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Increasing the efficiency of the separation and purification process for paclitaxel by pre-treatment with water
Process Biochemistry 51 (2016) 1738–1743
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Increasing the efficiency of the separation and purification process for paclitaxel by pre-treatment with water Chung-Gi Lee, Jin-Hyun Kim ∗ Department of Chemical Engineering, Kongju NationalUniversity, Cheonan 330-717, South Korea
a r t i c l e
i n f o
Article history: Received 14 January 2016 Received in revised form 6 July 2016 Accepted 27 July 2016 Available online 30 July 2016 Keywords: Paclitaxel Water pre-treatment Purification Process Development
a b s t r a c t In this study, a water pre-treatment method for the separation and purification of an anticancer agent paclitaxel from plant cell cultures was developed. When the methanol extract obtained by biomass extraction was pre-treated with water, a high yield (>99%) of high-purity (∼12.1%) paclitaxel was observed within a short period of time (∼10 min). In addition, in the follow-up pre-purification process, using a crude extract obtained by water pre-treatment, high-yield (∼87.9%), high-purity (50% or higher) paclitaxel was obtained in a short operating time (∼2.9 h). Thus, the efficiency of the separation and purification process for paclitaxel was improved dramatically by water pre-treatment, particularly in terms of the reduction of operating times and the simplification of processes. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Paclitaxel is a diterpenoid anticancer agent found in the bark of the yew tree. The chemical structure and the anticancer mechanism were investigated by Wani et al. [1] in 1971 and Schiff et al. [2] in 1979. Since its discovery, paclitaxel has been used as an important anticancer drug, with the approval of the U.S. Food and Drug Administration, to treat ovarian cancer, breast cancer, Kaposi’s sarcoma and non-small cell lung cancer. The main production methods for paclitaxel are; 1) paclitaxel is directly extracted from yew trees [3]; 2) precursors (baccatin III, 10-deacetylbaccatin III, 10-deacetylpaclitaxel, etc.) are obtained from the leaves of yew trees to combine side chains chemically for semi-synthesis [4]; and 3) callus is induced into yew trees and then the plants’ cells are cultured in a bioreactor after seed culture [5]. In the latter case, the plant cell culture is not affected by external factors, such as climate or the environment, and can be stably produced in a bioreactor. This makes the mass production of paclitaxel of a consistent quality possible, allowing production to meet with increasing demand [6]. Many separation and purification steps are required to obtain high-purity paclitaxel from plant cell cultures. In general, paclitaxel is extracted from the biomass by using organic solvents and then high-purity paclitaxel is obtained through pre-purification
∗ Corresponding author. E-mail address: [email protected] (J.-H. Kim). http://dx.doi.org/10.1016/j.procbio.2016.07.025 1359-5113/© 2016 Elsevier Ltd. All rights reserved.
and final purification steps. However, in previous studies, expensive chromatography has been applied to the pre-purification process for the final purification stage, or a crude extract has been directly used in final purification by HPLC. In particular, if a low-purity sample is used for final purification by HPLC without pre-purification, a large amount of organic solvent is consumed and the lifetime of column packing materials (resin) and throughput are reduced. This is very uneconomical, rendering this process unsuitable for mass production [7–10]. Therefore, efficient and economical pre-purification is definitely required to obtain a high-yield of high-purity paclitaxel. According to previous studies, the pre-purification process for the mass production of paclitaxel consists of liquid-liquid extraction, adsorbent treatment, hexane precipitation and fractional precipitation (Fig. 1) [11–14]. The purity of paclitaxel extracted from biomass using an organic solvent (methanol) generally ranges from 0.5 to 0.7%. If liquidliquid extraction, adsorbent treatment, hexane precipitation and fractional precipitation, which are included in the pre-purification process, are performed, the purity is improved to 6–9%, 9–10%, 21–27% and 46–61%, respectively [11,12,15–21]. Where a lot of impurities are removed by the pre-purification process, the purity of crude paclitaxel increases greatly. Thus, the sample becomes suitable for final purification using HPLC. However, many steps in the pre-purification process require a long operating time and a large amount of organic solvent, so the efficient mass production of paclitaxel is not easy and possible reductions in production costs are limited [11,12,16,22–24]. In this study, we adopted the water pre-treatment in an attempt to dramatically improve the efficiency
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Fig. 1. Traditional purification process of paclitaxel from biomass [14].
of the pre-purification stage for paclitaxel. In other words, we optimized the conditions for water pre-treatment and then evaluated various follow-up pre-purification processes in order to improve efficiency, in terms of the enhancement of yield and purity, the reduction of operating time, and the simplification of processes for the separation and purification of paclitaxel. 2. Materials and methods 2.1. Plant materials and culture conditions Suspension cells originating from Taxus chinensis were maintained under darkness at 24 ◦ C with shaking at 150 rpm. The suspension cells were cultured in a modified Gamborg’s B5 medium [25] supplemented with 30 g/l sucrose, 10 M naphthalene acetic acid, 0.2 M 6-benzylaminopurine, 1 g/l casein hydrolysate, and 1 g/l 2-(N-morpholino) ethanesulfonic acid. The cell cultures were transferred to a fresh medium every 2 weeks. In a prolonged culture for production, 1 and 2% (w/v) maltose were added to the culture medium on day 7 and day 21, respectively, and 4 M of AgNO3 was added on the initiation of a culture as an elicitor [26]. After plant cell culture, plant cells and cell debris (biomass) were recovered from the suspension using a decanter (Westfalia, CA150 Claritying Decanter) and high speed centrifuge (␣-Laval, BTPX205GD-35CDEEP). The biomass was provided by the Samyang Genex Company, South Korea.
tor (CCA-1100, EYELA, Japan) under vacuum and dried in a vacuum oven (UP-2000; EYELA) at 35 ◦ C for 24 h. The dried crude extract (purity: 2.5%) was crushed to powder for water-pre-treatment. 2.3. Water pre-treatment method Distilled water was added to the powdered extract (purity: 2.5%) and the mixture was agitated (∼300 rpm) for 10 min at room temperature. The crude extract/water ratio was changed to 1:5, 1:10, 1:20, 1:30, 1:40, 1:50 and 1:60 (w/v) in order to obtain the optimal water volume. After water pre-treatment, the precipitate was filtered with filter paper (Whatman Grade 5, 2.5 m, particle retention, 150 mm diameter) and dried. Then, it was used in the follow-up pre-purification process. 2.4. Liquid-liquid extraction The dried crude extract (purity: 12.1%) obtained by water pretreatment was dissolved in methanol (crude extract/methanol ratio, 1:80, w/v) and methylene chloride (25% of the amount of methanol) and distilled water (70% of the amount of methanol) were added. The liquid–liquid extraction was performed three times for 30 min. After the upper methanol-water layer containing polar impurities was removed, the lower methylene chloride layer containing paclitaxel was collected. The methylene chloride layers were concentrated and dried under vacuum by a concentrator (CCA-1100, EYELA, Japan).
2.2. Sample preparation for water pre-treatment 2.5. Adsorbent treatment The biomass was mixed with methanol at a ratio of 1:1 (w/v) and extracted at room temperature for 30 min. The mixture was filtered under vacuum in a Buchner funnel through filter paper. Extraction was repeated four times with new methanol. Following this, the extract was collected, pooled and concentrated in a rotary evapora-
The dried crude extract (purity: 12.1, 28.6%) obtained by water pre-treatment and liquid-liquid extraction was dissolved in methylene chloride at the ratio of 20% (v/w) and synthetic adsorbent sylopute (Fuji Silysia Chemical Ltd., Japan) was added. The tem-
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perature, time and crude extract/sylopute ratio for the adsorbent treatment were 40 ◦ C, 30 min and 1:1 (w/w), respectively. After adsorbent treatment, the extract was filtered with filtration paper (Whatman Grade 4, 20–25 m, particle retention, 150 mm diameter) and the filtrate was dried at 30 ◦ C under vacuum [27]. 2.6. Fractional precipitation The dried crude extract (purity: 30.0, 36.8%) obtained by the adsorbent treatment was dissolved in methanol (pure paclitaxel basis: 0.5%, w/v), while distilled water was added under agitation (180 rpm) until the methanol/water ratio became 20:80 (v/v). In addition, the solution was mixed for 10 min after distilled water was added. The precipitate was filtered and dried in the vacuum oven (UP-2000, EYELA, Japan) at 35 ◦ C for 24 h [27]. The step and overall yield of paclitaxel in the pre-purification process were defined as follows: Step (Overall) yield (%) = [quantity of paclitaxel after each (overall) step/ quantity of paclitaxel before each (overall) step] × 100
(1)
2.7. Paclitaxel analysis The dried residue was redissolved in methanol for a quantitative analysis using an HPLC system (Waters, USA) with a Capcell Pak C18 column (250 mm x 4.6 mm; Shiseido). The elution was performed based on a gradient using a distilled water–acetonitrile mixture varying from 65:35 to 35:65 within 40 min (flow rate = 1.0 mL/min). The injection volume was 20 L, and the effluent was monitored at 227 nm using a UV detector. Authentic paclitaxel (purity: 97%) was purchased from Sigma–Aldrich and used as the standard. Each sample was analysed in triplicate. 3. Results and discussion 3.1. Water pre-treatment method Several steps for extraction, pre-purification and final purification are needed to obtain high-purity paclitaxel from biomass by cell culture. An efficient pre-purification process should be developed for cost reduction. In general, if a low-purity sample from the pre-purification process is directly used in HPLC for final purification, a large amount of organic solvent is required and the lifetime of the column packing material (resin) and the throughput is reduced, which is uneconomical. In this way the mass production process is inefficient. Before final purification using HPLC, it is important to decrease impurities by the preparation of a high-purity (50% or higher) sample [12,13,24,28,29]. In this study, our objective was to dramatically improve the efficiency of the separation and purification process for biomass-derived paclitaxel through water pre-treatment. We investigated the effect of water pretreatment using the crude extract (purity: 2.5%) from the extraction of biomass. The crude extract/water ratio was changed to 1:5, 1:10, 1:20, 1:30, 1:40, 1:50 and 1:60 (w/v) for water pre-treatment and then the purity and the yield of paclitaxel were investigated. As shown in Fig. 2(A), the purity of paclitaxel was 7.2, 9.2, 10.2, 10.5, 12.1, 11.1 and 11.8% at the ratios of 1:5, 1:10, 1:20, 1:30, 1:40, 1:50 and 1:60 (w/v), respectively. The purity of paclitaxel slowly and steadily increased until 1:40 (w/v) of crude extract/water ratio, then showed little change after that. Hence, the purity of paclitaxel kept rising until a ratio of 1:40 (w/v) was achieved. In addition, the yield of paclitaxel was 99.99, 99.7, 99.6, 99.6, 99.4, 99.3 and 99.1% at a crude extract/water ratio of 1:5, 1:10, 1:20, 1:30, 1:40, 1:50 and 1:60 (w/v) as shown in Fig. 2(B). Thus, most of the paclitaxel (>99%) was recovered under all conditions. Therefore, the optimal crude extract/water ratio for water pre-treatment was 1:40 (w/v).
Fig. 2. Effect of crude extract/water ratio on the purity and yield of paclitaxel during water pre-treatment. (A): purity, (B): yield.
After water pre-treatment in optimum conditions, the filtrate was collected for an HPLC chromatogram. In the results, there were a lot of polar impurities and no paclitaxel (data not shown). Water pre-treatment, which was first adopted in this study, utilizes the insolubility of paclitaxel in water [13]. Unlike other pre-treatment methods, only water was used, without organic solvents, in pretreatment for 10 min, resulting in polar impurities being removed effectively. As a result, a high-yield (∼99.3%) and a high-purity (∼12.1%) paclitaxel were efficiently obtained. 3.2. The effect of water pre-treatment on the follow-up separation/purification process In this study, water pre-treatment improved the purity of paclitaxel at room temperature in a short period of time (∼10 min). Liquid-liquid extraction, adsorbent treatment and fractional precipitation were conducted to investigate the effect of water pre-treatment on the follow-up pre-purification process. At this time, the efficiency of the process was studied under two conditions; adsorbent treatment with liquid-liquid extraction (Fig. 3, Method I) and in the absence of liquid-liquid extraction (Fig. 3, Method II). Liquid-liquid extraction, adsorbent treatment and fractional precipitation, in order, were conducted on the dried crude extract (Fig. 4(B), purity: 12.1%) obtained by biomass extraction (Fig. 4(A),
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Fig. 3. Strategy of pre-purification of paclitaxel after water pre-treatment. Method I: With liquid-liquid extraction, Method II: Without liquid-liquid extraction.
purity: 2.5%) and water pre-treatment, to investigate the effect of water pre-treatment on the follow-up process when liquid-liquid extraction was performed (Fig. 3, Method I). The result of liquidliquid extraction for the crude extract (purity: 12.1%) obtained by water pre-treatment was that the purity of the paclitaxel was 28.6%, as shown in Fig. 4(C), and a lot of polar impurities were removed simultaneously. The result of adsorbent treatment for the crude extract (purity: 28.6%) obtained by liquid-liquid extraction, was that the purity of the paclitaxel was 36.8%, as shown in Fig. 4(D). When water pre-treatment was used, a large amount of polar impurities were effectively removed by liquid-liquid extraction and adsorbent treatment, which were the follow-up pre-purification processes. Also, high-purity (∼36.8%) paclitaxel was obtained. In the result of fractional precipitation for the sample (purity: 36.8%) obtained by adsorbent treatment, paclitaxel, at a purity of 56.5%, was obtained in a short period of time (∼10 min) as shown in Fig. 4(E). In the previous studies, the cost of final purification using HPLC was reduced by increasing the purity of the sample (50% or higher) in the pre-purification process [12,13,24,28,29]. Therefore, a high-purity (purity: 56.5%) sample suitable for final purification using HPLC was obtained just by liquid-liquid extraction, adsorbent treatment and fractional precipitation, which were the follow-up pre-purification processes when water pre-treatment was used. As a result, we obtained high-purity paclitaxel through adsorbent treatment as we adopted water pre-treatment, and improved the process in terms of cost reduction due to the significant simplification of the traditional pre-purification process. Adsorbent treatment and fractional precipitation were also conducted for the dried crude extract (Fig. 4(B), purity: 12.1%) obtained by biomass extraction (Fig. 4(A), purity: 2.5%) to investigate the effect of water pre-treatment on the follow-up process when liquid-liquid extraction had not been conducted (Fig. 3, Method II). Where adsorbent treatment was used for the crude extract obtained by water pre-treatment, the purity of the paclitaxel was 30.0%, as shown in Fig. 4(F). Despite the absence of liquid-liquid extraction, which generally removes polar impurities, a lot of polar impurities besides deep color were successfully removed from the sample in adsorbent treatment after water pre-treatment. Also, the purity was dramatically improved (12.1% → 30.0%). In previous
studies, the purity was commonly less than 10% in the traditional pre-purification process [11,12,16]. However, when adsorbent treatment following water pre-treatment was conducted, we could obtain high-purity (∼30.0%) paclitaxel. Furthermore, in the result of fractional precipitation for the sample (purity: 30.0%) obtained by adsorbent treatment, paclitaxel at a purity was 50.0% was obtained in a short period of time (∼10 min) as shown in Fig. 4(G). When water pre-treatment was used, only adsorbent treatment and fractional precipitation (without liquid-liquid extraction and hexane precipitation) facilitated high-purity (50% or higher) paclitaxel suitable for final purification. Based on the result, the possibility of removing liquid-liquid extraction from the pre-purification process was confirmed. The required amount of solvents and the treatment time for the follow-up process would be reduced due to this process simplification. Therefore, this method would be appropriate for the mass production of paclitaxel. 3.3. Evaluation of the efficiency of water pre-treatment In order to investigate the effect of water pre-treatment on the efficiency of pre-purification for paclitaxel, the pre-purification process when adopting water pre-treatment was compared to the pre-purification process for paclitaxel reported in previous studies [11,12,17,19,26–29]. The results are shown in Table 1. When liquid-liquid extraction, adsorbent treatment, hexane precipitation and fractional precipitation were conducted in that order in the traditional process without water pre-treatment after the extraction of biomass (Fig. 1), the final purity, overall yield, and operating time were 46.5–61.0%, 69.3–75.4% and 18.2–76.2 h, respectively. Among these steps, the fractional precipitation in particular has a significant impact on the total operating time [12,28]. However, when liquid-liquid extraction, adsorbent treatment and fractional precipitation were conducted in that order after the extraction of biomass and water pre-treatment (Fig. 3, Method I), the final purity, overall yield and operating time were 56.5%, 86.2% and 4.4 h, respectively. In addition, when adsorbent treatment and fractional precipitation in order were conducted after extraction of biomass and water pre-treatment without liquid-liquid extraction (Fig. 3, Method II), the final purity, overall yield and operating time were
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Fig. 4. Chromatogram of pre-purification steps analysed by RP-HPLC. (A): Biomass extraction, (B): Water pre-treatment, (C): Liquid-liquid extraction, (D): Adsorbent treatment with liquid-liquid extraction, (E): Fractional precipitation with liquid-liquid extraction, (F): Adsorbent treatment without liquid-liquid extraction, (G): Fractional precipitation without liquid-liquid extraction, respectively.
50.0%, 87.9% and 2.9 h, respectively. The operating time required for the fractional precipitation has improved dramatically. Based on the result, it was confirmed that water pre-treatment facilitated the extraction of a high-yield and high-purity crude extract from the biomass in a short period of time. In addition, the process was very effective, considering the aspects of purity, yield and operating time, when compared to the traditional pre-purification process for the separation and purification of paclitaxel. In particular, a lot of polar impurities besides deep color were successfully removed in the adsorbent treatment (Figs. 4(B) and (F)) and a high-purity paclitaxel was obtained when compared to adsorbent treatment in the previous pre-purification processes [12,19]. As a result, the pre-purification process with water pre-treatment dramatically shortened the traditional pre-purification process and also pro-
vided a high yield of high-purity (50% or higher) paclitaxel suitable for final purification using HPLC in a short period of time. 4. Conclusions Several steps of extraction, pre-purification and final purification are required to obtain high-purity paclitaxel from the biomass recovered from plant cell cultures. In particular, the preparation of high-purity (50% or higher) paclitaxel in the pre-purification process is important from the aspect of reduction of production costs. In the study, we dramatically improved the efficiency of the process for separation and purification of paclitaxel by water pre-treatment. A high yield (∼99.3%) and high-purity (∼12.1%) crude extract was obtained only by adopting water pre-treatment
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Table 1 Summary of pre-purification for paclitaxel from biomass (1 kg). Process
Purity (%)a
Step yield(%)a
Overall yield (%)a
Step operating time (hr)
Overall operating time (hr)
References
Traditional method Biomass Biomass extraction Liquid-liquid extraction Adsorbent treatment Hexane precipitation Fractional precipitation
– 0.5 – 0.7 6.0– 8.8 8.8–9.8 21.0–27.1 46.5–61.0
100.0 97.0–99.0 96.5–99.0 95.6 95.2– 99.0 81.3
100.0 97.0–99.0 93.6–98.0 89.5–93.7 85.2–92.7 69.3–75.4
– 2.0 1.5 0.5 0.2 14.0–72.0
– 2.0 3.5 4.0 4.2 18.2–76.2
– [11,12,25] [12,19,26,27] [12,19] [12,27–29] [12,28]
Method I in this study Biomass Biomass extraction Water pre-treatment Liquid-liquid extraction Adsorbent treatment Fractional precipitation
– 2.5 ± 0.3 12.1 ± 1.0 28.6 ± 2.1 36.8 ± 1.8 56.5 ± 1.5
100.0 99.2 ± 0.4 99.4 ± 0.8 98.2 ± 1.7 93.9 ± 3.1 94.8 ± 3.0
100.0 99.2 ± 0.4 98.6 ± 1.2 96.8 ± 2.9 90.9 ± 5.6 86.2 ± 8.4
– 2.0 0.2 1.5 0.5 0.2
– 2.0 2.2 3.7 4.2 4.4
– – – – – –
Method II in this study Biomass Biomass extraction Water pre-treatment Adsorbent treatment Fractional precipitation
– 2.5 ± 0.3 12.1 ± 1.0 30.0 ± 1.2 50.0 ± 2.3
100.0 99.2 ± 0.4 99.4 ± 0.8 92.7 ± 2.7 96.2 ± 2.0
100.0 99.2 ± 0.4 98.6 ± 1.2 91.4 ± 3.8 87.9 ± 5.4
– 2.0 0.2 0.5 0.2
– 2.0 2.2 2.7 2.9
– – – – –
a
Data are shown as purity, step yield, and overall yield (%) ± SD.
without the use of organic solvents in a short period of time (∼10 min). Furthermore, when the follow-up separation and purification processes following water pre-treatment were conducted, the efficiency of the process was highly increased compared to that in the traditional pre-purification process. In adsorbent treatment, a lot of polar impurities were removed effectively. In addition, a high yield (∼87.9%) of high-purity (50% or higher) paclitaxel suitable for final purification using HPLC was also obtained in brief time frame (∼2.9 h) through fractional precipitation. As a result, the adoption of water pre-treatment by industry could dramatically improve the efficiency of the process being used, particularly in terms of the enhancement of yield and purity and the reduction of operating time, for the separation and purification of paclitaxel. Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) and funded by the Ministry of Education, Science and Technology (Grant Number: 2015016271). References [1] M.C. Wani, H.L. Taylor, M.E. Wall, P. Coggon, A.T. McPhail, Plant antitumor agents: VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia, J. Am. Chem. Soc. 93 (1971) 2325–2327. [2] P.B. Schiff, J. Fant, S.B. Horwitz, Promotion of microtubule assembly in vitro by taxol, Nature 277 (1979) 655–667. [3] K.V. Rao, J.B. Hanuman, C. Alvarez, M. Stoy, J. Juchum, R.M. Davies, R. Baxley, A new large-scale process for taxol and related taxanes from Taxus brevifolia, Pharm. Res. 12 (1995) 1003–1010. [4] Y. Fu, Y. Zu, S. Li, R. Sun, T. Efferth, W. Liu, S. Jiang, H. Luo, Y. Wang, Separation of 7-xylosyl-10-deacetyl paclitaxel and 10-deacetylbaccatin III from the remainder extracts free of paclitaxel using macroporous resins, J. Chromatogr. A 1177 (2008) 77–86. [5] H.K. Choi, T.L. Adams, R.W. Stahlhut, S.I. Kim, J.H. Yun, B.K. Song, J.H. Kim, S.S. Hong, H.S. Lee, Method for mass production of taxol by semi-continuous culture with Taxus chinensis cell culture, US Patent, 5 (871)(1999) 979. [6] G.J. Kim, J.H. Kim, A simultaneous microwave-assisted extraction and adsorbent treatment process under acidic conditions for recovery and separation of paclitaxel from plant cell cultures, Korean J. Chem. Eng. 32 (2015) 1023–1028. [7] K.M. Witherup, S.A. Look, M.W. Stasko, T.J. Ghiorzi, G.M. Muschik, G.M. Cragg, Taxus spp. needles contain amounts of taxol comparable to the bark of Taxus brevifolia: analysis and isolation, J. Nat. Prod. 53 (1990) 1249–1255. [8] H.N. ElSohly, E.M. Croom Jr., M.A. ElSohly, J.D. McChesney, Methods and compositions for isolating taxanes, US Patent 5 (618) (1997) 538.
[9] K.V. Rao, Method for the isolation and purification of taxol and its natural analogues, US Patent 5 (670) (1997) 673. [10] T.P. Castor, Method and apparatus for isolating therapeutic compositions from source materials, US Patent 5 (750) (1998) 709. [11] 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. [12] S.H. Pyo, H.B. Park, B.K. Song, B.H. Han, J.H. Kim, A large-scale purification of paclitaxel from cell cultures of Taxus chinensis, Process Biochem. 39 (2004) 1985–1991. [13] J.H. Kim, Paclitaxel: recovery and purification in commercialization step, Korean J. Biotechnol. Bioeng. 21 (1) (2006) 1–10. [14] J.Y. Lee, J.H. Kim, Microwave-assisted drying of paclitaxel for removal of residual solvents, Process Biochem. 48 (2013) 545–550. [15] J.H. Kim, S.S. Hong, Optimization of extraction process for mass production of paclitaxel from plant cell cultures, Korean J. Biotechnol. Bioeng. 15 (4) (2000) 346–351. [16] M.G. Han, K.Y. Jeon, S. Mun, J.H. Kim, Development of a micelle-fractional precipitation hybrid process for the pre-purification of paclitaxel from plant cell cultures, Process Biochem. 45 (2010) 1368–1374. [17] J.H. Kim, Prepurification of paclitaxel by micelle and precipitation, Process Biochem. 39 (2004) 1567–1571. [18] K.Y. Jeon, J.H. Kim, Effect of surfactant on the micelle process for the pre-purification of paclitaxel, Korean J. Biotechnol. Bioeng. 23 (2008) 557–560. [19] J.H. Kim, Optimization of liquid-liquid extraction conditions for paclitaxel separation from plant cell cultures, KSBB J. 24 (2009) 212–215. [20] D.H. Lee, S.G. Kim, S.Y. Mun, J.H. Kim, Evaluation of feeding and mixing conditions for fractional precipitation of paclitaxel from plant cell cultures, Process Biochem. 45 (2010) 1134–1140. [21] H.A. Sim, J.Y. Lee, J.H. Kim, Evaluation of a high surface area acetone/pentane precipitation process for the purification of paclitaxel from plant cell cultures, Sep. Purif. Technol. 89 (2012) 112–116. [22] S.H. Pyo, B.K. Song, C.H. Ju, B.H. Han, H.J. Choi, Effects of absorbent treatment on the purification of pacliatxel from cell cultures of Taxus chinensis and yew tree, Process Biochem. 40 (2005) 1113–1117. [23] G.Y. Park, G.J. Kim, J.H. Kim, Effect of tar compounds on the purification efficiency of paclitaxel from Taxus chinensis, J. Ind. Eng. Chem. 21 (2015) 151–154. [24] H.J. Oh, H.R. Jang, K.Y. Jung, J.H. Kim, Evaluation of adsorbents for separation and purification of paclitaxel from plant cell cultures, Process Biochem. 47 (2012) 331–334. [25] O.L. Gamborg, R.A. Miller, K. Ojima, Nutrient requirements of suspension cultures of soybean root cells, Exp. Cell Res. 50 (1968) 151–158. [26] H.K. Choi, S.J. Son, G.H. Na, S.S. Hong, Y.S. Park, J.Y. Song, Mass production of paclitaxel by plant cell culture, Korean J. Plant Biotechnol. 29 (2002) 59–62. [27] C.G. Lee, J.H. Kim, Improved fractional precipitation method for purification of paclitaxel, Process Biochem. 49 (2014) 1370–1376. [28] K.Y. Jeon, J.H. Kim, Improvement of fractional precipitation process for pre-purification of paclitaxel, Process Biochem. 44 (2009) 736–741. [29] S.H. Pyo, H.J. Choi, An improved high-performance liquid chromatography process for the large-scale production of paclitaxel, Sep. Purif. Technol. 76 (2011) 378–384.
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Short communication
Increasing the efficiency of the separation and purification process for paclitaxel by pre-treatment with water Chung-Gi Lee, Jin-Hyun Kim ∗ Department of Chemical Engineering, Kongju NationalUniversity, Cheonan 330-717, South Korea
a r t i c l e
i n f o
Article history: Received 14 January 2016 Received in revised form 6 July 2016 Accepted 27 July 2016 Available online 30 July 2016 Keywords: Paclitaxel Water pre-treatment Purification Process Development
a b s t r a c t In this study, a water pre-treatment method for the separation and purification of an anticancer agent paclitaxel from plant cell cultures was developed. When the methanol extract obtained by biomass extraction was pre-treated with water, a high yield (>99%) of high-purity (∼12.1%) paclitaxel was observed within a short period of time (∼10 min). In addition, in the follow-up pre-purification process, using a crude extract obtained by water pre-treatment, high-yield (∼87.9%), high-purity (50% or higher) paclitaxel was obtained in a short operating time (∼2.9 h). Thus, the efficiency of the separation and purification process for paclitaxel was improved dramatically by water pre-treatment, particularly in terms of the reduction of operating times and the simplification of processes. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Paclitaxel is a diterpenoid anticancer agent found in the bark of the yew tree. The chemical structure and the anticancer mechanism were investigated by Wani et al. [1] in 1971 and Schiff et al. [2] in 1979. Since its discovery, paclitaxel has been used as an important anticancer drug, with the approval of the U.S. Food and Drug Administration, to treat ovarian cancer, breast cancer, Kaposi’s sarcoma and non-small cell lung cancer. The main production methods for paclitaxel are; 1) paclitaxel is directly extracted from yew trees [3]; 2) precursors (baccatin III, 10-deacetylbaccatin III, 10-deacetylpaclitaxel, etc.) are obtained from the leaves of yew trees to combine side chains chemically for semi-synthesis [4]; and 3) callus is induced into yew trees and then the plants’ cells are cultured in a bioreactor after seed culture [5]. In the latter case, the plant cell culture is not affected by external factors, such as climate or the environment, and can be stably produced in a bioreactor. This makes the mass production of paclitaxel of a consistent quality possible, allowing production to meet with increasing demand [6]. Many separation and purification steps are required to obtain high-purity paclitaxel from plant cell cultures. In general, paclitaxel is extracted from the biomass by using organic solvents and then high-purity paclitaxel is obtained through pre-purification
∗ Corresponding author. E-mail address: [email protected] (J.-H. Kim). http://dx.doi.org/10.1016/j.procbio.2016.07.025 1359-5113/© 2016 Elsevier Ltd. All rights reserved.
and final purification steps. However, in previous studies, expensive chromatography has been applied to the pre-purification process for the final purification stage, or a crude extract has been directly used in final purification by HPLC. In particular, if a low-purity sample is used for final purification by HPLC without pre-purification, a large amount of organic solvent is consumed and the lifetime of column packing materials (resin) and throughput are reduced. This is very uneconomical, rendering this process unsuitable for mass production [7–10]. Therefore, efficient and economical pre-purification is definitely required to obtain a high-yield of high-purity paclitaxel. According to previous studies, the pre-purification process for the mass production of paclitaxel consists of liquid-liquid extraction, adsorbent treatment, hexane precipitation and fractional precipitation (Fig. 1) [11–14]. The purity of paclitaxel extracted from biomass using an organic solvent (methanol) generally ranges from 0.5 to 0.7%. If liquidliquid extraction, adsorbent treatment, hexane precipitation and fractional precipitation, which are included in the pre-purification process, are performed, the purity is improved to 6–9%, 9–10%, 21–27% and 46–61%, respectively [11,12,15–21]. Where a lot of impurities are removed by the pre-purification process, the purity of crude paclitaxel increases greatly. Thus, the sample becomes suitable for final purification using HPLC. However, many steps in the pre-purification process require a long operating time and a large amount of organic solvent, so the efficient mass production of paclitaxel is not easy and possible reductions in production costs are limited [11,12,16,22–24]. In this study, we adopted the water pre-treatment in an attempt to dramatically improve the efficiency
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Fig. 1. Traditional purification process of paclitaxel from biomass [14].
of the pre-purification stage for paclitaxel. In other words, we optimized the conditions for water pre-treatment and then evaluated various follow-up pre-purification processes in order to improve efficiency, in terms of the enhancement of yield and purity, the reduction of operating time, and the simplification of processes for the separation and purification of paclitaxel. 2. Materials and methods 2.1. Plant materials and culture conditions Suspension cells originating from Taxus chinensis were maintained under darkness at 24 ◦ C with shaking at 150 rpm. The suspension cells were cultured in a modified Gamborg’s B5 medium [25] supplemented with 30 g/l sucrose, 10 M naphthalene acetic acid, 0.2 M 6-benzylaminopurine, 1 g/l casein hydrolysate, and 1 g/l 2-(N-morpholino) ethanesulfonic acid. The cell cultures were transferred to a fresh medium every 2 weeks. In a prolonged culture for production, 1 and 2% (w/v) maltose were added to the culture medium on day 7 and day 21, respectively, and 4 M of AgNO3 was added on the initiation of a culture as an elicitor [26]. After plant cell culture, plant cells and cell debris (biomass) were recovered from the suspension using a decanter (Westfalia, CA150 Claritying Decanter) and high speed centrifuge (␣-Laval, BTPX205GD-35CDEEP). The biomass was provided by the Samyang Genex Company, South Korea.
tor (CCA-1100, EYELA, Japan) under vacuum and dried in a vacuum oven (UP-2000; EYELA) at 35 ◦ C for 24 h. The dried crude extract (purity: 2.5%) was crushed to powder for water-pre-treatment. 2.3. Water pre-treatment method Distilled water was added to the powdered extract (purity: 2.5%) and the mixture was agitated (∼300 rpm) for 10 min at room temperature. The crude extract/water ratio was changed to 1:5, 1:10, 1:20, 1:30, 1:40, 1:50 and 1:60 (w/v) in order to obtain the optimal water volume. After water pre-treatment, the precipitate was filtered with filter paper (Whatman Grade 5, 2.5 m, particle retention, 150 mm diameter) and dried. Then, it was used in the follow-up pre-purification process. 2.4. Liquid-liquid extraction The dried crude extract (purity: 12.1%) obtained by water pretreatment was dissolved in methanol (crude extract/methanol ratio, 1:80, w/v) and methylene chloride (25% of the amount of methanol) and distilled water (70% of the amount of methanol) were added. The liquid–liquid extraction was performed three times for 30 min. After the upper methanol-water layer containing polar impurities was removed, the lower methylene chloride layer containing paclitaxel was collected. The methylene chloride layers were concentrated and dried under vacuum by a concentrator (CCA-1100, EYELA, Japan).
2.2. Sample preparation for water pre-treatment 2.5. Adsorbent treatment The biomass was mixed with methanol at a ratio of 1:1 (w/v) and extracted at room temperature for 30 min. The mixture was filtered under vacuum in a Buchner funnel through filter paper. Extraction was repeated four times with new methanol. Following this, the extract was collected, pooled and concentrated in a rotary evapora-
The dried crude extract (purity: 12.1, 28.6%) obtained by water pre-treatment and liquid-liquid extraction was dissolved in methylene chloride at the ratio of 20% (v/w) and synthetic adsorbent sylopute (Fuji Silysia Chemical Ltd., Japan) was added. The tem-
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perature, time and crude extract/sylopute ratio for the adsorbent treatment were 40 ◦ C, 30 min and 1:1 (w/w), respectively. After adsorbent treatment, the extract was filtered with filtration paper (Whatman Grade 4, 20–25 m, particle retention, 150 mm diameter) and the filtrate was dried at 30 ◦ C under vacuum [27]. 2.6. Fractional precipitation The dried crude extract (purity: 30.0, 36.8%) obtained by the adsorbent treatment was dissolved in methanol (pure paclitaxel basis: 0.5%, w/v), while distilled water was added under agitation (180 rpm) until the methanol/water ratio became 20:80 (v/v). In addition, the solution was mixed for 10 min after distilled water was added. The precipitate was filtered and dried in the vacuum oven (UP-2000, EYELA, Japan) at 35 ◦ C for 24 h [27]. The step and overall yield of paclitaxel in the pre-purification process were defined as follows: Step (Overall) yield (%) = [quantity of paclitaxel after each (overall) step/ quantity of paclitaxel before each (overall) step] × 100
(1)
2.7. Paclitaxel analysis The dried residue was redissolved in methanol for a quantitative analysis using an HPLC system (Waters, USA) with a Capcell Pak C18 column (250 mm x 4.6 mm; Shiseido). The elution was performed based on a gradient using a distilled water–acetonitrile mixture varying from 65:35 to 35:65 within 40 min (flow rate = 1.0 mL/min). The injection volume was 20 L, and the effluent was monitored at 227 nm using a UV detector. Authentic paclitaxel (purity: 97%) was purchased from Sigma–Aldrich and used as the standard. Each sample was analysed in triplicate. 3. Results and discussion 3.1. Water pre-treatment method Several steps for extraction, pre-purification and final purification are needed to obtain high-purity paclitaxel from biomass by cell culture. An efficient pre-purification process should be developed for cost reduction. In general, if a low-purity sample from the pre-purification process is directly used in HPLC for final purification, a large amount of organic solvent is required and the lifetime of the column packing material (resin) and the throughput is reduced, which is uneconomical. In this way the mass production process is inefficient. Before final purification using HPLC, it is important to decrease impurities by the preparation of a high-purity (50% or higher) sample [12,13,24,28,29]. In this study, our objective was to dramatically improve the efficiency of the separation and purification process for biomass-derived paclitaxel through water pre-treatment. We investigated the effect of water pretreatment using the crude extract (purity: 2.5%) from the extraction of biomass. The crude extract/water ratio was changed to 1:5, 1:10, 1:20, 1:30, 1:40, 1:50 and 1:60 (w/v) for water pre-treatment and then the purity and the yield of paclitaxel were investigated. As shown in Fig. 2(A), the purity of paclitaxel was 7.2, 9.2, 10.2, 10.5, 12.1, 11.1 and 11.8% at the ratios of 1:5, 1:10, 1:20, 1:30, 1:40, 1:50 and 1:60 (w/v), respectively. The purity of paclitaxel slowly and steadily increased until 1:40 (w/v) of crude extract/water ratio, then showed little change after that. Hence, the purity of paclitaxel kept rising until a ratio of 1:40 (w/v) was achieved. In addition, the yield of paclitaxel was 99.99, 99.7, 99.6, 99.6, 99.4, 99.3 and 99.1% at a crude extract/water ratio of 1:5, 1:10, 1:20, 1:30, 1:40, 1:50 and 1:60 (w/v) as shown in Fig. 2(B). Thus, most of the paclitaxel (>99%) was recovered under all conditions. Therefore, the optimal crude extract/water ratio for water pre-treatment was 1:40 (w/v).
Fig. 2. Effect of crude extract/water ratio on the purity and yield of paclitaxel during water pre-treatment. (A): purity, (B): yield.
After water pre-treatment in optimum conditions, the filtrate was collected for an HPLC chromatogram. In the results, there were a lot of polar impurities and no paclitaxel (data not shown). Water pre-treatment, which was first adopted in this study, utilizes the insolubility of paclitaxel in water [13]. Unlike other pre-treatment methods, only water was used, without organic solvents, in pretreatment for 10 min, resulting in polar impurities being removed effectively. As a result, a high-yield (∼99.3%) and a high-purity (∼12.1%) paclitaxel were efficiently obtained. 3.2. The effect of water pre-treatment on the follow-up separation/purification process In this study, water pre-treatment improved the purity of paclitaxel at room temperature in a short period of time (∼10 min). Liquid-liquid extraction, adsorbent treatment and fractional precipitation were conducted to investigate the effect of water pre-treatment on the follow-up pre-purification process. At this time, the efficiency of the process was studied under two conditions; adsorbent treatment with liquid-liquid extraction (Fig. 3, Method I) and in the absence of liquid-liquid extraction (Fig. 3, Method II). Liquid-liquid extraction, adsorbent treatment and fractional precipitation, in order, were conducted on the dried crude extract (Fig. 4(B), purity: 12.1%) obtained by biomass extraction (Fig. 4(A),
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Fig. 3. Strategy of pre-purification of paclitaxel after water pre-treatment. Method I: With liquid-liquid extraction, Method II: Without liquid-liquid extraction.
purity: 2.5%) and water pre-treatment, to investigate the effect of water pre-treatment on the follow-up process when liquid-liquid extraction was performed (Fig. 3, Method I). The result of liquidliquid extraction for the crude extract (purity: 12.1%) obtained by water pre-treatment was that the purity of the paclitaxel was 28.6%, as shown in Fig. 4(C), and a lot of polar impurities were removed simultaneously. The result of adsorbent treatment for the crude extract (purity: 28.6%) obtained by liquid-liquid extraction, was that the purity of the paclitaxel was 36.8%, as shown in Fig. 4(D). When water pre-treatment was used, a large amount of polar impurities were effectively removed by liquid-liquid extraction and adsorbent treatment, which were the follow-up pre-purification processes. Also, high-purity (∼36.8%) paclitaxel was obtained. In the result of fractional precipitation for the sample (purity: 36.8%) obtained by adsorbent treatment, paclitaxel, at a purity of 56.5%, was obtained in a short period of time (∼10 min) as shown in Fig. 4(E). In the previous studies, the cost of final purification using HPLC was reduced by increasing the purity of the sample (50% or higher) in the pre-purification process [12,13,24,28,29]. Therefore, a high-purity (purity: 56.5%) sample suitable for final purification using HPLC was obtained just by liquid-liquid extraction, adsorbent treatment and fractional precipitation, which were the follow-up pre-purification processes when water pre-treatment was used. As a result, we obtained high-purity paclitaxel through adsorbent treatment as we adopted water pre-treatment, and improved the process in terms of cost reduction due to the significant simplification of the traditional pre-purification process. Adsorbent treatment and fractional precipitation were also conducted for the dried crude extract (Fig. 4(B), purity: 12.1%) obtained by biomass extraction (Fig. 4(A), purity: 2.5%) to investigate the effect of water pre-treatment on the follow-up process when liquid-liquid extraction had not been conducted (Fig. 3, Method II). Where adsorbent treatment was used for the crude extract obtained by water pre-treatment, the purity of the paclitaxel was 30.0%, as shown in Fig. 4(F). Despite the absence of liquid-liquid extraction, which generally removes polar impurities, a lot of polar impurities besides deep color were successfully removed from the sample in adsorbent treatment after water pre-treatment. Also, the purity was dramatically improved (12.1% → 30.0%). In previous
studies, the purity was commonly less than 10% in the traditional pre-purification process [11,12,16]. However, when adsorbent treatment following water pre-treatment was conducted, we could obtain high-purity (∼30.0%) paclitaxel. Furthermore, in the result of fractional precipitation for the sample (purity: 30.0%) obtained by adsorbent treatment, paclitaxel at a purity was 50.0% was obtained in a short period of time (∼10 min) as shown in Fig. 4(G). When water pre-treatment was used, only adsorbent treatment and fractional precipitation (without liquid-liquid extraction and hexane precipitation) facilitated high-purity (50% or higher) paclitaxel suitable for final purification. Based on the result, the possibility of removing liquid-liquid extraction from the pre-purification process was confirmed. The required amount of solvents and the treatment time for the follow-up process would be reduced due to this process simplification. Therefore, this method would be appropriate for the mass production of paclitaxel. 3.3. Evaluation of the efficiency of water pre-treatment In order to investigate the effect of water pre-treatment on the efficiency of pre-purification for paclitaxel, the pre-purification process when adopting water pre-treatment was compared to the pre-purification process for paclitaxel reported in previous studies [11,12,17,19,26–29]. The results are shown in Table 1. When liquid-liquid extraction, adsorbent treatment, hexane precipitation and fractional precipitation were conducted in that order in the traditional process without water pre-treatment after the extraction of biomass (Fig. 1), the final purity, overall yield, and operating time were 46.5–61.0%, 69.3–75.4% and 18.2–76.2 h, respectively. Among these steps, the fractional precipitation in particular has a significant impact on the total operating time [12,28]. However, when liquid-liquid extraction, adsorbent treatment and fractional precipitation were conducted in that order after the extraction of biomass and water pre-treatment (Fig. 3, Method I), the final purity, overall yield and operating time were 56.5%, 86.2% and 4.4 h, respectively. In addition, when adsorbent treatment and fractional precipitation in order were conducted after extraction of biomass and water pre-treatment without liquid-liquid extraction (Fig. 3, Method II), the final purity, overall yield and operating time were
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Fig. 4. Chromatogram of pre-purification steps analysed by RP-HPLC. (A): Biomass extraction, (B): Water pre-treatment, (C): Liquid-liquid extraction, (D): Adsorbent treatment with liquid-liquid extraction, (E): Fractional precipitation with liquid-liquid extraction, (F): Adsorbent treatment without liquid-liquid extraction, (G): Fractional precipitation without liquid-liquid extraction, respectively.
50.0%, 87.9% and 2.9 h, respectively. The operating time required for the fractional precipitation has improved dramatically. Based on the result, it was confirmed that water pre-treatment facilitated the extraction of a high-yield and high-purity crude extract from the biomass in a short period of time. In addition, the process was very effective, considering the aspects of purity, yield and operating time, when compared to the traditional pre-purification process for the separation and purification of paclitaxel. In particular, a lot of polar impurities besides deep color were successfully removed in the adsorbent treatment (Figs. 4(B) and (F)) and a high-purity paclitaxel was obtained when compared to adsorbent treatment in the previous pre-purification processes [12,19]. As a result, the pre-purification process with water pre-treatment dramatically shortened the traditional pre-purification process and also pro-
vided a high yield of high-purity (50% or higher) paclitaxel suitable for final purification using HPLC in a short period of time. 4. Conclusions Several steps of extraction, pre-purification and final purification are required to obtain high-purity paclitaxel from the biomass recovered from plant cell cultures. In particular, the preparation of high-purity (50% or higher) paclitaxel in the pre-purification process is important from the aspect of reduction of production costs. In the study, we dramatically improved the efficiency of the process for separation and purification of paclitaxel by water pre-treatment. A high yield (∼99.3%) and high-purity (∼12.1%) crude extract was obtained only by adopting water pre-treatment
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Table 1 Summary of pre-purification for paclitaxel from biomass (1 kg). Process
Purity (%)a
Step yield(%)a
Overall yield (%)a
Step operating time (hr)
Overall operating time (hr)
References
Traditional method Biomass Biomass extraction Liquid-liquid extraction Adsorbent treatment Hexane precipitation Fractional precipitation
– 0.5 – 0.7 6.0– 8.8 8.8–9.8 21.0–27.1 46.5–61.0
100.0 97.0–99.0 96.5–99.0 95.6 95.2– 99.0 81.3
100.0 97.0–99.0 93.6–98.0 89.5–93.7 85.2–92.7 69.3–75.4
– 2.0 1.5 0.5 0.2 14.0–72.0
– 2.0 3.5 4.0 4.2 18.2–76.2
– [11,12,25] [12,19,26,27] [12,19] [12,27–29] [12,28]
Method I in this study Biomass Biomass extraction Water pre-treatment Liquid-liquid extraction Adsorbent treatment Fractional precipitation
– 2.5 ± 0.3 12.1 ± 1.0 28.6 ± 2.1 36.8 ± 1.8 56.5 ± 1.5
100.0 99.2 ± 0.4 99.4 ± 0.8 98.2 ± 1.7 93.9 ± 3.1 94.8 ± 3.0
100.0 99.2 ± 0.4 98.6 ± 1.2 96.8 ± 2.9 90.9 ± 5.6 86.2 ± 8.4
– 2.0 0.2 1.5 0.5 0.2
– 2.0 2.2 3.7 4.2 4.4
– – – – – –
Method II in this study Biomass Biomass extraction Water pre-treatment Adsorbent treatment Fractional precipitation
– 2.5 ± 0.3 12.1 ± 1.0 30.0 ± 1.2 50.0 ± 2.3
100.0 99.2 ± 0.4 99.4 ± 0.8 92.7 ± 2.7 96.2 ± 2.0
100.0 99.2 ± 0.4 98.6 ± 1.2 91.4 ± 3.8 87.9 ± 5.4
– 2.0 0.2 0.5 0.2
– 2.0 2.2 2.7 2.9
– – – – –
a
Data are shown as purity, step yield, and overall yield (%) ± SD.
without the use of organic solvents in a short period of time (∼10 min). Furthermore, when the follow-up separation and purification processes following water pre-treatment were conducted, the efficiency of the process was highly increased compared to that in the traditional pre-purification process. In adsorbent treatment, a lot of polar impurities were removed effectively. In addition, a high yield (∼87.9%) of high-purity (50% or higher) paclitaxel suitable for final purification using HPLC was also obtained in brief time frame (∼2.9 h) through fractional precipitation. As a result, the adoption of water pre-treatment by industry could dramatically improve the efficiency of the process being used, particularly in terms of the enhancement of yield and purity and the reduction of operating time, for the separation and purification of paclitaxel. Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) and funded by the Ministry of Education, Science and Technology (Grant Number: 2015016271). References [1] M.C. Wani, H.L. Taylor, M.E. Wall, P. Coggon, A.T. McPhail, Plant antitumor agents: VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia, J. Am. Chem. Soc. 93 (1971) 2325–2327. [2] P.B. Schiff, J. Fant, S.B. Horwitz, Promotion of microtubule assembly in vitro by taxol, Nature 277 (1979) 655–667. [3] K.V. Rao, J.B. Hanuman, C. Alvarez, M. Stoy, J. Juchum, R.M. Davies, R. Baxley, A new large-scale process for taxol and related taxanes from Taxus brevifolia, Pharm. Res. 12 (1995) 1003–1010. [4] Y. Fu, Y. Zu, S. Li, R. Sun, T. Efferth, W. Liu, S. Jiang, H. Luo, Y. Wang, Separation of 7-xylosyl-10-deacetyl paclitaxel and 10-deacetylbaccatin III from the remainder extracts free of paclitaxel using macroporous resins, J. Chromatogr. A 1177 (2008) 77–86. [5] H.K. Choi, T.L. Adams, R.W. Stahlhut, S.I. Kim, J.H. Yun, B.K. Song, J.H. Kim, S.S. Hong, H.S. Lee, Method for mass production of taxol by semi-continuous culture with Taxus chinensis cell culture, US Patent, 5 (871)(1999) 979. [6] G.J. Kim, J.H. Kim, A simultaneous microwave-assisted extraction and adsorbent treatment process under acidic conditions for recovery and separation of paclitaxel from plant cell cultures, Korean J. Chem. Eng. 32 (2015) 1023–1028. [7] K.M. Witherup, S.A. Look, M.W. Stasko, T.J. Ghiorzi, G.M. Muschik, G.M. Cragg, Taxus spp. needles contain amounts of taxol comparable to the bark of Taxus brevifolia: analysis and isolation, J. Nat. Prod. 53 (1990) 1249–1255. [8] H.N. ElSohly, E.M. Croom Jr., M.A. ElSohly, J.D. McChesney, Methods and compositions for isolating taxanes, US Patent 5 (618) (1997) 538.
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