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Ultrasound-assisted fractional precipitation of paclitaxel from Taxus chinensis cell cultures
Process Biochemistry xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Ultrasound-assisted fractional precipitation of paclitaxel from Taxus chinensis cell cultures Hye-Won Seo, Jin-Hyun Kim
⁎
Department of Chemical Engineering, Kongju National University, Cheonan 330-717, South Korea
ARTICLE INFO
ABSTRACT
Keywords: Paclitaxel Ultrasound-assisted fractional precipitation Kinetics Johnson-Mehl-Avrami-Kolmogorov model
In this study, we developed an ultrasound-assisted fractional precipitation for the purification of poorly watersoluble anticancer agent paclitaxel. The effects of ultrasound power (80, 180, 250, and 380 W) were investigated in samples of different purity (20.4, 63.6, and 92.7%) for precipitation. Higher precipitation rate and shorter precipitation time were found with ultrasound treated solutions than with those without ultrasound. When the experimental data were applied to the Johnson-Mehl-Avrami-Komolgorov (JMAK) equation, the rate constant increased to 200–500-fold for 20.4% of sample purity, 150–250-fold for 63.6% of sample purity, and 60–120fold for 92.7% of sample purity by treatment with ultrasound. In addition, the lower the purity of the sample, the greater was the decrease in activation energy compared with the control, which means that the lower the sample purity, the more effective the fractional precipitation using ultrasound.
1. Introduction Paclitaxel is a natural diterpene alkaloid that is used for treatment of non-small cell lung cancer, ovarian cancer, Kaposi’s sarcoma, breast cancer, and so forth. It is expected that demand for paclitaxel will increase in reflection of indications for the use of paclitaxel as well as the development of relevant formulae [1]. Paclitaxel is produced mainly by extraction, semi-synthesis, and plant cell culture [2], among which plant cell culture is advantageous because there is little influence from external factors (climate, environment, etc.) and it is suitable for stable mass-production in a bioreactor [3]. In order to obtain high-purity paclitaxel from plant cell culture, multiple steps of isolation and purification are required. Specifically, solvent extraction of paclitaxel from the raw material biomass (plant cells) is followed by pre-purification, final purification, and production [4]. In particular, it is of great importance to increase the purity of samples in the pre-purification process as much as possible because the pre-purification process has a great effect on the cost of final purification [5,6]. Fractional precipitation is a simple and efficient pre-treatment process that is based on the solubility difference of paclitaxel. In 2000, a fractional precipitation method through which paclitaxel of high purity could be obtained from biomass was first reported [7]. Thereafter, research has been conducted regarding the optimal precipitation solvent, precipitation temperature, and sample characteristics (purity and content) for fractional precipitation of paclitaxel [8–10]. However, it takes
⁎
much operating time (∼ 3 days) for fractional precipitation of paclitaxel, which limits economical mass production [7,9,10]. In order to overcome such challenges, specific research has been conducted to improve the efficiency of fractional precipitation by increasing the surface area per working volume (S/V) in use of surface area-increasing materials (glass bead or ion exchange resin) [11–13]. While this method contributed to some reduction in the time required for fractional precipitation, it was still not suitable for efficient operation. In 2014, the time of fractional precipitation was shortened further by increasing the content of distilled water in a fractional precipitation solution (methanol-distilled water mixture) of paclitaxel. However, excessive addition of distilled water decreased the purity of the precipitate [6]. According to previous studies [14,15], the rate of nucleation was increased by precipitation of poorly water-soluble drugs such as itraconazole, griseofulvin, ibuprofen, and sulfamethoxazole in the presence of ultrasound. Furthermore, sonication in the precipitation process reduced the size of the paclitaxel nanoparticles and improved their bioavailability [16]. Based on this idea, we tried to develop a method to dramatically shorten the time required for fractional precipitation of paclitaxel using ultrasound. To this end, the efficiency of fractional precipitation in relation to ultrasonic power was investigated by means of three kinds of samples of different purity. In addition, kinetic analysis was also performed using the Johnson-Mehl-Avrami-Komolgorov (JMAK) equation, which is widely used in precipitation and crystallization processes. Furthermore, the activation energy change (ΔEa) was
Corresponding author. E-mail address: [email protected] (J.-H. Kim).
https://doi.org/10.1016/j.procbio.2019.09.019 Received 11 August 2019; Received in revised form 3 September 2019; Accepted 13 September 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Hye-Won Seo and Jin-Hyun Kim, Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.09.019
Process Biochemistry xxx (xxxx) xxx–xxx
H.-W. Seo and J.-H. Kim
calculated and the reaction behavior determined quantitatively in an ultrasound-assisted fractional precipitation process.
Fractional precipitation was performed at a fixed temperature (5℃) by varying the ultrasonic power (80, 180, 250, 380 W). After precipitation, the precipitate was filtered (Whatman Grade 4, 20–25 μm particle retention, 150 mm diameter) and dried in a vacuum oven (UP-2000, EYELA, Japan) for 24 h at 35 °C to examine the paclitaxel yield and purity over time. The dried precipitate was analyzed by high performance liquid chromatography (HPLC). Each experiment was performed three times.
2. Materials and methods 2.1. Sample preparation Biomass recovered from plant cell cultures of Taxus chinensis and methanol were mixed at a 1:1 ratio (w/v). After extraction at room temperature for 30 min and then filtering, the methanol extract was concentrated using a rotary evaporator (CCA-1100, EYELA, Japan) under reduced pressure. For liquid-liquid extraction, methylene chloride was added to the extract, which was then stirred for 30 min. After phase separation, the methylene chloride phase was collected, concentrated, and dried. The dried crude extract from liquid-liquid extraction was dissolved in methylene chloride at a ratio of 20% (v/w), and adsorbent Sylopute (Fuji Silysia Chemical Ltd., Japan) was added to the dried crude extract at the rate of 100% (w/w). The mixture was stirred at 40℃ for 30 min, then filtered. The filtrate was dried at 30℃ under reduced pressure and then purified by means of a silica-gel 60 N (Timely, Japan) column (elution: 1.5% (v/v) methanol in dichloromethane). The purified samples (sample purity: 20.4%) were then dissolved in methylene chloride, and the solution was dropped into n-hexane (methylene chloride/hexane = 1:10, v/v) with mixing (∼500 rpm) for precipitation. After hexane precipitation, the precipitate obtained through filtration was dried at 35℃ under vacuum for 24 h (sample purity: 63.6%). Further purification with C18 ODS-HPLC (elution: methanol/water = 65:35, v/v) gave paclitaxel with a purity of 92.7% [17]. Three types of samples (purity: 20.4, 63.6, 92.7%) obtained in the above isolation and purification process were used in this study.
2.3. Paclitaxel analysis The paclitaxel content was analyzed by means of an HPLC system (Waters, USA) equipped with a Capcell Pak C18 (250 × 4.6 mm, Shiseido, Japan) column. Elution was performed by gradient using a mixture of distilled water and acetonitrile from 65:35 to 35:65 within 40 min (flow rate: 1.0 mL/min). The injection volume was 20 μL, and the effluent was monitored at 227 nm using an ultraviolet detector [18]. Authentic sample (paclitaxel purity: 95%) was purchased from Sigma-Aldrich and used as a standard. Each sample was analyzed three times. 2.4. Kinetic model The equation of Johnson-Mehl-Avrami-Komolgorov (JMAK), which is mainly used for the crystallization or precipitation process, deals with the kinetics of isothermal phase-transformation during nucleation and particle growth [5,19]. The JMAK equation is expressed as Eqs. (1) and (2).
2.2. Fractional precipitation
(1)
ktn
X= 1
e
log ln
1 1 X
= nlogt+ logk
(2)
( ( ) ) and log t, the values of n and k were
From the plot of log ln
A schematic diagram of ultrasound-assisted fractional precipitation is illustrated in Fig. 1. Three types of samples (purity: 20.4, 63.6, 92.7%) were dissolved in methanol (pure paclitaxel basis: 0.5%, w/v), and then distilled water was added dropwise while mixing (∼180 rpm) until the methanol/distilled water ratio became 61.5:38.5 (v/v).
1
1 X
calculated based on the slope and intercept. where X, t, k, and n indicate the fraction of precipitation, precipitation time, rate constant, and Avrami exponent, respectively. Both n and k reflect the nucleation and growth mechanisms of the sample. The coefficient of determination (r2) was used to validate the kinetic
Fig. 1. Schematic diagram of ultrasound-assisted fractional precipitation for pre-purification of paclitaxel. 2
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H.-W. Seo and J.-H. Kim
Fig. 2. Effect of ultrasound power on the yield of paclitaxel obtained by fractional precipitation at various sample purities. (A) Paclitaxel purity 20.4%; (B) Paclitaxel purity: 63.6%; (C) Paclitaxel purity 92.7%.
model (JMAK model) in the fractional precipitation process of paclitaxel. The closer the coefficient of determination is to 1, the smaller the error between the experimental value and the calculated value.
3. Results and discussion 3.1. Development of ultrasound-assisted precipitation process for paclitaxel Fractional precipitation is a simple and efficient pre-treatment process for paclitaxel that is based on differences of solubility [7]. However, the precipitation process takes such a long time that there is a 3
Process Biochemistry xxx (xxxx) xxx–xxx
H.-W. Seo and J.-H. Kim
limitation in applying it to a mass production process for paclitaxel [1,7–9]. In order to address this challenge, a process for ultrasoundassisted fractional precipitation was developed as part of this study. First, the effect of ultrasound power (80, 180, 250, 380 W) was investigated using three kinds of samples (purity: 20.4, 63.6, 92.7%). The paclitaxel yields were 36.7–68.4% (sample purity: 20.4%), 55.0–69.8% (sample purity: 63.6%), and 76.1–85.6% (sample purity: 92.7%) at an ultrasonic power of 80–380 W (Fig. 2). The yield of paclitaxel increased with increasing ultrasonic power in all samples; however, in the control group the yield was relatively low in comparison with that of ultrasound-assisted fractional precipitation at 59% (sample purity: 92.7%), 44% (sample purity: 63.6%), and 10% (sample purity: 20.4%), even after 300 min of fractional precipitation. In addition, the fractional precipitation time was 5–6 min (sample purity: 92.7%), 6–8 min (sample purity: 63.6%), and 30–40 min (sample purity: 20.4%) at 80–380 W of ultrasonic power. Compared with the control (> 300 min), the operating time required for fractional precipitation was dramatically shortened. The higher the purity of the sample under the same conditions (ultrasonic power, precipitation time), the higher was the yield and the shorter the precipitation time. The purity of the precipitates was not significantly different in relation to ultrasonic power, with a purity of 32, 74, and 95% at a sample purity of 20.4, 63.6, and 92.7%, respectively (data not shown). The results also showed that paclitaxel purity remained almost unchanged during fractional precipitation, and these results are similar to those of previous studies [20]. The time required for fractional precipitation in conventional fractional precipitation [7–9] and in fractional precipitation using surface area-increasing materials [11,12] was ∼72 h and 6–12 h, respectively. Meanwhile, the fractional precipitation using ultrasonic waves developed in this study only took 0.08–1.5 h depending on the sample type. Therefore, the method of ultrasound-assisted fractional precipitation shortened the operating time drastically and thus addressed the problem in existing fractional precipitation. It is considered that the uniform micromixing of the precipitate solution and the transfer of solute molecules are facilitated by the ultrasonic wave, and the nucleation is accelerated by the local temperature decrease stemming from the instant energy release due to the bubble destruction caused by the ultrasonic wave (Fig. 3) [14,15,21].
log t was performed based on Eq. (2) as illustrated in Fig. 4. The JMAK exponent (n) and the rate constant (k) calculated from the slope of intercept are shown in Table 1 along with the coefficient of determination (r2). The value of n was found to be 1.319 (control) and 0.9895–1.014 (80–380 W) for sample purity of 20.4%, 0.9685 (control) and 0.51580.4432 (80–380 W) for sample purity of 63.6%, and 0.9046 (control) and 0.8220-0.7220 (80–380 W) for sample purity of 92.7%. For the control, the value of n was similar or slightly smaller than that in the existing fractionation precipitation of paclitaxel (1.912 at 4 °C) [5]. The value of k was found to be 6.310 × 10−5 (control) and 1.374 × QUOTE10−23.213 × 10−2 (80–380 W) for sample purity of 20.4%, 2.014 × 10−3 (control) and 3.020 × QUOTE10−1-4.989 × 10−1 (80–380 W) for sample purity of 63.6%, and 4.355 × 10-3 (control) and 2.716 × QUOTE10−1-5.070 × 10−1 (80–380 W) for sample purity of 92.7%. In the case of ultrasound-assisted fractional precipitation (ultrasound power: 80–380 W) compared with the control, the rate constants increased by 200–500, 150–250, and 60–120 times at the sample purity of 20.4, 63.6, and 92.7%, respectively. Especially, the rate constant increase was found to be larger at lower sample purity (20.4%). In the case of the control group, it was similar or slightly larger than the k value (1.810 × 10−5 at 4 °C) in the existing fractional precipitation of paclitaxel [5]. The result of the calculation of activation energy change (ΔEa=ΔEa,ultrasound-ΔEa,control) in relation to ultrasonic power (80–380 W) using the Arrhenius equation [7] showed that the ΔEa value was −12.442 ∼ −14.406 kJ/mol for sample purity 20.4%, −11.580 ∼ −12.740 kJ/mol for sample purity 63.6.4%, and −9.552 ∼ −10.995 kJ/mol for sample purity of 92.7%. In other words, fractional precipitation utilizing ultrasonic waves was effective in reducing activation energy. At a low sample purity, the activation energy decrease rate was higher than that of the control group. This indicates that ultrasound-assisted fractional precipitation was more effective when the sample purity was relatively low. These results show that, by introducing ultrasonic waves into the fractional precipitation process, it is possible to increase the precipitation rate by reducing the activation energy, which is the minimum energy required for the reaction. The kinetic analysis result shows that the JMAK model has a high r2 value (> 0.917) and is suitable for fractional precipitation of paclitaxel. 4. Conclusions
3.2. Kinetic analysis of ultrasound-assisted precipitation process for paclitaxel
In this study, we developed a fractional precipitation method for paclitaxel using ultrasound. Ultrasound-assisted fractional precipitation with different sample purity (20.4, 63.6, and 92.7%) resulted in paclitaxel yields of 36.7–68.4%, 55.0–69.8%, and 76.1–85.6%, respectively, at ultrasound power of 80–380 W. The time required for fractional precipitation was 5–6 min (sample purity: 92.7%), 6–8 min (sample purity: 63.6%), and 30–40 min (sample purity: 20.4%). The fractional precipitation time was drastically shortened compared with the control (> 300 min). The higher the purity of the sample under the same conditions (ultrasonic power, precipitation time), the higher was the yield and the shorter the precipitation time. As a result of kinetic analysis using the JMAK equation, the rate constants at 80–380 W of ultrasonic power compared with the control increased by 200–500 times at sample purity of 20.4%, 150–250 times at sample purity of 63.6%, and 60–120 times at sample purity of 92.7%. Particularly when the sample purity (20.4%) was low, the increase rate of the rate constant was greater than that of the control group. The activation energy change was −12.442 ∼ −14.406 kJ/mol (80–380 W) at the sample purity of 20.4%, −11.580 ∼ −12.740 kJ/mol (80–380 W) at the sample purity of 63.6%, and −9.552 ∼ −10.995 kJ/mol (80–380 W) at the sample purity of 92.7%. In other words, fractional precipitation utilizing ultrasonic waves proved to be effective in reducing activation energy. At a low sample purity, the activation energy decrease rate was higher than that of the control group. This indicates that ultrasoundassisted fractional precipitation was more effective when the sample purity was relatively low.
Generally, the formation of particles involves nucleation and growth in precipitation [5,18]. Hence, the experimental data (Fig. 2) was applied to the JMAK equation in order for kinetic analysis of the paclitaxel precipitation patterns over time of precipitation. In order to apply the 1 JMAK equation to fractional precipitation, plotting of log(ln( 1 X )) and
Fig. 3. Principle of ultrasound-assisted fractional precipitation of paclitaxel. 4
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H.-W. Seo and J.-H. Kim
Fig. 4. Johnson-Mehl-Avrami-Komolgorov (JMAK) plots at various sample purities. (A) Paclitaxel purity 20.4%; (B) Paclitaxel purity: 63.6%; (C) Paclitaxel purity 92.7%; (D) Control. Table 1 Values of kinetic parameters for the fractional precipitation of paclitaxel at different ultrasound powers. Sample purity (%)
Ultrasound power (W)
na
20.4
Control 80 180 250 380 Control 80 180 250 380 Control 80 180 250 380
1.319 0.990 1.095 1.212 1.014 0.969 0.516 0.573 0.481 0.443 0.905 0.820 0.846 0.794 0.722
63.6
92.7
a b
ka ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.237 0.001 0.041 0.049 0.014 0.049 0.014 0.013 0.012 0.007 0.039 0.004 0.001 0.015 0.001
(6.310 (1.374 (1.527 (1.762 (3.213 (2.014 (3.020 (3.436 (4.477 (4.989 (4.355 (2.716 (3.420 (4.018 (5.070
Data are shown as n, k, and ΔEa ± SD. ΔEa = (Ea,ultrasound - Ea,control).
5
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.220)×10−5 0.080)×10−2 0.220)×10−2 0.180)×10−2 0.250)×10−2 0.810)×10−3 0.093)×10−1 0.120)×10−1 0.190)×10−1 0.150)×10−1 1.300)×10−3 0.039)×10−1 0.160)×10−1 0.190)×10−1 0.160)×10−1
ΔEa (J/mol)a,b
r2
– −12442 ± 592 −12687 ± 848 −13017 ± 692 −14406 ± 631 – −11580 ± 835 −11878 ± 842 −12490 ± 865 −12740 ± 835 – −9552 ± 640 −10085 ± 715 −10458 ± 719 −10995 ± 679
0.929 0.987 0.981 0.976 0.972
0.934 0.968 0.996 0.971 0.990 0.966 0.919 0.917 0.931 0.990
H.-W. Seo and J.-H. Kim
Process Biochemistry xxx (xxxx) xxx–xxx
Acknowledgments
(2013) 8–14. [11] K.Y. Jeon, J.H. Kim, Improvement of fractional precipitation process for pre-purification of paclitaxel, Process Biochem. 44 (2009) 736–741. [12] I.S. Kang, J.H. Kim, Effect of reactor type on the purification efficiency of paclitaxel in the increased surface area fractional precipitation process, Sep. Purif. Technol. 99 (2012) 14–19. [13] J.Y. Lee, J.H. Kim, Evaluation of the effect of crude extract purity and pure paclitaxel content on the increased surface area fractional precipitation process for the purification of paclitaxel, Process Biochem. 47 (2012) 2388–2397. [14] S.V. Dalvi, M.D. Yadav, Effect of ultrasound and stabilizers on nucleation kinetics of curcumin during liquid antisolvent precipitation, Ultrason. Sonochem. 24 (2015) 114–122. [15] S.V. Dalvi, R.N. Dave, Analysis of nucleation kinetics of poorly water-soluble drugs in presence of ultrasound and hydroxypropyl methyl cellulose during antisolvent precipitation, Int. J. Pharm. 387 (2010) 172–179. [16] F. Madani, S.S. Esnaashari, B. Mujokoro, F. Dorkoosh, M. Khosravani, M. Adabi, Investigation of effective parameters on size of paclitaxel loaded PLGA nanoparticles, Adv. Pharm. Bull. 8 (2018) 77–84. [17] S.H. Pyo, H.J. Choi, B.H. Han, Large-scale purification of 13-dehydroxybaccatin III and 10-deacetylpaclitaxel, semi-synthetic precursors of paclitaxel, from cell cultures of Taxus chinensis, J. Chromatogr. A 1123 (2006) 15–21. [18] W.S. Jang, J.H. Kim, Characteristics and mechanism of microwave-assisted drying of amorphous paclitaxel for removal of residual solvent, Biotechnol. Bioprocess Eng. 24 (2019) 529–535. [19] J.N. Park, J.H. Kim, Kinetic and thermodynamic characteristics of fractional precipitationof (+)-dihydromyricetin, Process Biochem. 53 (2017) 224–231. [20] M.J. Kim, J.H. Kim, Decreasing particle size of paclitaxel using polymer in fractional precipitation process, Korean Chem. Eng. Res. 54 (2016) 278–283. [21] A.T. Ayodele, A. Valizadeh, M. Adabi, S.S. Esnaashari, F. Madani, M. Khosravani, M. Adabi, Ultrasound nanobubbles and their applications as theranostic agents in cancer therapy: a review, Biointerface Res. Appl. Chem. 7 (2017) 2253–2262.
This work was supported by a research grant of Kongju National University in 2019. References [1] K.W. Yoo, J.H. Kim, Kinetics and mechanism of ultrasound-assisted extraction of paclitaxel from Taxus chinensis, Biotechnol. Bioprocess Eng. 23 (2018) 532–540. [2] Y.S. Kim, J.H. Kim, Isotherm, kinetic and thermodynamic studies on the adsorption of paclitaxel onto Sylopute, J. Chem. Thermodyn. 130 (2019) 104–113. [3] S.H. Lee, J.H. Kim, Kinetic and thermodynamic characteristics of microwave-assisted extraction for the recovery of paclitaxel from Taxus chinensis, Process Biochem. 76 (2019) 187–193. [4] H.J. Kang, J.H. Kim, Adsorption kinetics, mechanism, isotherm, and thermodynamic analysis of paclitaxel from extracts of Taxus chinensis cell cultures onto Sylopute, Biotechnol. Bioprocess Eng. 24 (2019) 513–521. [5] C.G. Lee, J.H. Kim, A kinetic and thermodynamic study of fractional precipitation of paclitaxel from Taxus chinensis, Process Biochem. 59 (2017) 216–222. [6] C.G. Lee, J.H. Kim, Improved fractional precipitation method for purification of paclitaxel, Process Biochem. 49 (2014) 1370–1376. [7] J.H. Kim, I.S. Kang, H.K. Choi, S.S. Hong, H.S. Lee, Fractional precipitation for paclitaxel pre-purification from plant cell cultures of Taxus chinensis, Biotechnol. Lett. 22 (2000) 1753–1756. [8] J.H. Kim, I.S. Kang, H.K. Choi, S.S. Hong, H.S. Lee, A novel prepurification for paclitaxel from plant cell cultures, Process Biochem. 37 (2002) 679–682. [9] S.I. Jeon, S. Mun, J.H. Kim, Optimal temperature control in fractional precipitation for paclitaxel pre-purification, Process Biochem. 41 (2006) 276–280. [10] J.Y. Lee, J.H. Kim, Influence of crude extract purity and pure paclitaxel content on fractional precipitation for purification of paclitaxel, Sep. Purif. Technol. 103
6
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Ultrasound-assisted fractional precipitation of paclitaxel from Taxus chinensis cell cultures Hye-Won Seo, Jin-Hyun Kim
⁎
Department of Chemical Engineering, Kongju National University, Cheonan 330-717, South Korea
ARTICLE INFO
ABSTRACT
Keywords: Paclitaxel Ultrasound-assisted fractional precipitation Kinetics Johnson-Mehl-Avrami-Kolmogorov model
In this study, we developed an ultrasound-assisted fractional precipitation for the purification of poorly watersoluble anticancer agent paclitaxel. The effects of ultrasound power (80, 180, 250, and 380 W) were investigated in samples of different purity (20.4, 63.6, and 92.7%) for precipitation. Higher precipitation rate and shorter precipitation time were found with ultrasound treated solutions than with those without ultrasound. When the experimental data were applied to the Johnson-Mehl-Avrami-Komolgorov (JMAK) equation, the rate constant increased to 200–500-fold for 20.4% of sample purity, 150–250-fold for 63.6% of sample purity, and 60–120fold for 92.7% of sample purity by treatment with ultrasound. In addition, the lower the purity of the sample, the greater was the decrease in activation energy compared with the control, which means that the lower the sample purity, the more effective the fractional precipitation using ultrasound.
1. Introduction Paclitaxel is a natural diterpene alkaloid that is used for treatment of non-small cell lung cancer, ovarian cancer, Kaposi’s sarcoma, breast cancer, and so forth. It is expected that demand for paclitaxel will increase in reflection of indications for the use of paclitaxel as well as the development of relevant formulae [1]. Paclitaxel is produced mainly by extraction, semi-synthesis, and plant cell culture [2], among which plant cell culture is advantageous because there is little influence from external factors (climate, environment, etc.) and it is suitable for stable mass-production in a bioreactor [3]. In order to obtain high-purity paclitaxel from plant cell culture, multiple steps of isolation and purification are required. Specifically, solvent extraction of paclitaxel from the raw material biomass (plant cells) is followed by pre-purification, final purification, and production [4]. In particular, it is of great importance to increase the purity of samples in the pre-purification process as much as possible because the pre-purification process has a great effect on the cost of final purification [5,6]. Fractional precipitation is a simple and efficient pre-treatment process that is based on the solubility difference of paclitaxel. In 2000, a fractional precipitation method through which paclitaxel of high purity could be obtained from biomass was first reported [7]. Thereafter, research has been conducted regarding the optimal precipitation solvent, precipitation temperature, and sample characteristics (purity and content) for fractional precipitation of paclitaxel [8–10]. However, it takes
⁎
much operating time (∼ 3 days) for fractional precipitation of paclitaxel, which limits economical mass production [7,9,10]. In order to overcome such challenges, specific research has been conducted to improve the efficiency of fractional precipitation by increasing the surface area per working volume (S/V) in use of surface area-increasing materials (glass bead or ion exchange resin) [11–13]. While this method contributed to some reduction in the time required for fractional precipitation, it was still not suitable for efficient operation. In 2014, the time of fractional precipitation was shortened further by increasing the content of distilled water in a fractional precipitation solution (methanol-distilled water mixture) of paclitaxel. However, excessive addition of distilled water decreased the purity of the precipitate [6]. According to previous studies [14,15], the rate of nucleation was increased by precipitation of poorly water-soluble drugs such as itraconazole, griseofulvin, ibuprofen, and sulfamethoxazole in the presence of ultrasound. Furthermore, sonication in the precipitation process reduced the size of the paclitaxel nanoparticles and improved their bioavailability [16]. Based on this idea, we tried to develop a method to dramatically shorten the time required for fractional precipitation of paclitaxel using ultrasound. To this end, the efficiency of fractional precipitation in relation to ultrasonic power was investigated by means of three kinds of samples of different purity. In addition, kinetic analysis was also performed using the Johnson-Mehl-Avrami-Komolgorov (JMAK) equation, which is widely used in precipitation and crystallization processes. Furthermore, the activation energy change (ΔEa) was
Corresponding author. E-mail address: [email protected] (J.-H. Kim).
https://doi.org/10.1016/j.procbio.2019.09.019 Received 11 August 2019; Received in revised form 3 September 2019; Accepted 13 September 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Hye-Won Seo and Jin-Hyun Kim, Process Biochemistry, https://doi.org/10.1016/j.procbio.2019.09.019
Process Biochemistry xxx (xxxx) xxx–xxx
H.-W. Seo and J.-H. Kim
calculated and the reaction behavior determined quantitatively in an ultrasound-assisted fractional precipitation process.
Fractional precipitation was performed at a fixed temperature (5℃) by varying the ultrasonic power (80, 180, 250, 380 W). After precipitation, the precipitate was filtered (Whatman Grade 4, 20–25 μm particle retention, 150 mm diameter) and dried in a vacuum oven (UP-2000, EYELA, Japan) for 24 h at 35 °C to examine the paclitaxel yield and purity over time. The dried precipitate was analyzed by high performance liquid chromatography (HPLC). Each experiment was performed three times.
2. Materials and methods 2.1. Sample preparation Biomass recovered from plant cell cultures of Taxus chinensis and methanol were mixed at a 1:1 ratio (w/v). After extraction at room temperature for 30 min and then filtering, the methanol extract was concentrated using a rotary evaporator (CCA-1100, EYELA, Japan) under reduced pressure. For liquid-liquid extraction, methylene chloride was added to the extract, which was then stirred for 30 min. After phase separation, the methylene chloride phase was collected, concentrated, and dried. The dried crude extract from liquid-liquid extraction was dissolved in methylene chloride at a ratio of 20% (v/w), and adsorbent Sylopute (Fuji Silysia Chemical Ltd., Japan) was added to the dried crude extract at the rate of 100% (w/w). The mixture was stirred at 40℃ for 30 min, then filtered. The filtrate was dried at 30℃ under reduced pressure and then purified by means of a silica-gel 60 N (Timely, Japan) column (elution: 1.5% (v/v) methanol in dichloromethane). The purified samples (sample purity: 20.4%) were then dissolved in methylene chloride, and the solution was dropped into n-hexane (methylene chloride/hexane = 1:10, v/v) with mixing (∼500 rpm) for precipitation. After hexane precipitation, the precipitate obtained through filtration was dried at 35℃ under vacuum for 24 h (sample purity: 63.6%). Further purification with C18 ODS-HPLC (elution: methanol/water = 65:35, v/v) gave paclitaxel with a purity of 92.7% [17]. Three types of samples (purity: 20.4, 63.6, 92.7%) obtained in the above isolation and purification process were used in this study.
2.3. Paclitaxel analysis The paclitaxel content was analyzed by means of an HPLC system (Waters, USA) equipped with a Capcell Pak C18 (250 × 4.6 mm, Shiseido, Japan) column. Elution was performed by gradient using a mixture of distilled water and acetonitrile from 65:35 to 35:65 within 40 min (flow rate: 1.0 mL/min). The injection volume was 20 μL, and the effluent was monitored at 227 nm using an ultraviolet detector [18]. Authentic sample (paclitaxel purity: 95%) was purchased from Sigma-Aldrich and used as a standard. Each sample was analyzed three times. 2.4. Kinetic model The equation of Johnson-Mehl-Avrami-Komolgorov (JMAK), which is mainly used for the crystallization or precipitation process, deals with the kinetics of isothermal phase-transformation during nucleation and particle growth [5,19]. The JMAK equation is expressed as Eqs. (1) and (2).
2.2. Fractional precipitation
(1)
ktn
X= 1
e
log ln
1 1 X
= nlogt+ logk
(2)
( ( ) ) and log t, the values of n and k were
From the plot of log ln
A schematic diagram of ultrasound-assisted fractional precipitation is illustrated in Fig. 1. Three types of samples (purity: 20.4, 63.6, 92.7%) were dissolved in methanol (pure paclitaxel basis: 0.5%, w/v), and then distilled water was added dropwise while mixing (∼180 rpm) until the methanol/distilled water ratio became 61.5:38.5 (v/v).
1
1 X
calculated based on the slope and intercept. where X, t, k, and n indicate the fraction of precipitation, precipitation time, rate constant, and Avrami exponent, respectively. Both n and k reflect the nucleation and growth mechanisms of the sample. The coefficient of determination (r2) was used to validate the kinetic
Fig. 1. Schematic diagram of ultrasound-assisted fractional precipitation for pre-purification of paclitaxel. 2
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Fig. 2. Effect of ultrasound power on the yield of paclitaxel obtained by fractional precipitation at various sample purities. (A) Paclitaxel purity 20.4%; (B) Paclitaxel purity: 63.6%; (C) Paclitaxel purity 92.7%.
model (JMAK model) in the fractional precipitation process of paclitaxel. The closer the coefficient of determination is to 1, the smaller the error between the experimental value and the calculated value.
3. Results and discussion 3.1. Development of ultrasound-assisted precipitation process for paclitaxel Fractional precipitation is a simple and efficient pre-treatment process for paclitaxel that is based on differences of solubility [7]. However, the precipitation process takes such a long time that there is a 3
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limitation in applying it to a mass production process for paclitaxel [1,7–9]. In order to address this challenge, a process for ultrasoundassisted fractional precipitation was developed as part of this study. First, the effect of ultrasound power (80, 180, 250, 380 W) was investigated using three kinds of samples (purity: 20.4, 63.6, 92.7%). The paclitaxel yields were 36.7–68.4% (sample purity: 20.4%), 55.0–69.8% (sample purity: 63.6%), and 76.1–85.6% (sample purity: 92.7%) at an ultrasonic power of 80–380 W (Fig. 2). The yield of paclitaxel increased with increasing ultrasonic power in all samples; however, in the control group the yield was relatively low in comparison with that of ultrasound-assisted fractional precipitation at 59% (sample purity: 92.7%), 44% (sample purity: 63.6%), and 10% (sample purity: 20.4%), even after 300 min of fractional precipitation. In addition, the fractional precipitation time was 5–6 min (sample purity: 92.7%), 6–8 min (sample purity: 63.6%), and 30–40 min (sample purity: 20.4%) at 80–380 W of ultrasonic power. Compared with the control (> 300 min), the operating time required for fractional precipitation was dramatically shortened. The higher the purity of the sample under the same conditions (ultrasonic power, precipitation time), the higher was the yield and the shorter the precipitation time. The purity of the precipitates was not significantly different in relation to ultrasonic power, with a purity of 32, 74, and 95% at a sample purity of 20.4, 63.6, and 92.7%, respectively (data not shown). The results also showed that paclitaxel purity remained almost unchanged during fractional precipitation, and these results are similar to those of previous studies [20]. The time required for fractional precipitation in conventional fractional precipitation [7–9] and in fractional precipitation using surface area-increasing materials [11,12] was ∼72 h and 6–12 h, respectively. Meanwhile, the fractional precipitation using ultrasonic waves developed in this study only took 0.08–1.5 h depending on the sample type. Therefore, the method of ultrasound-assisted fractional precipitation shortened the operating time drastically and thus addressed the problem in existing fractional precipitation. It is considered that the uniform micromixing of the precipitate solution and the transfer of solute molecules are facilitated by the ultrasonic wave, and the nucleation is accelerated by the local temperature decrease stemming from the instant energy release due to the bubble destruction caused by the ultrasonic wave (Fig. 3) [14,15,21].
log t was performed based on Eq. (2) as illustrated in Fig. 4. The JMAK exponent (n) and the rate constant (k) calculated from the slope of intercept are shown in Table 1 along with the coefficient of determination (r2). The value of n was found to be 1.319 (control) and 0.9895–1.014 (80–380 W) for sample purity of 20.4%, 0.9685 (control) and 0.51580.4432 (80–380 W) for sample purity of 63.6%, and 0.9046 (control) and 0.8220-0.7220 (80–380 W) for sample purity of 92.7%. For the control, the value of n was similar or slightly smaller than that in the existing fractionation precipitation of paclitaxel (1.912 at 4 °C) [5]. The value of k was found to be 6.310 × 10−5 (control) and 1.374 × QUOTE10−23.213 × 10−2 (80–380 W) for sample purity of 20.4%, 2.014 × 10−3 (control) and 3.020 × QUOTE10−1-4.989 × 10−1 (80–380 W) for sample purity of 63.6%, and 4.355 × 10-3 (control) and 2.716 × QUOTE10−1-5.070 × 10−1 (80–380 W) for sample purity of 92.7%. In the case of ultrasound-assisted fractional precipitation (ultrasound power: 80–380 W) compared with the control, the rate constants increased by 200–500, 150–250, and 60–120 times at the sample purity of 20.4, 63.6, and 92.7%, respectively. Especially, the rate constant increase was found to be larger at lower sample purity (20.4%). In the case of the control group, it was similar or slightly larger than the k value (1.810 × 10−5 at 4 °C) in the existing fractional precipitation of paclitaxel [5]. The result of the calculation of activation energy change (ΔEa=ΔEa,ultrasound-ΔEa,control) in relation to ultrasonic power (80–380 W) using the Arrhenius equation [7] showed that the ΔEa value was −12.442 ∼ −14.406 kJ/mol for sample purity 20.4%, −11.580 ∼ −12.740 kJ/mol for sample purity 63.6.4%, and −9.552 ∼ −10.995 kJ/mol for sample purity of 92.7%. In other words, fractional precipitation utilizing ultrasonic waves was effective in reducing activation energy. At a low sample purity, the activation energy decrease rate was higher than that of the control group. This indicates that ultrasound-assisted fractional precipitation was more effective when the sample purity was relatively low. These results show that, by introducing ultrasonic waves into the fractional precipitation process, it is possible to increase the precipitation rate by reducing the activation energy, which is the minimum energy required for the reaction. The kinetic analysis result shows that the JMAK model has a high r2 value (> 0.917) and is suitable for fractional precipitation of paclitaxel. 4. Conclusions
3.2. Kinetic analysis of ultrasound-assisted precipitation process for paclitaxel
In this study, we developed a fractional precipitation method for paclitaxel using ultrasound. Ultrasound-assisted fractional precipitation with different sample purity (20.4, 63.6, and 92.7%) resulted in paclitaxel yields of 36.7–68.4%, 55.0–69.8%, and 76.1–85.6%, respectively, at ultrasound power of 80–380 W. The time required for fractional precipitation was 5–6 min (sample purity: 92.7%), 6–8 min (sample purity: 63.6%), and 30–40 min (sample purity: 20.4%). The fractional precipitation time was drastically shortened compared with the control (> 300 min). The higher the purity of the sample under the same conditions (ultrasonic power, precipitation time), the higher was the yield and the shorter the precipitation time. As a result of kinetic analysis using the JMAK equation, the rate constants at 80–380 W of ultrasonic power compared with the control increased by 200–500 times at sample purity of 20.4%, 150–250 times at sample purity of 63.6%, and 60–120 times at sample purity of 92.7%. Particularly when the sample purity (20.4%) was low, the increase rate of the rate constant was greater than that of the control group. The activation energy change was −12.442 ∼ −14.406 kJ/mol (80–380 W) at the sample purity of 20.4%, −11.580 ∼ −12.740 kJ/mol (80–380 W) at the sample purity of 63.6%, and −9.552 ∼ −10.995 kJ/mol (80–380 W) at the sample purity of 92.7%. In other words, fractional precipitation utilizing ultrasonic waves proved to be effective in reducing activation energy. At a low sample purity, the activation energy decrease rate was higher than that of the control group. This indicates that ultrasoundassisted fractional precipitation was more effective when the sample purity was relatively low.
Generally, the formation of particles involves nucleation and growth in precipitation [5,18]. Hence, the experimental data (Fig. 2) was applied to the JMAK equation in order for kinetic analysis of the paclitaxel precipitation patterns over time of precipitation. In order to apply the 1 JMAK equation to fractional precipitation, plotting of log(ln( 1 X )) and
Fig. 3. Principle of ultrasound-assisted fractional precipitation of paclitaxel. 4
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Fig. 4. Johnson-Mehl-Avrami-Komolgorov (JMAK) plots at various sample purities. (A) Paclitaxel purity 20.4%; (B) Paclitaxel purity: 63.6%; (C) Paclitaxel purity 92.7%; (D) Control. Table 1 Values of kinetic parameters for the fractional precipitation of paclitaxel at different ultrasound powers. Sample purity (%)
Ultrasound power (W)
na
20.4
Control 80 180 250 380 Control 80 180 250 380 Control 80 180 250 380
1.319 0.990 1.095 1.212 1.014 0.969 0.516 0.573 0.481 0.443 0.905 0.820 0.846 0.794 0.722
63.6
92.7
a b
ka ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.237 0.001 0.041 0.049 0.014 0.049 0.014 0.013 0.012 0.007 0.039 0.004 0.001 0.015 0.001
(6.310 (1.374 (1.527 (1.762 (3.213 (2.014 (3.020 (3.436 (4.477 (4.989 (4.355 (2.716 (3.420 (4.018 (5.070
Data are shown as n, k, and ΔEa ± SD. ΔEa = (Ea,ultrasound - Ea,control).
5
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.220)×10−5 0.080)×10−2 0.220)×10−2 0.180)×10−2 0.250)×10−2 0.810)×10−3 0.093)×10−1 0.120)×10−1 0.190)×10−1 0.150)×10−1 1.300)×10−3 0.039)×10−1 0.160)×10−1 0.190)×10−1 0.160)×10−1
ΔEa (J/mol)a,b
r2
– −12442 ± 592 −12687 ± 848 −13017 ± 692 −14406 ± 631 – −11580 ± 835 −11878 ± 842 −12490 ± 865 −12740 ± 835 – −9552 ± 640 −10085 ± 715 −10458 ± 719 −10995 ± 679
0.929 0.987 0.981 0.976 0.972
0.934 0.968 0.996 0.971 0.990 0.966 0.919 0.917 0.931 0.990
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Acknowledgments
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