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Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures
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Process Biochemistry xxx (2014) xxx–xxx
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Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures Gun-Joong Kim, Jin-Hyun Kim ∗ Department of Chemical Engineering, Kongju National University, Cheonan 330-717, South Korea
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Article history: Received 11 July 2014 Received in revised form 22 November 2014 Accepted 25 November 2014 Available online xxx Keywords: Plant cell culture Paclitaxel Microwave-assisted extraction (MAE) Acid hydrolysis Simultaneous process
a b s t r a c t In this study, a microwave-assisted extraction (MAE)/acid hydrolysis simultaneous process was developed for recovering the anticancer agent paclitaxel from plant cell cultures of Taxus chinensis. The optimal pH of the extraction solution (90% aqueous methanol) for hydrolysis was 2.2 at fixed extraction time (6 min), ratio of extraction solution to biomass (1:1, v/w), and extraction temperature (40 ◦ C). In the MAE/acid hydrolysis simultaneous process, the paclitaxel recovery was 2.2-fold higher than in the existing extraction methods. Regarding changes in the content of glycoside (7-xylosyl paclitaxel as sugar-binding paclitaxel) and paclitaxel depending on the inclusion of acid hydrolysis in the MAE process, the content of 7-xylosyl paclitaxel decreased after acid hydrolysis whereas the content of paclitaxel increased. Based on this result, it was confirmed that acid hydrolysis breaks down a glycosidic bond of glycoside (sugar-binding paclitaxel), and so the recovery of paclitaxel increases. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Paclitaxel is an anticancer agent that is a diterpenoid found in the bark of the yew tree [1,2]. It is an FDA (Food and Drug Administration)-approved anticancer drug most widely used for treatment of ovarian cancer, breast cancer, Kaposi’s sarcoma and non-small cell lung cancer [3]. Its application has also been expanded to the treatment of acute rheumatoid arthritis and Alzheimer’s disease. Since clinical trials regarding its combined prescription with various other treatments are underway, the demand for paclitaxel is expected to increase [4,5]. The main paclitaxel production methods are direct extraction from the yew tree, semisynthesis, and plant cell culture [6–8]. Among these methods, plant cell culture enables stable mass production of paclitaxel of consistent quality in a bioreactor without being affected by such external factors as climate and environment [7]. Most of the paclitaxel produced by plant cell culture is contained in plant cells and the debris [9], and it is important to effectively extract paclitaxel from cells in regard to increasing the recovery. The existing extraction methods used most frequently use an organic solvent to recover paclitaxel from the biomass, a plant cell [10–12]. However, the conventional solvent extraction (CSE) method requires a long extraction time and large amounts of organic solvents and has a low extraction
∗ Corresponding author. Tel.: +82 41 521 9361; fax: +82 41 554 2640. E-mail address: [email protected] (J.-H. Kim).
efficiency. To overcome these disadvantages, microwave-assisted extraction (MAE) has been studied [13]. During MAE, microwaves heat the solvent or solvent mixture directly. In addition, the direct interaction of microwaves with free water molecules present in glands and vascular systems results in the subsequent rupture of plant tissue and the release of active compounds into the organic solvent, which increases recovery. Compared with CSE, MAE has many advantages, which include a shorter extraction time, a lower solvent requirement, a higher extraction rate, and production of a higher-quality product at lower cost [14]. Recently, research studies have been conducted on the development of MAE methods for the extraction of saponin from Ganoderma atrum [15], camptothecin from Nothapodytes foetida [16], essential oil from Elettaria cardamomum [17], ginsenoside from ginseng root [18], glycyrrhizic acid from licorice root [19] and polyphenol and caffeine from green tea leaves [20]. In our previous study [21], we confirmed the possibility that the extraction of paclitaxel from plant cell cultures using MAE could overcome the aforementioned problems with CSE. In 2000, Kim et al. [22] reported that the recovery of paclitaxel from the supernatant of plant cell culture increased by hydrolysis. It was assumed that the increase in recovery of paclitaxel in the supernatant of plant cell culture by acid hydrolysis was caused by the cleavage of 7-xylosyl paclitaxel and 7-xylosyl-10deacetylpaclitaxel, which are glycosides (sugar-binding paclitaxel) found in the cell culture supernatant (the cleavage of glycosidic bonds between sugar and paclitaxel) [23]. However, the
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Please cite this article in press as: Kim G-J, Kim J-H. Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.11.009
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cause of the increase in recovery of paclitaxel by acid hydrolysis has still not been clarified. Therefore, in this study, we developed the simultaneous MAE and acid hydrolysis process to increase the recovery of paclitaxel from plant cells of Taxus chinensis. In particular, the efficiency of the process was maximized by investigating and understanding the mechanism of acid hydrolysis of glycosides in the MAE/acid hydrolysis simultaneous process. 2. Materials and methods 2.1. Plant materials A suspension of plant cells originating from Taxus chinensis was cultured in a bioreactor [24]. Following cultivation, biomass (plant cell and debris) was recovered using a decanter (CA150 Clarifying Decanter; Westfalia, Germany) and a high-speed centrifuge (BTPX 205GD – 35CDEFP; Alfa Laval, Sweden). The biomass was provided by Samyang Genex Company, South Korea. 2.2. Paclitaxel and glycoside (7-xylosyl paclitaxel, 7-xylosyl-10-deacetylpaclitaxel) analysis An HPLC system (SCL-10AVP, Shimadzu, Japan) and a Capcell Pak C18 column (250 × 4.6 mm, Shiseido) were used to analyze the paclitaxel and glycoside contents. Acetonitrile/water (35:65–65:35, v/v gradient) for paclitaxel and acetonitrile/water (25:75–65:35, v/v gradient) for glycoside were used as the mobile phase. Using a UV detector, paclitaxel and glycoside were analyzed at 227 nm and 228 nm, respectively [25]. In addition, the flow rate and sample injection volume were 1.0 mL/min and 20 L, respectively. Authentic paclitaxel, 7-xylosyl paclitaxel and the 7xylosyl-10-deacetylpaclitaxel were purchased from Sigma–Aldrich (purity: 95%), Quality Phytochemicals (purity: 99%), and Santa Cruz Biotechnology (purity: 60%), respectively, and used as standards. Each sample was analyzed in triplicate.
Fig. 1. Schematic diagram of the microwave-assisted extraction (MAE) process.
concentrated and dried at 40 ◦ C under vacuum (635 mm Hg) for HPLC analysis.
2.5. Simultaneous MAE and acid hydrolysis process In our previous studies [21,26,27], the optimal extraction time, ratio of extraction solution to biomass, extraction temperature, and extraction solvent in MAE were found to be 6 min, 1:1 (v/w), 40 ◦ C, and 90% aqueous methanol, respectively. The pH of extraction solution in the simultaneous process was optimized under these optimal conditions. The pH of the extraction solution (90% aqueous methanol) was adjusted to 1.8, 2.2, 2.6, and 3.0 for the experiment. Hydrochloric acid (HCl), sulfuric acid (H2 SO4 ), or acetic acid (CH3 COOH) was used to adjust the pH of the extraction solution. In addition, the effects of the extraction time (3, 6, 9, 12 min), ratio of extraction solution to biomass (1:1, 2:1, 3:1, 4:1, v/w), extraction temperature (30, 35, 40, 45, 50 ◦ C), and methanol concentration in the extraction solution (85, 90, 95, 100%, v/v) in the simultaneous process were investigated based on the optimal conditions in our previous studies [21,26,27].
2.3. Conventional solvent extraction (CSE) The biomass from plant cell cultures was mixed with 90% aqueous methanol and stirred at a room temperature for 30 min. The mixture was filtered under vacuum in a Buchner funnel through filter paper (150 mm, Whatman), where the 90% aqueous methanol was preferably added to biomass at a ratio of 1:1 (mL/g, v/w). Extraction was performed four times with new 90% aqueous methanol [10]. Each methanol extract was collected, pooled, concentrated and dried at 40 ◦ C under vacuum (635 mm Hg) for HPLC analysis. 2.4. Microwave-assisted extraction (MAE) The microwave facility (2450 MHz Model 1501, Korea Microwave Instrument Co., Korea) used for MAE consisted of a microwave generator, cooling system, and extraction unit (Fig. 1) [14]. A thermocouple was installed to measure temperature changes continuously during extraction. The cooling system condensed vaporized solvent, thereby returning it to the reactor. The microwave power (150 W) supply was operated by a computer program to control the temperature. The extraction temperature, extraction time, and extraction solution (90% aqueous methanol)/biomass ratio were 40 ◦ C, 6 min, and 1:1 (v/w), respectively [21]. After extraction, the mixture was filtered under vacuum in a Buchner funnel through filter paper (150 mm, Whatman). The methanol extract was collected,
Fig. 2. Effect of type of acid on the simultaneous microwave-assisted extraction and acid hydrolysis process. The extraction solvent, temperature, time, and solvent/biomass ratio were 90% aqueous methanol, 40 ◦ C, 6 min, and 1:1 (v/w), respectively. The control means the experimental result of CSE without acid hydrolysis.
Please cite this article in press as: Kim G-J, Kim J-H. Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.11.009
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3. Results and discussion 3.1. Development of simultaneous MAE/acid hydrolysis process The effect of acid addition for acid hydrolysis in MAE on the extraction efficiency was investigated. Based on the paclitaxel recovery obtained by extracting four times with the existing CSE method, the paclitaxel recovery in MAE was investigated. The MAE was performed under the optimum extraction temperature (40 ◦ C) and time (6 min) determined in a previous study [21]. HCl, H2 SO4 , or CH3 COOH was added to the extraction solution (90% aqueous methanol) and the pH of the extraction solution was adjusted to 2.2 for extraction. When the acid was HCl, H2 SO4 , and CH3 COOH, the relative percentage of paclitaxel increase was 220%, 205%, and 185%, respectively (Fig. 2). The highest paclitaxel recovery obtained under H2 SO4 or CH3 COOH hydrolysis was lower than that under HCl hydrolysis, implying that HCl could break glycoside in biomass better than H2 SO4 and CH3 COOH. However, the reason is not clear. This result was also observed in the acid hydrolysis of sugarcane bagasse for obtaining the hydrolysate, where the highest xylose concentration obtained under HCl hydrolysis was higher than that under H2 SO4 hydrolysis [28]. Among these acids, HCl gave the highest paclitaxel recovery in MAE, and so HCl was used for this study. To confirm the effect of pH of extraction solution, HCl was added to the extraction solution (90% aqueous methanol) and the pH of the extraction solution was adjusted to 1.8, 2.2, 2.6, and 3.0 for extraction. As shown in Fig. 3, the extraction recovery of paclitaxel increased as the pH increased from 1.8 to 2,2, and decreased as the pH increased from 2.2 to 3.0, which may due to the decrease of hydrolysis efficiency at high pH of extraction solution. When the pH of the extraction solution was 2.2, the paclitaxel recovery increased 2.2-fold compared to control (CSE). A common form of anticancer agents existing in natural substances is glycoside, and the recovery of paclitaxel seems to have increased due to acid hydrolysis of glycoside contained in the biomass. In other words, glycoside is divided into sugar bound to a glycoside and paclitaxel by acid hydrolysis (Fig. 4). Therefore, the paclitaxel recovery increased. This result can be confirmed by investigating changes in the content of glycoside (sugar-binding paclitaxel; 7xylosyl paclitaxel, 7-xylosyl-10-deacetylpaclitaxel) and paclitaxel in the biomass before and after acid hydrolysis (Fig. 5). These compounds were identified and confirmed by comparing their retention times (RTs) with those of authentic standards. Each compound also spiked with the standard. The retention times of 7-xylosyl paclitaxel (RT: 27 min) and paclitaxel (RT: 32 min) were perfectly matched those of authentic standards by HPLC analysis. The content of 7-xylosyl paclitaxel in the biomass decreased after acid hydrolysis whereas the paclitaxel content increased. It was found that the increase of paclitaxel was perfectly proportional to the decrease of glycoside by HPLC analysis. On the other hand, 7-xylosyl-10-deacetylpaclitaxel was not found in the biomass.
Fig. 3. Effect of pH on the simultaneous microwave-assisted extraction and acid hydrolysis process. The extraction solvent, temperature, time, and solvent/biomass ratio were 90% aqueous methanol, 40 ◦ C, 6 min, and 1:1 (v/w), respectively. The control means the experimental result of CSE without acid hydrolysis.
Therefore, it was confirmed that the paclitaxel recovery increased because the glycosidic bonds of a glycoside (sugar-binding paclitaxel; 7-xylosyl paclitaxel) in the biomass broke down due to acid hydrolysis. To optimize major process variables in the simultaneous process, the pH of the extraction solution was adjusted to 2.2 and the effect of the extraction time (3, 6, 9, 12 min) was investigated. As shown in Fig. 6, the maximum recovery of paclitaxel was obtained at 6 min. The paclitaxel recovery rapidly decreased after 6 min of extraction, the reason for which appears to be that the product decomposed due to overexposure to microwaves during the increased extraction time [21]. This result was also observed in the extraction of triterpenoid saponins from Ganoderma atrum, where the recovery of some saponins decreased with increasing irradiation time [15]. In the case of the simultaneous process, the highest recovery was obtained after 6 min of extraction at an extraction solution pH of 2.2, which agrees with the results of a previous study [21]. Thus, the optimal extraction time was set to 6 min to maximize the efficiency of the simultaneous MAE/acid hydrolysis process. The ratio of the extraction solution and biomass was adjusted to 1:1, 2:1, 3:1, and 4:1 (v/w). The simultaneous process was performed at a ratio of extraction solution to biomass of 1:1 (v/w) or higher because there was difficulty in mixing at lower ratios.
Fig. 4. Theory of acid hydrolysis.
Please cite this article in press as: Kim G-J, Kim J-H. Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.11.009
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Fig. 5. Comparison of HPLC chromatograms of 7-xylosyl paclitaxel and paclitaxel in MAE method (A) and simultaneous MAE/acid hydrolysis method (B).
Fig. 6. Effect of extraction time on the simultaneous microwave-assisted extraction and acid hydrolysis process. The extraction solvent, temperature, pH of solvent, and solvent/biomass ratio were 90% aqueous methanol, 40 ◦ C, 2.2, and 1:1 (v/w), respectively. The control means the experimental result of CSE without acid hydrolysis.
Paclitaxel recovery decreased as the ratio of extraction solution to biomass was increased, and the highest recovery was obtained at a ratio of 1:1 (v/w) (Fig. 7). This result was probably due to inadequate stirring of the solvent when the microwaves were applied to larger volumes [15,26]. Also, microwave energy is absorbed and dispersed by larger amounts of plant materials, an effect which is disadvantageous for the extraction process [26]. If the extraction was carried out under a low solid-to-liquid ratio, the concentration of the extraction solution was low, thus requiring more energy and time to condense the extraction solution in the later separation and purification process. Consequently, it was found that the optimal ratio of extraction solution to biomass is 1:1 (v/w), which is in agreement with that of a previous study [26]. To optimize the extraction temperature (30, 35, 40, 45, and 50 ◦ C), the simultaneous MAE/acid hydrolysis process was performed at a fixed pH of extraction solution (2.2), extraction time (6 min), and ratio of extraction solution to biomass (1:1, v/w). As shown in Fig. 8, the maximum recovery of paclitaxel was obtained at 40 ◦ C, but when the temperature was increased to 45 ◦ C and 50 ◦ C, the paclitaxel recovery rapidly decreased. This phenomenon appears to be due to the decomposition of paclitaxel at higher extraction temperatures [26]. The polarity index of an organic solvent plays a very important role in MAE because a polar solvent that has a higher dielectric constant can absorb more microwave energy [29]. Thus, the effects of
Please cite this article in press as: Kim G-J, Kim J-H. Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.11.009
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Fig. 7. Effect of solvent/biomass ratio on the simultaneous microwave-assisted extraction and acid hydrolysis process. The extraction solvent, temperature, pH of solvent, and extraction time were 90% aqueous methanol, 40 ◦ C, 2.2, and 6 min, respectively. The control means the experimental result of CSE without acid hydrolysis.
solvent water content were investigated by conducting MAE with methanol at a concentration of 85, 90, 95, and 100% (v/v) under the following optimized conditions for the simultaneous process: pH of the extraction solution, 2.2; extraction time, 6 min; extraction temperature, 40 ◦ C; ratio of extraction solution and biomass, 1:1 (v/w). As shown in Fig. 9, the paclitaxel recovery was the highest at 90% aqueous methanol. Such as with the existing extraction process [27], it was confirmed that 90% aqueous methanol was appropriate for the MAE/acid hydrolysis simultaneous process. This result suggests that the water, itself a polar solvent, improved the efficiency of MAE, apparently by efficiently absorbing microwave energy and heating up. Thus, the addition of a small amount of water may
Fig. 8. Effect of extraction temperature on the simultaneous microwave-assisted extraction and acid hydrolysis process. The extraction solvent, time, pH of solvent, and solvent/biomass ratio were 90% aqueous methanol, 6 min, 2.2, and 1:1 (v/w), respectively. The control means the experimental result of CSE without acid hydrolysis.
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Fig. 9. Effect of aqueous solvent concentration on paclitaxel recovery in the simultaneous microwave-assisted extraction and acid hydrolysis process. The extraction time, temperature, pH of solvent, and solvent/biomass ratio were 6 min, 40 ◦ C, 2.2, and 1:1 (v/w), respectively. The control means the experimental result of CSE without acid hydrolysis.
have improved paclitaxel extraction efficiency by swelling the plant material and increasing the contact surface area between the plant matrix and the solvent [30]. If the water content was too high, however, the recovery decreased, a result which is in agreement with those of the studies by Hemwimon et al. [29] and Xiao et al. [30]. Thus, recovery can be increased by using an extraction solvent of appropriate water content, but if the water content is higher than the optimal mixing composition of water and the organic solvent, it could interfere with the contact between the plant matrix and the solvent, thereby lowering the solubility of the target substance. 3.2. Comparison of CSE/acid hydrolysis and MAE/acid hydrolysis with CSE and MAE To improve the existing extraction process for paclitaxel, the recovery in CSE, CSE/acid hydrolysis simultaneous process, MAE, and MAE/acid hydrolysis simultaneous process was compared. In CSE [10], extraction was performed four times using 90% aqueous methanol (extraction solution) at room temperature with 30 min of extraction time and a 1:1 (v/w) extraction solution/biomass ratio. In MAE, extraction was performed once using 90% aqueous methanol at 40 ◦ C with 6 min of extraction time and a 1:1 (v/w) extraction solution/biomass ratio. In the CSE/acid hydrolysis simultaneous process and MAE/acid hydrolysis simultaneous process, HCl was added to 90% aqueous methanol (extraction solution) to adjust the pH of the solution to the optimized pH (2.2). As shown in Fig. 10, the relative percentage of paclitaxel increase in the MAE/acid hydrolysis simultaneous process and CSE/acid hydrolysis simultaneous process increased by >220% and >170% compared to the MAE process and CSE process, respectively. In addition, in regard to the time required for extraction, extraction in CSE was performed four times, requiring 30 min per extraction. However, extraction in MAE was performed once only for 6 min and most of paclitaxel was recovered from the biomass. Thus, the process proposed herein provides advantages in terms of efficiency in that the extraction time is shorter and less solvent is required. Therefore, it is expected that the cost of the paclitaxel separation/purification process could be dramatically reduced by maximizing the recovery of paclitaxel from plant cell cultures using the process developed in this study.
Please cite this article in press as: Kim G-J, Kim J-H. Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.11.009
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References
Fig. 10. Comparison of CSE, CSE/acid hydrolysis, MAE, and MAE/acid hydrolysis. In CSE, extraction was performed four times using 90% aqueous methanol at room temperature with 30 min of extraction time and a 1:1 (v/w) extraction solution/biomass ratio. In MAE, extraction was performed once using 90% aqueous methanol at 40 ◦ C with 6 min of extraction time and a 1:1 (v/w) extraction solution/biomass ratio. In CSE/acid hydrolysis and MAE/acid hydrolysis, the pH of extraction solution was 2.2.
4. Conclusions In this study, the MAE/acid hydrolysis simultaneous process was developed to improve the recovery of the anticancer agent paclitaxel from plant cell cultures of Taxus chinensis. In addition, the efficiency of the process was maximized by investigating the mechanism of acid hydrolysis and by optimizing the major process variables, such as the pH of the extraction solution, extraction temperature, extraction time, and ratio of the extraction solution and biomass in the MAE/acid hydrolysis simultaneous process. The paclitaxel recovery was 2.2-fold higher than that of MAE under the following conditions: pH of the extraction solution, 2.2; extraction temperature, 40 ◦ C; extraction time, 6 min; ratio of the extraction solution and biomass, 1:1 (v/w); extraction solution, 90% aqueous methanol. To determine the cause of the increase in paclitaxel recovery, changes in the content of glycoside (sugar-binding paclitaxel; 7-xylosyl paclitaxel and 7-xylosyl-10-deacetyl paclitaxel) and paclitaxel were investigated after acid hydrolysis in the MAE process. The content of 7-xylosyl paclitaxel after acid hydrolysis decreased whereas the content of paclitaxel increased. Based on this result, it was confirmed that acid hydrolysis increased paclitaxel recovery by the cleavage of glycosidic bonds in a glycoside (sugar-binding paclitaxel). As a result, the paclitaxel recovery increased by 1.7- and 2.2-fold when applying acid hydrolysis to CSE and MAE, respectively. In addition, in regard to the time required for extraction, extraction in CSE was performed four times, requiring 30 min per extraction. However, most of the paclitaxel could be recovered from the biomass by MAE in a 6 min extraction, making it very economical. Acknowledgment This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST) (No. 2011-0010907).
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Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures Gun-Joong Kim, Jin-Hyun Kim ∗ Department of Chemical Engineering, Kongju National University, Cheonan 330-717, South Korea
a r t i c l e
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Article history: Received 11 July 2014 Received in revised form 22 November 2014 Accepted 25 November 2014 Available online xxx Keywords: Plant cell culture Paclitaxel Microwave-assisted extraction (MAE) Acid hydrolysis Simultaneous process
a b s t r a c t In this study, a microwave-assisted extraction (MAE)/acid hydrolysis simultaneous process was developed for recovering the anticancer agent paclitaxel from plant cell cultures of Taxus chinensis. The optimal pH of the extraction solution (90% aqueous methanol) for hydrolysis was 2.2 at fixed extraction time (6 min), ratio of extraction solution to biomass (1:1, v/w), and extraction temperature (40 ◦ C). In the MAE/acid hydrolysis simultaneous process, the paclitaxel recovery was 2.2-fold higher than in the existing extraction methods. Regarding changes in the content of glycoside (7-xylosyl paclitaxel as sugar-binding paclitaxel) and paclitaxel depending on the inclusion of acid hydrolysis in the MAE process, the content of 7-xylosyl paclitaxel decreased after acid hydrolysis whereas the content of paclitaxel increased. Based on this result, it was confirmed that acid hydrolysis breaks down a glycosidic bond of glycoside (sugar-binding paclitaxel), and so the recovery of paclitaxel increases. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Paclitaxel is an anticancer agent that is a diterpenoid found in the bark of the yew tree [1,2]. It is an FDA (Food and Drug Administration)-approved anticancer drug most widely used for treatment of ovarian cancer, breast cancer, Kaposi’s sarcoma and non-small cell lung cancer [3]. Its application has also been expanded to the treatment of acute rheumatoid arthritis and Alzheimer’s disease. Since clinical trials regarding its combined prescription with various other treatments are underway, the demand for paclitaxel is expected to increase [4,5]. The main paclitaxel production methods are direct extraction from the yew tree, semisynthesis, and plant cell culture [6–8]. Among these methods, plant cell culture enables stable mass production of paclitaxel of consistent quality in a bioreactor without being affected by such external factors as climate and environment [7]. Most of the paclitaxel produced by plant cell culture is contained in plant cells and the debris [9], and it is important to effectively extract paclitaxel from cells in regard to increasing the recovery. The existing extraction methods used most frequently use an organic solvent to recover paclitaxel from the biomass, a plant cell [10–12]. However, the conventional solvent extraction (CSE) method requires a long extraction time and large amounts of organic solvents and has a low extraction
∗ Corresponding author. Tel.: +82 41 521 9361; fax: +82 41 554 2640. E-mail address: [email protected] (J.-H. Kim).
efficiency. To overcome these disadvantages, microwave-assisted extraction (MAE) has been studied [13]. During MAE, microwaves heat the solvent or solvent mixture directly. In addition, the direct interaction of microwaves with free water molecules present in glands and vascular systems results in the subsequent rupture of plant tissue and the release of active compounds into the organic solvent, which increases recovery. Compared with CSE, MAE has many advantages, which include a shorter extraction time, a lower solvent requirement, a higher extraction rate, and production of a higher-quality product at lower cost [14]. Recently, research studies have been conducted on the development of MAE methods for the extraction of saponin from Ganoderma atrum [15], camptothecin from Nothapodytes foetida [16], essential oil from Elettaria cardamomum [17], ginsenoside from ginseng root [18], glycyrrhizic acid from licorice root [19] and polyphenol and caffeine from green tea leaves [20]. In our previous study [21], we confirmed the possibility that the extraction of paclitaxel from plant cell cultures using MAE could overcome the aforementioned problems with CSE. In 2000, Kim et al. [22] reported that the recovery of paclitaxel from the supernatant of plant cell culture increased by hydrolysis. It was assumed that the increase in recovery of paclitaxel in the supernatant of plant cell culture by acid hydrolysis was caused by the cleavage of 7-xylosyl paclitaxel and 7-xylosyl-10deacetylpaclitaxel, which are glycosides (sugar-binding paclitaxel) found in the cell culture supernatant (the cleavage of glycosidic bonds between sugar and paclitaxel) [23]. However, the
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Please cite this article in press as: Kim G-J, Kim J-H. Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.11.009
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cause of the increase in recovery of paclitaxel by acid hydrolysis has still not been clarified. Therefore, in this study, we developed the simultaneous MAE and acid hydrolysis process to increase the recovery of paclitaxel from plant cells of Taxus chinensis. In particular, the efficiency of the process was maximized by investigating and understanding the mechanism of acid hydrolysis of glycosides in the MAE/acid hydrolysis simultaneous process. 2. Materials and methods 2.1. Plant materials A suspension of plant cells originating from Taxus chinensis was cultured in a bioreactor [24]. Following cultivation, biomass (plant cell and debris) was recovered using a decanter (CA150 Clarifying Decanter; Westfalia, Germany) and a high-speed centrifuge (BTPX 205GD – 35CDEFP; Alfa Laval, Sweden). The biomass was provided by Samyang Genex Company, South Korea. 2.2. Paclitaxel and glycoside (7-xylosyl paclitaxel, 7-xylosyl-10-deacetylpaclitaxel) analysis An HPLC system (SCL-10AVP, Shimadzu, Japan) and a Capcell Pak C18 column (250 × 4.6 mm, Shiseido) were used to analyze the paclitaxel and glycoside contents. Acetonitrile/water (35:65–65:35, v/v gradient) for paclitaxel and acetonitrile/water (25:75–65:35, v/v gradient) for glycoside were used as the mobile phase. Using a UV detector, paclitaxel and glycoside were analyzed at 227 nm and 228 nm, respectively [25]. In addition, the flow rate and sample injection volume were 1.0 mL/min and 20 L, respectively. Authentic paclitaxel, 7-xylosyl paclitaxel and the 7xylosyl-10-deacetylpaclitaxel were purchased from Sigma–Aldrich (purity: 95%), Quality Phytochemicals (purity: 99%), and Santa Cruz Biotechnology (purity: 60%), respectively, and used as standards. Each sample was analyzed in triplicate.
Fig. 1. Schematic diagram of the microwave-assisted extraction (MAE) process.
concentrated and dried at 40 ◦ C under vacuum (635 mm Hg) for HPLC analysis.
2.5. Simultaneous MAE and acid hydrolysis process In our previous studies [21,26,27], the optimal extraction time, ratio of extraction solution to biomass, extraction temperature, and extraction solvent in MAE were found to be 6 min, 1:1 (v/w), 40 ◦ C, and 90% aqueous methanol, respectively. The pH of extraction solution in the simultaneous process was optimized under these optimal conditions. The pH of the extraction solution (90% aqueous methanol) was adjusted to 1.8, 2.2, 2.6, and 3.0 for the experiment. Hydrochloric acid (HCl), sulfuric acid (H2 SO4 ), or acetic acid (CH3 COOH) was used to adjust the pH of the extraction solution. In addition, the effects of the extraction time (3, 6, 9, 12 min), ratio of extraction solution to biomass (1:1, 2:1, 3:1, 4:1, v/w), extraction temperature (30, 35, 40, 45, 50 ◦ C), and methanol concentration in the extraction solution (85, 90, 95, 100%, v/v) in the simultaneous process were investigated based on the optimal conditions in our previous studies [21,26,27].
2.3. Conventional solvent extraction (CSE) The biomass from plant cell cultures was mixed with 90% aqueous methanol and stirred at a room temperature for 30 min. The mixture was filtered under vacuum in a Buchner funnel through filter paper (150 mm, Whatman), where the 90% aqueous methanol was preferably added to biomass at a ratio of 1:1 (mL/g, v/w). Extraction was performed four times with new 90% aqueous methanol [10]. Each methanol extract was collected, pooled, concentrated and dried at 40 ◦ C under vacuum (635 mm Hg) for HPLC analysis. 2.4. Microwave-assisted extraction (MAE) The microwave facility (2450 MHz Model 1501, Korea Microwave Instrument Co., Korea) used for MAE consisted of a microwave generator, cooling system, and extraction unit (Fig. 1) [14]. A thermocouple was installed to measure temperature changes continuously during extraction. The cooling system condensed vaporized solvent, thereby returning it to the reactor. The microwave power (150 W) supply was operated by a computer program to control the temperature. The extraction temperature, extraction time, and extraction solution (90% aqueous methanol)/biomass ratio were 40 ◦ C, 6 min, and 1:1 (v/w), respectively [21]. After extraction, the mixture was filtered under vacuum in a Buchner funnel through filter paper (150 mm, Whatman). The methanol extract was collected,
Fig. 2. Effect of type of acid on the simultaneous microwave-assisted extraction and acid hydrolysis process. The extraction solvent, temperature, time, and solvent/biomass ratio were 90% aqueous methanol, 40 ◦ C, 6 min, and 1:1 (v/w), respectively. The control means the experimental result of CSE without acid hydrolysis.
Please cite this article in press as: Kim G-J, Kim J-H. Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.11.009
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3. Results and discussion 3.1. Development of simultaneous MAE/acid hydrolysis process The effect of acid addition for acid hydrolysis in MAE on the extraction efficiency was investigated. Based on the paclitaxel recovery obtained by extracting four times with the existing CSE method, the paclitaxel recovery in MAE was investigated. The MAE was performed under the optimum extraction temperature (40 ◦ C) and time (6 min) determined in a previous study [21]. HCl, H2 SO4 , or CH3 COOH was added to the extraction solution (90% aqueous methanol) and the pH of the extraction solution was adjusted to 2.2 for extraction. When the acid was HCl, H2 SO4 , and CH3 COOH, the relative percentage of paclitaxel increase was 220%, 205%, and 185%, respectively (Fig. 2). The highest paclitaxel recovery obtained under H2 SO4 or CH3 COOH hydrolysis was lower than that under HCl hydrolysis, implying that HCl could break glycoside in biomass better than H2 SO4 and CH3 COOH. However, the reason is not clear. This result was also observed in the acid hydrolysis of sugarcane bagasse for obtaining the hydrolysate, where the highest xylose concentration obtained under HCl hydrolysis was higher than that under H2 SO4 hydrolysis [28]. Among these acids, HCl gave the highest paclitaxel recovery in MAE, and so HCl was used for this study. To confirm the effect of pH of extraction solution, HCl was added to the extraction solution (90% aqueous methanol) and the pH of the extraction solution was adjusted to 1.8, 2.2, 2.6, and 3.0 for extraction. As shown in Fig. 3, the extraction recovery of paclitaxel increased as the pH increased from 1.8 to 2,2, and decreased as the pH increased from 2.2 to 3.0, which may due to the decrease of hydrolysis efficiency at high pH of extraction solution. When the pH of the extraction solution was 2.2, the paclitaxel recovery increased 2.2-fold compared to control (CSE). A common form of anticancer agents existing in natural substances is glycoside, and the recovery of paclitaxel seems to have increased due to acid hydrolysis of glycoside contained in the biomass. In other words, glycoside is divided into sugar bound to a glycoside and paclitaxel by acid hydrolysis (Fig. 4). Therefore, the paclitaxel recovery increased. This result can be confirmed by investigating changes in the content of glycoside (sugar-binding paclitaxel; 7xylosyl paclitaxel, 7-xylosyl-10-deacetylpaclitaxel) and paclitaxel in the biomass before and after acid hydrolysis (Fig. 5). These compounds were identified and confirmed by comparing their retention times (RTs) with those of authentic standards. Each compound also spiked with the standard. The retention times of 7-xylosyl paclitaxel (RT: 27 min) and paclitaxel (RT: 32 min) were perfectly matched those of authentic standards by HPLC analysis. The content of 7-xylosyl paclitaxel in the biomass decreased after acid hydrolysis whereas the paclitaxel content increased. It was found that the increase of paclitaxel was perfectly proportional to the decrease of glycoside by HPLC analysis. On the other hand, 7-xylosyl-10-deacetylpaclitaxel was not found in the biomass.
Fig. 3. Effect of pH on the simultaneous microwave-assisted extraction and acid hydrolysis process. The extraction solvent, temperature, time, and solvent/biomass ratio were 90% aqueous methanol, 40 ◦ C, 6 min, and 1:1 (v/w), respectively. The control means the experimental result of CSE without acid hydrolysis.
Therefore, it was confirmed that the paclitaxel recovery increased because the glycosidic bonds of a glycoside (sugar-binding paclitaxel; 7-xylosyl paclitaxel) in the biomass broke down due to acid hydrolysis. To optimize major process variables in the simultaneous process, the pH of the extraction solution was adjusted to 2.2 and the effect of the extraction time (3, 6, 9, 12 min) was investigated. As shown in Fig. 6, the maximum recovery of paclitaxel was obtained at 6 min. The paclitaxel recovery rapidly decreased after 6 min of extraction, the reason for which appears to be that the product decomposed due to overexposure to microwaves during the increased extraction time [21]. This result was also observed in the extraction of triterpenoid saponins from Ganoderma atrum, where the recovery of some saponins decreased with increasing irradiation time [15]. In the case of the simultaneous process, the highest recovery was obtained after 6 min of extraction at an extraction solution pH of 2.2, which agrees with the results of a previous study [21]. Thus, the optimal extraction time was set to 6 min to maximize the efficiency of the simultaneous MAE/acid hydrolysis process. The ratio of the extraction solution and biomass was adjusted to 1:1, 2:1, 3:1, and 4:1 (v/w). The simultaneous process was performed at a ratio of extraction solution to biomass of 1:1 (v/w) or higher because there was difficulty in mixing at lower ratios.
Fig. 4. Theory of acid hydrolysis.
Please cite this article in press as: Kim G-J, Kim J-H. Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.11.009
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Fig. 5. Comparison of HPLC chromatograms of 7-xylosyl paclitaxel and paclitaxel in MAE method (A) and simultaneous MAE/acid hydrolysis method (B).
Fig. 6. Effect of extraction time on the simultaneous microwave-assisted extraction and acid hydrolysis process. The extraction solvent, temperature, pH of solvent, and solvent/biomass ratio were 90% aqueous methanol, 40 ◦ C, 2.2, and 1:1 (v/w), respectively. The control means the experimental result of CSE without acid hydrolysis.
Paclitaxel recovery decreased as the ratio of extraction solution to biomass was increased, and the highest recovery was obtained at a ratio of 1:1 (v/w) (Fig. 7). This result was probably due to inadequate stirring of the solvent when the microwaves were applied to larger volumes [15,26]. Also, microwave energy is absorbed and dispersed by larger amounts of plant materials, an effect which is disadvantageous for the extraction process [26]. If the extraction was carried out under a low solid-to-liquid ratio, the concentration of the extraction solution was low, thus requiring more energy and time to condense the extraction solution in the later separation and purification process. Consequently, it was found that the optimal ratio of extraction solution to biomass is 1:1 (v/w), which is in agreement with that of a previous study [26]. To optimize the extraction temperature (30, 35, 40, 45, and 50 ◦ C), the simultaneous MAE/acid hydrolysis process was performed at a fixed pH of extraction solution (2.2), extraction time (6 min), and ratio of extraction solution to biomass (1:1, v/w). As shown in Fig. 8, the maximum recovery of paclitaxel was obtained at 40 ◦ C, but when the temperature was increased to 45 ◦ C and 50 ◦ C, the paclitaxel recovery rapidly decreased. This phenomenon appears to be due to the decomposition of paclitaxel at higher extraction temperatures [26]. The polarity index of an organic solvent plays a very important role in MAE because a polar solvent that has a higher dielectric constant can absorb more microwave energy [29]. Thus, the effects of
Please cite this article in press as: Kim G-J, Kim J-H. Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.11.009
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Fig. 7. Effect of solvent/biomass ratio on the simultaneous microwave-assisted extraction and acid hydrolysis process. The extraction solvent, temperature, pH of solvent, and extraction time were 90% aqueous methanol, 40 ◦ C, 2.2, and 6 min, respectively. The control means the experimental result of CSE without acid hydrolysis.
solvent water content were investigated by conducting MAE with methanol at a concentration of 85, 90, 95, and 100% (v/v) under the following optimized conditions for the simultaneous process: pH of the extraction solution, 2.2; extraction time, 6 min; extraction temperature, 40 ◦ C; ratio of extraction solution and biomass, 1:1 (v/w). As shown in Fig. 9, the paclitaxel recovery was the highest at 90% aqueous methanol. Such as with the existing extraction process [27], it was confirmed that 90% aqueous methanol was appropriate for the MAE/acid hydrolysis simultaneous process. This result suggests that the water, itself a polar solvent, improved the efficiency of MAE, apparently by efficiently absorbing microwave energy and heating up. Thus, the addition of a small amount of water may
Fig. 8. Effect of extraction temperature on the simultaneous microwave-assisted extraction and acid hydrolysis process. The extraction solvent, time, pH of solvent, and solvent/biomass ratio were 90% aqueous methanol, 6 min, 2.2, and 1:1 (v/w), respectively. The control means the experimental result of CSE without acid hydrolysis.
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Fig. 9. Effect of aqueous solvent concentration on paclitaxel recovery in the simultaneous microwave-assisted extraction and acid hydrolysis process. The extraction time, temperature, pH of solvent, and solvent/biomass ratio were 6 min, 40 ◦ C, 2.2, and 1:1 (v/w), respectively. The control means the experimental result of CSE without acid hydrolysis.
have improved paclitaxel extraction efficiency by swelling the plant material and increasing the contact surface area between the plant matrix and the solvent [30]. If the water content was too high, however, the recovery decreased, a result which is in agreement with those of the studies by Hemwimon et al. [29] and Xiao et al. [30]. Thus, recovery can be increased by using an extraction solvent of appropriate water content, but if the water content is higher than the optimal mixing composition of water and the organic solvent, it could interfere with the contact between the plant matrix and the solvent, thereby lowering the solubility of the target substance. 3.2. Comparison of CSE/acid hydrolysis and MAE/acid hydrolysis with CSE and MAE To improve the existing extraction process for paclitaxel, the recovery in CSE, CSE/acid hydrolysis simultaneous process, MAE, and MAE/acid hydrolysis simultaneous process was compared. In CSE [10], extraction was performed four times using 90% aqueous methanol (extraction solution) at room temperature with 30 min of extraction time and a 1:1 (v/w) extraction solution/biomass ratio. In MAE, extraction was performed once using 90% aqueous methanol at 40 ◦ C with 6 min of extraction time and a 1:1 (v/w) extraction solution/biomass ratio. In the CSE/acid hydrolysis simultaneous process and MAE/acid hydrolysis simultaneous process, HCl was added to 90% aqueous methanol (extraction solution) to adjust the pH of the solution to the optimized pH (2.2). As shown in Fig. 10, the relative percentage of paclitaxel increase in the MAE/acid hydrolysis simultaneous process and CSE/acid hydrolysis simultaneous process increased by >220% and >170% compared to the MAE process and CSE process, respectively. In addition, in regard to the time required for extraction, extraction in CSE was performed four times, requiring 30 min per extraction. However, extraction in MAE was performed once only for 6 min and most of paclitaxel was recovered from the biomass. Thus, the process proposed herein provides advantages in terms of efficiency in that the extraction time is shorter and less solvent is required. Therefore, it is expected that the cost of the paclitaxel separation/purification process could be dramatically reduced by maximizing the recovery of paclitaxel from plant cell cultures using the process developed in this study.
Please cite this article in press as: Kim G-J, Kim J-H. Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.11.009
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References
Fig. 10. Comparison of CSE, CSE/acid hydrolysis, MAE, and MAE/acid hydrolysis. In CSE, extraction was performed four times using 90% aqueous methanol at room temperature with 30 min of extraction time and a 1:1 (v/w) extraction solution/biomass ratio. In MAE, extraction was performed once using 90% aqueous methanol at 40 ◦ C with 6 min of extraction time and a 1:1 (v/w) extraction solution/biomass ratio. In CSE/acid hydrolysis and MAE/acid hydrolysis, the pH of extraction solution was 2.2.
4. Conclusions In this study, the MAE/acid hydrolysis simultaneous process was developed to improve the recovery of the anticancer agent paclitaxel from plant cell cultures of Taxus chinensis. In addition, the efficiency of the process was maximized by investigating the mechanism of acid hydrolysis and by optimizing the major process variables, such as the pH of the extraction solution, extraction temperature, extraction time, and ratio of the extraction solution and biomass in the MAE/acid hydrolysis simultaneous process. The paclitaxel recovery was 2.2-fold higher than that of MAE under the following conditions: pH of the extraction solution, 2.2; extraction temperature, 40 ◦ C; extraction time, 6 min; ratio of the extraction solution and biomass, 1:1 (v/w); extraction solution, 90% aqueous methanol. To determine the cause of the increase in paclitaxel recovery, changes in the content of glycoside (sugar-binding paclitaxel; 7-xylosyl paclitaxel and 7-xylosyl-10-deacetyl paclitaxel) and paclitaxel were investigated after acid hydrolysis in the MAE process. The content of 7-xylosyl paclitaxel after acid hydrolysis decreased whereas the content of paclitaxel increased. Based on this result, it was confirmed that acid hydrolysis increased paclitaxel recovery by the cleavage of glycosidic bonds in a glycoside (sugar-binding paclitaxel). As a result, the paclitaxel recovery increased by 1.7- and 2.2-fold when applying acid hydrolysis to CSE and MAE, respectively. In addition, in regard to the time required for extraction, extraction in CSE was performed four times, requiring 30 min per extraction. However, most of the paclitaxel could be recovered from the biomass by MAE in a 6 min extraction, making it very economical. Acknowledgment This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST) (No. 2011-0010907).
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Please cite this article in press as: Kim G-J, Kim J-H. Development of a simultaneous extraction and acid hydrolysis process for recovery of paclitaxel from plant cell cultures. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.11.009