutescens leaves are frequently used as a fresh vegetable in Asian cooking as a garnish and to process pickles. However, it is most often used as an herbal medicine to treat a wide variety of ailments such as asthma, coprostasis and sitotoxism [2,3]. P. frutescens leaves are not only used in medicine
⇑ Corresponding author. E-mail address: [email protected] (K. Mishima). http://dx.doi.org/10.1016/j.ultsonch.2015.08.016 1350-4177/Ó 2015 Elsevier B.V. All rights reserved.
or food but also in skin creams and soap preparations. Previous investigations have reported P. frutescens to be one of the richest sources of natural polyphenols that exhibit significant pharmacological activities such as anti-allergy, anti-inflammatory [4–6] and anti-oxidant [3] properties. Therefore, worldwide interest in the consumption and use of P. frutescens leaves and products has dramatically and increased. Among the natural polyphenols of low molecular weight, luteolin (30 ,40 ,5,7-tetrahydroxyflavone) and apigenin (40 ,5,7,-trihydroxyflavone) are reportedly the main flavones that exhibit those pharmacological effects [3]. However, as these compounds are usually present in low concentrations, a great deal of research needs to be conducted to develop more effective and selective extraction methods for the recovery of these compounds from the raw materials.
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Various elution and separation methods have been developed for the separation of luteolin and apigenin from plant materials, such as solvent, Soxhlet, or heat reflux extraction [7,8]. Although these methods are effective, they can easily lead to the degradation of heat-sensitive compounds and leave traces of toxic solvent in the solute. Alternatives to conventional solvent extraction, such as supercritical carbon dioxide (supercritical CO2) extraction, have provoked considerable interest in many fields [9–17]. In our previous paper [10,11,17], the possibility of the extraction and separation of flavonoids using SC-CO2 was demonstrated. Although carbon dioxide (CO2) is nonflammable, nontoxic, inexpensive and environmentally benign with an easily accessible critical condition, the extraction of flavones from leaves using SC-CO2 has been little reported on an industrial scale. The reason why SC-CO2 is not used is the excessive pressure sensitivity of the solvent power of SC-CO2 and the low solubility of flavones in SC-CO2. Generally, the pressure sensitivity of the solvent power of the SC-CO2 comes from the change in density of the solvent. However, when the gas phase and the liquid phase coexist in an extraction vessel, the density change in the liquid CO2 is very small with a change in the operating pressure. In the liquid CO2 extraction process, the excessive sensitivity of the solvent power to changes in the operating pressure can thereby be avoided. Therefore, we used liquid CO2 instead of SC-CO2 as the extraction solvent and added ethanol as a cosolvent [14]. Recently, supercritical fluid technology enhanced by ultrasonic treatment has been proposed to produce a greater yield at lower temperatures together with a shorter extraction time for several applications, allowing the extraction of temperature-sensitive components [18]. This can be simply achieved by the induction of an ultrasonic transducer into the extraction unit. The ultrasound-assisted extraction can be used with any solvent, but a literature survey [19] has shown that ultrasound more efficiently enhances solvent extractions involving neat or aqueous ethanol than when applied to any other classical method. The optimization process is also taken into consideration, as various parameters significantly affect the success of the ultrasound-enhanced extraction process. The optimum condition can be obtained by investigating multiple parameters such as particle size, temperature, pressure, ultrasonic power and irradiation time, and cosolvent concentration. This condition was considered achieved when a high yield was obtained. In the present study, we describe the possibility of the ultrasonic-assisted enhancement of the liquid CO2 extraction of luteolin and apigenin from P. frutescens leaves, where liquid CO2 is used as an extraction solvent, and ethanol is added as a cosolvent. We also determined the effect of various parameters (particle size, temperature, pressure, ultrasonic power and irradiation time, and cosolvent (ethanol) concentration) on the extraction yield using the single-factor method. 2. Materials and methods 2.1. Raw materials and reagents P. frutescens leaves were purchased from local markets in Fukuoka, dried at room temperature, and ground into a fine powder by using a freezer mill 6750 (SPEX CentriPrep Co. Ltd., New Jersey, USA) and a mortar and pestle. The particles were ground by using the mortar and pestle and passed through sieves with openings of 0.18 mm and 0.8 mm to ensure their homogeneity. Afterwards, the mean particle diameter (Dpm) was determined by a laser diffraction particle size analyzer (SALD-2000, Shimadzu Co. Ltd., Kyoto, Japan). Finally, powder particles with an average diameter of 0.015 mm (obtained using a freezer mill), assigned as size 1; an average diameter of 0.12 mm, assigned as size 2
(obtained by using a mortar and pestle); and an average diameter of 0.234 mm, assigned as size 3 (obtained by using a mortar and pestle), were separated for the experiments. Samples were stored in a cold and dark place because of their photosensitivity. Luteolin, apigenin, methanol, ethanol and acetic acid were purchased from Wako Co., Ltd., Osaka, Japan. Analytical grade methanol and ethanol were used as solvents for the conventional liquid extraction and the HPLC analysis. High-purity CO2 (more than 99%, Fukuoka Sanso Co., Ltd., Fukuoka, Japan) was used as received. 2.2. Equipment and procedure A schematic diagram of the experimental apparatus used for the liquid CO2 extractions with and without ultrasonic irradiation is shown in Fig. 1. The extraction cell was approximately 150 cm3 in volume (34 mm i.d. 165 mm long, Toyo Koatsu Co., Ltd., HIroshima, Japan). A titanium ultrasound horn is installed inside the upper part of the extraction cell and driven by electrical signals from an ultrasonic processor (VC-750, Sonic and Materials, Inc., Suffolk, UK). Liquid CO2 was pumped from a gas cylinder at a constant flow rate (2 cm3/min) through the dryer and cooling unit into the extraction cell by an HPLC pump (SCF-get, JASCO, Tokyo, Japan) up to the desired pressure. The cooling unit is used to cool the pump-head, which keeps the CO2 liquid. The back pressure regulator (880-81, JASCO), located at the outlet of the cell, is adjusted to maintain the desired pressure within an accuracy of ±0.3 MPa and monitored by a digital pressure gauge (Shinwa Electronics Co., Tokyo, Japan; model DD-501; accuracy ±0.3%). Prior to the pressurization, 0.3 g of a P. frutescens leaf sample was loaded directly into the high-pressure cell, and ultrasound was delivered to the pressurized fluid. The ultrasonic processor produced ultrasonic waves at a frequency of 20 kHz with a maximum power capability of 750 W. During the ultrasonic irradiation, waves of constant amplitude were delivered from the ultrasonic processor by automatically adjusting the power supply. The maximum amplitude was 61 lm, and the amplitude control was set at 100%. The extraction experiments were performed at a constant amplitude of 15.3 lm (the amplitude control was set at 25%). To prevent a rapid increase in the system temperature from the ultrasonic irradiation, the ultrasonic irradiation was performed intermittently (on time: 5 s; off time: 2 s), and the ultrasonic horn was cooled using a cooling jacket. The high-pressure cell is immersed in a water bath to monitor and control the temperature to an accuracy of ±0.1 °C. Ethanol was injected through a co-solvent feeder. The solvent composition xEtOH was defined by the mole fraction of ethanol in the solution of CO2 and ethanol and was controlled by adjusting the amount of ethanol. After the pressure and temperature of the high-pressure cell reached the desired values, the ultrasonic irradiation was performed using the ultrasonic processor. And in case of without ultrasonic irradiation, the high-pressure cell was set on a magnetic stirrer. After each experiment, the cell was slowly depressurized to atmospheric pressure. The extract was collected from the high-pressure cell through an expansion valve, tubes, and U-tube cold traps maintained at atmospheric pressure. The amount of CO2 used was measured by a gas flow meter. 2.2.1. Experimental design In our study, the liquid CO2 extraction of luteolin and apigenin from P. frutescens leaves was performed with and without ultrasound. A time-dependent assay was performed under atmospheric pressure to determine the enhancing effect of the ultrasonic irradiation on the yield. The extraction process was essentially affected by the solute solubility, which was sensitive to parameters such as the particle size, temperature, pressure, ultrasound irradiation time and power, and cosolvent (ethanol) concentration. Therefore,
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Fig. 1. Schematic diagram of the experimental apparatus for liquid CO2 extraction using ultrasonic irradiation.
the effects of particle size (sizes 1, 2 and 3), temperature (5, 20 and 25 °C), liquid CO2 pressure (8, 10 and 14 MPa), ultrasonic irradiation time (75, 125, 200 and 250 s) and power (25, 30 and 40% of nominal power), and mole fraction of ethanol in liquid CO2 (xEtOH = 0, 0.06, 0.131, 0.262, 0.393 and 1) on the extraction yield were investigated. 0.3 g of P. frutescens leaf sample was used in all experiments. The optimum condition was considered achieved when the highest yield was obtained. One parameter was changed at a time, while the other parameters were kept constant under the condition in which the highest yield achieved. Some of the analyses was carried out in triplicate. The compositions of the collected samples were analyzed using HPLC, and a scanning electron microscope (SEM, JEOL JSM6060, Tokyo, Japan) was used to image. The sample particles were stuck on a specimen holder and examined by a scanning electron microscope in a vacuum at an accelerating voltage of 10 kV. 2.2.2. HPLC Analysis Qualitative and quantitative information was obtained by HPLC. We modified the HPLC profiles that Cristea et al. originally developed [20] to more accurately separate the components in the extraction composite. This HPLC system consisted of a Tosoh LC-8010 system equipped with a UV detector. The detection wavelength was set at 343 nm. The separation of the extract was achieved using a TSK-GEL (Tosoh Co., Tokyo, Japan) ODS-80Ts column (150 4.6 mm) set to 40 °C. The injection volume was 100 lL. Two mobile phases were employed during the separations, (A) HPLC-grade methanol and (B) 1% acetic acid in water. The methanol gradient profile is shown in Table 1. The flow rate of the solvent was set at 1.0 mL/min. Table 1 Time course of methanol gradient for HPLC analysis. Time [min]
0
25
40
50
60
65
75
Methanol [vol%]
25
40
60
90
90
5
5
3. Results and discussion 3.1. Enhancing effect of ultrasound on extraction yield The enhancing effect of ultrasound irradiation on the yield of the P. frutescens leaves was determined under atmospheric pressure. Methanol and ethanol were used as the solvents, and the temperature was controlled at 25 °C. The total time of ultrasonic irradiation was 125 s. The experimental extraction curves for the yields of luteolin and apigenin with and without ultrasound are presented in Fig. 2a and b, respectively. It is shown that using either methanol or ethanol, the yields of luteolin and apigenin gradually increased over time until 60 min, at which point saturation occurred, indicating that the optimum time for extraction is approximately 60 min. Although the extract yield was greater with methanol, it is apparent that for both methanol or ethanol extractions, the total yield with ultrasound was higher than that without ultrasound at each identical time. The yields of luteolin with and without ultrasound were found to be 68.76 lg/g and 34.38 lg/g with methanol and 44.85 lg/g and 14.95 lg/g with ethanol, respectively. The yields of apigenin with and without ultrasound were found to be 71.11 lg/g and 61.80 lg/g with methanol and 48.14 lg/g and 25.36 lg/g with ethanol, respectively. These results imply that ultrasonic irradiation clearly improves the extraction yield of luteolin and apigenin. Based on this result, experiments on ultrasound-enhanced liquid CO2 extractions were conducted with different operating parameters to further the investigation. 3.2. Effect of extraction parameters on yield 3.2.1. Effect of particle size Ground P. frutescens leaves with sizes 1, 2, and 3 (Dpm of 0.015, 0.12 and 0.234 mm, respectively) were used to determine the effect of particle size on the extraction yield. Extractions were performed at 10 MPa and 25 °C. The total time of ultrasonic irradiation was 125 s. Fig. 3 shows the yields of luteolin and apigenin
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Fig. 2. Effect of ultrasound on extraction yields. The rates of extraction of luteolin (a) and apigenin (b) from leaves of P. frutescens powder using methanol or ethanol as the solvent at 25 °C, with and without ultrasound under atmospheric pressure. The total time of ultrasonic irradiation was 125 s. Symbols N and 4 show the mass extracted with and without ultrasound using ethanol, whereas j and h show the mass extracted with and without ultrasound using methanol.
extracted by liquid CO2 with and without ultrasound after 60 min. Both yields slightly decreased when the particle size changed from 0.015 to 0.2 mm, but there is no significant difference in the extraction yields among the different sizes. On the other hand, Fig. 3 shows a clear increase in the yield when ultrasound was applied. Generally, the extraction yields increase with a decrease in the particle size because of the shorter diffusion paths and the expanded contact area [21]. We considered that the grinding process increases the surface area, may disrupt the cell walls, reducing mass transfer resistance, and facilitates solvent access, consequently immediately increasing the transfer rate upon exposure to ultrasonic irradiation. Therefore, it is reasonable to use the reduced size particles for the rest of the experiments. 3.2.2. Effects of extraction temperature and pressure The effect of temperature on the total amounts of luteolin and apigenin extracted using liquid CO2 extraction with ethanol (xEtOH = 0.131) was studied with and without ultrasonic irradiation. For the calculation of the liquid CO2 density at various temperatures and pressures, we used the Peng–Robinson–Stryjek–Vera (PRSV) equations of state [22]. The pressure was set at 10 MPa, and the extraction was performed for 60 min. The total time of ultrasonic irradiation was 125 s. The temperature of the air bath was set to 5, 20 and 25 °C. As changing the temperature significantly affects the density of liquid CO2, changing the mole fraction ratio of ethanol to liquid CO2 (xEtOH) in the cell, additional ethanol was added to the extraction cell as a cosolvent before the extraction to balance the mole fraction ratio. Fig. 4a shows the effect of temperature on the yields of luteolin and apigenin. In the case of luteolin, the extraction yields gradually increased with the increasing temperature, and they were significantly further enhanced with ultrasound (44.85 lg/g and 48.14 lg/g, respectively at 25 °C), whereas the yields obtained for apigenin at 5 °C and 20 °C were clearly lower, and significant extraction was only achieved
at 25 °C and further enhanced by ultrasound. The explanation for this lies in the fact that, under the extraction conditions (temperature = 25 °C, pressure = 10 MPa), gas and liquid phases coexist in the extraction vessel, which was near the critical temperature of CO2, such as 32 °C. In this state, changing the temperature significantly affects the density of liquid CO2, which also changes the mole fraction ratio of ethanol to liquid CO2 (xEtOH) in the cell. We should take account of the high density gas–phase, which exist over liquid phase of CO2, thus a compromise between temperature and density must be achieved that may result into obtain the highest concentration at 25 °C. This result also implies that the extraction yields of luteolin or apigenin, are very much temperature specific. In addition, the effects of operating temperature on the extraction yield are similar to the results of other liquid CO2 extractions [13,16,17,22,23]. However, the tendency for elution at the extraction temperature negligibly differed between luteolin and apigenin. Moreover, in the presence of ultrasound, the yields of luteolin and apigenin at 5 °C and 20 °C were notably higher than those at 25 °C without ultrasound when the other extraction parameters were identical. This implies that the same yields of luteolin and apigenin could be obtained at a lower extraction temperature by applying ultrasound to the traditional liquid CO2 extraction. These results imply that ultrasonic irradiation improves the yields of the extractions. Furthermore, we examined the effect of the extraction pressure on the yields of luteolin and apigenin with and without ultrasonic irradiation using the liquid CO2 extraction with ethanol (xEtOH = 0.131) at 25 °C. The pressure was maintained at 8, 10 and 14 MPa. The temperature was set at 25 °C, as the highest yield was obtained at this temperature and also to avoid the unstable phases of gas and liquid that exist near the critical temperature of CO2, 32 °C. The total time of ultrasonic irradiation was 125 s. The densities of liquid CO2 at 8, 10, and 14 MPa at 25 °C are 766, 812, and 871 kg/m3, respectively [23]. As shown in Fig. 4b, the
Fig. 3. Effect of particle size on the extraction yields of luteolin (a) and apigenin (b) from the leaves of P. frutescens powder obtained by liquid CO2 extraction with ethanol (xEtOH = 0.131) at 10 MPa with ( ) and without ( ) ultrasonic irradiation.
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Fig. 4. Effects of temperature (a) and pressure (b) on the extraction yields of luteolin and apigenin from leaves of P. frutescens powder obtained by liquid CO2 extraction with ethanol (xEtOH = 0.131) at 10 MPa with and without ultrasonic irradiation. Symbols N and 4 show the mass extracted with and without ultrasound for luteolin, whereas j and h show the mass extracted with and without ultrasound for apigenin.
highest yields of luteolin and apigenin in ethanol were obtained at 10 MPa, and they were further successfully enhanced by ultrasonic irradiation. This shows that the eluent obtained at these pressures gave similar concentrations, implying that the yields of luteolin and apigenin were insensitive to the operating pressure of liquid CO2, and the extraction rate was clearly enhanced by ultrasonic irradiation. Although the yields of luteolin and apigenin were expected to be higher at 14 MPa, which gave a higher density of CO2 than at 10 MPa, the yields of luteolin and apigenin at 14 MPa were just slightly lower than that at 10 MPa. It is considered that the amount of ethanol, as the cosolvent, decreases in the liquid phase of CO2 because the ethanol distributed more to the vaporphase at 14 MPa. The results of the other liquid CO2 extractions are also similar for the effects of the operating pressure on the extraction yield to [13,16,17,22,23]. Under these extraction conditions (temperature = 25 °C, pressure = 8, 10, and 14 MPa), gas and liquid phases coexist in the extraction vessel. The change in the density of liquid CO2 is very small with a change in the operating pressure, so the solvent power of liquid CO2 is considered to be almost constant under these conditions. 3.2.3. Effect of ultrasonic power and irradiation time The effect of the ultrasonic power was studied using liquid CO2 extraction with ethanol (xEtOH = 0.131) at 25 °C and 10 MPa. The powers of ultrasound used were 0%, 10%, 25%, and 40% of the nominal power at a frequency of 20 kHz. The total time of ultrasonic irradiation was 125 s. As shown in Fig. 5a, the luteolin and apigenin yields first increased slightly with the change of ultrasonic power up to 40%. However, the luteolin and apigenin yields increased significantly when the ultrasonic irradiation time changed. To further examine the efficacy of ultrasound, different ultrasonic irradiation times were investigated - 0, 75, 125, 200 and 250 s – at 25% of the nominal power at a frequency of 20 kHz. The yields of luteolin and apigenin increased with an increase in the total time of
ultrasonic irradiation (Fig. 5), almost saturating at 200 s. Therefore, it is considered that the ultrasonic irradiation can improve the extraction yield, and 125 s of ultrasonic irradiation time gives a sufficient extraction effect. It is not easy to control the temperature when ultrasonic irradiation is applied using an ultrasonic horn. However, the temperature change inside the extractor is negligible. This may be because of the use of two strategies to control the temperature: the use of dedicated vessels that are specially designed to dissipate warming and the use of the pulse mode of ultrasonic application. Therefore, the temperature can be controlled inside the extractor. 3.2.4. Effect of mole fraction of cosolvent To elucidate the effect of changing the mole fraction (xEtOH) of ethanol on the yields of luteolin and apigenin, we performed the extraction using ethanol and liquid CO2 alone and in combination; xEtOH = 0, 1 denotes pure CO2 and ethanol, respectively, as shown in Fig. 6. The pressure was set at 10 MPa, the temperature was set at 25 °C, and the extraction was performed for 60 min. The total time of the ultrasonic irradiation was 125 s. To change the mole fraction of ethanol in the mixtures of liquid CO2 and ethanol over the range of 0 to 1, we used three experimental schemes: xEtOH = 0 uses only liquid CO2, and xEtOH = 1 is the traditional extraction with ethanol without liquid CO2. For other values of xEtOH, the extraction was performed with mixtures of liquid CO2 and ethanol with ethanol mole fractions set at 0.131, 0.26 and 0.39 to reveal the effects of the mole fraction of ethanol more precisely. The maximum yields of luteolin and apigenin, as shown in Fig. 6a and b, were obtained at xEtOH = 0.131, and the values were further enhanced by ultrasound. However, the extraction yield declined at higher ethanol concentrations (>0.131). This result implies that lower or higher mole fractions of ethanol reduce the yields of luteolin and apigenin under our extraction conditions. This result is consistent with the results of our previous CO2 extraction of Resveratrol [24]. One possible explanation is that mixtures of liquid CO2 with a low mole
Fig. 5. Effects of power (a) and irradiation time (b) of ultrasound on the yields of luteolin and apigenin from the leaves of P. frutescens powder using liquid CO2 extraction with ethanol (xEtOH = 0.131) at 25 °C and 10 MPa. Symbol N shows the mass extracted for luteolin, whereas j shows the mass extracted for apigenin.
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Fig. 6. Effect of the mole fraction of ethanol in the liquid CO2 and ethanol solvent on the yields of luteolin (a) and apigenin (b) extracted from the leaves of P. frutescens at 25 °C and 10 MPa with and without ultrasound. Symbols N and 4 show the mass extracted with and without ultrasound for luteolin, whereas j and h show the mass extracted with and without ultrasound for apigenin.
fraction of ethanol may facilitate the swelling of the leaf powder, which increases the surface area of solvent-sample contact [25]. 3.3. Extraction mechanism Many experiments have been conducted to optimize extraction conditions, but little work has been performed to elucidate the mechanism of ultrasonic extraction. In this paper, ultrasonic irradiation enhanced the extraction yield by approximately two times for both luteolin and apigenin compared to the extraction yield under mechanically stirred condition. Ultrasound may give a disturbance of the mixture of solid and liquid by a couple of routes, cavitation or vibration of the horn. We initially assumed that the most probable mechanism for the ultrasonic enhancement of extraction was through cavitation. Usually, ultrasound produces cavitation that generates bubbles, and the collapse of cavitation bubbles produces high-speed jets that drive the release of the cell contents [26,27]. Therefore, the production of cavitation bubbles near the cell walls is expected to wash out the disrupted cell contents together with enabling good penetration of the solvent into the cells through the ultrasonic jet. Ultrasonic cavitation also facilitates the collision of the particles, resulting in an exchange of energy and enhancing the mass transfer [28]. We also considered the intensification of mass transfer through cell disruption by milling the material before extraction. Even theoretically, the mechanism for the ultrasonic enhancement of extraction was more likely corresponding with cavitation than only vibration, it is difficult to prove. However, an aluminum foil test is the easiest method to apply in the laboratory to check whether ultrasonic irradiation generates enough cavitation or not. As a result of cavitation, it is known to cause the aluminum foils perforation. For this experiment, a series of aluminum foil sheets were placed in the most intense zones of sonication inside
the cell. A preliminary investigation was made to check the stability of the aluminum foils against the effects of pressurizing and depressurizing the cell with liquid CO2. After this experiment, no damage was observed on the aluminum foils (Fig. 7a). However, after exposure under specific conditions (20 kHz sonication frequency and 25% sonication amplitude) and defined length of time (125 s), round holes were examined on the foil (Fig. 7b). It is known that, the maximum perforations occur at the maximum intensity. In this experiment, the holes on the foil proves that the ultrasonic cavitation is working well. The pattern of foil damage indicates the distribution of ultrasonic energy within the liquid while the severity of damage and deformation is indicating the intensity of the ultrasonic cavitation field. To further clarify these arguments, we conducted additional observations by scanning electron microscopy (SEM) of the surface of the ground P. frutescens leaf particles with and without ultrasound. Direct evidence of the ultrasound effects on extraction yield can be observed. Images at 1000 magnification of the surface of the ground P. frutescens leaf particles (particle size 1) before and after extraction with and without ultrasonic irradiation are given in Fig. 8 a, b and c, respectively. In Fig. 8a, destructed and broken cell contents are observed on the surface of the untreated leaf particle sample surface. There was a small change observed in the sample without ultrasonic irradiation (Fig. 8b), which might be caused by the dissolution and denaturation of some of the cell walls from the continuous stirring. However, after the ultrasound-assisted extraction, the broken cell contents of the sample particle surface were completely washed out, and the texture was shrunken (Fig. 8c). This phenomenon can be explained by the collapse of cavitation bubbles near the cell walls, which may result in the production of an ultrasound jet, is expected to wash out the disrupted cell contents. On the other hand, this ultrasound jet can also facilitate better penetration of solvents into the cells by
Fig. 7. Aluminum foil test. (a) after pressurizing and de-pressurising the cell with liquid CO2 (b) after sonicated at 20 kHz sonication frequency and 25% sonication amplitude; Sonication time used for 125 s.
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Fig. 8. SEM images (1000, 10.0 kV) of untreated raw milled sample of leaves of P. frutescens (a) and of milled sample after liquid CO2 extraction without ultrasound (b) and with ultrasound (c).
Fig. 9. Comparison of a-mangostin yields of luteolin (a) and apigenin (b) from leaves of P. frutescen powder at 25 °C and 10 MPa with (j) and without ultrasound (h).
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driving the solvent towards the walls acting as a solvent micropump. This forces solvent into the cell dissolving the components which may cause the texture shrunken. Therefore, it is reasonable to infer that the effectiveness of the cleaning process is related to the intensity of the ultrasonic irradiation and resulting implosions of cavitation bubbles, which facilitates the mass transfer of luteolin and apigenin and improves the extraction efficiency. 3.4. Comparison of mass of extract in yield A comparison of the mass of extracts in the yields of luteolin and apigenin with and without ultrasound with ethanol and methanol and with mixtures of CO2 and ethanol (mole fraction of ethanol, xEtOH = 0.131) at 25 °C and 10 MPa is shown in Fig. 9a and b, respectively. Extractions were performed for 60 min in each case. This figure clearly shows that the highest yields of luteolin and apigenin in each condition are enhanced by ultrasonic irradiation than the extraction yield under mechanically stirred condition. Therefore, these comparisons are important to see the clear effect of ultrasound irradiation on the extraction yields. However, the highest improvement by ultrasonic irradiation were obtained with the mixtures of liquid CO2 and ethanol (mole fraction of ethanol, xEtOH = 0.131). 4. Conclusions Luteolin and apigenin were successfully extracted from the leaves of P. frutescens using a ultrasound-assisted liquid extraction method. Carbon dioxide and ethanol were used as the liquid solvent and cosolvent, respectively. Ultrasonic irradiation enhanced the extraction yields by approximately two times for both luteolin and apigenin. The yields of luteolin and apigenin in the liquid CO2 extraction were significantly improved by adjusting the operating temperature and pressure (25 °C, 10 MPa), irradiation time (total time = 125 s, amplitude = 15.3 lm, frequency = 20 kHz) and mole fraction of ethanol in the liquid CO2 solution (xEtOH = 0.131). Acknowledgment This work was partially supported by a Grant-in-Aid for Scientific Research (Grant Nos. 26420770 and 23560913). References [1] Y. Heci, Valuable ingredients from herb perilla: a mini review, Innov. Food Technol. 29–30 (2001) 32–33. [2] M. Asif, A. Kumar, Nutritional and functional characterization of Perilla frutescens seed oil and evaluation of its effect on gastrointestinal motility, Malaysian J. Pharm. Sci. 8 (2010) 1–15. [3] L. Meng, Y.F. Lozano, E.M. Gaydou, B. Li, Antioxidant activities of polyphenols extracted from Perilla frutescens Varieties, Molecules 14 (2009) 133–140. [4] H. Ueda, C. Yamazaki, M. Yamazaki, Luteolin as an anti-inflammatory and antiallergic constituent of Perilla frutescens, Biol. Pharm. Bull. 25 (2002) 1197– 1202. [5] T. Makino, Y. Furata, H. Wakushima, H. Fujii, K. Saito, Y. Kano, Anti-allergic effect of Perilla frutescens and its active constituents, Phytother. Res. 17 (2003) 240–243.
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