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Effects of absorbent treatment on the purification of paclitaxel from cell cultures of Taxus chinensis and yew tree
Process Biochemistry 40 (2005) 1113–1117
Effects of absorbent treatment on the purification of paclitaxel from cell cultures of Taxus chinensis and yew tree. Sang-Hyun Pyo a , Bong-Kyu Song a , Chang-Hun Ju a , Byung-Hee Han b , Ho-Joon Choi a,∗ b
a Samyang Genex Biotech Research Institute, 63-2 Hwaam-Dong, Yuseong-Gu, Daejeon, 305-348, South Korea Department of Chemistry, Chungnam National University, 220 Gung-Dong, Yuseong-Gu, Daejeon, 305-764, South Korea
Received 1 February 2004; accepted 27 March 2004
Abstract A new synthetic absorbent, sylopute was applied for the purification of paclitaxel from plant cell cultures and yew tree. There was selective absorption of impurities in the treatment of sylopute. Therefore, higher purity paclitaxel could be obtained simply in the following process with a higher yield than with active clay or activated carbon. The purity of crude paclitaxel after sylopute treatment followed by precipitation was increased up to 63%, while the purities for active clay and activated carbon were 57.7 and 40%, respectively. The overall yields from sylopute, active clay, and activated carbon were 80.2, 67.7, and 35.5%, respectively. The best result in removing impurities was obtained from the combination of sylopute treatment followed by 1st and 2nd precipitation processes. A similar result was observed in the case of absorbent treatment of crude extract from yew tree. Consequently, sylopute can be applied for production scale purification of other natural products as an alternative to synthetic absorbents, activated carbon, and other alternative processes. © 2004 Elsevier Ltd. All rights reserved. Keywords: Sylopute; Paclitaxel; Isolation; Purification; Absorbent; Adsorption
1. Introduction Paclitaxel, first reported in 1971 [1], is one of the most important anticancer agents in the treatment of refractory ovarian, breast, and other cancers [2–4]. Supplies of paclitaxel are potentially limited and not affordable from the environmental point of view, because the original and major source material of the drug is the bark of Taxus brevifolia [5]. The yield of purification of paclitaxel from T. brevifolia is about 0.01% of the dry weight of bark [6]. It is known that there are significant differences in the contents according to location, season, and tissue variations [7–9]. In addition, chemical semi or total synthesis of paclitaxel [10,11] is very complex with a very low yield and may not be feasible commercially on an industrial scale. Paclitaxel production in plant cell and tissue cultures has the potential to provide sufficient quantities of paclitaxel for chemotherapeutic use
∗
Correspondence author. Fax: +82 42 865 8398. E-mail address: [email protected] (H.-J. Choi).
0032-9592/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.03.004
[12–14]. Meanwhile, cell culture offers the potential advantage of availability and reliable production of using a renewable resource whereas bark-stripping methods [5,6] lead to the destruction of scarce plant material. Nevertheless, the development of a more effective process for separation and purification are important subject. An efficient procedure for large-scale productionof paclitaxel from plant cell cultures has been developed [15–17]. To develop more efficient production methods on a commercial scale, absorption selectivity using several absorbents including, active clay, sylopute, and activated carbon were studied. Generally, activated carbon is usually used as absorbent and has been used broadly to remove color for decolorization of natural product and water as well as the removal of impurities in the crude product [16–19]. However, some synthetic absorbents show better efficiency in purity and yield than activated carbon. In this investigation, we have developed a large-scale and efficient method for removal of impurities in crude extracts using sylopute followed by precipitation. This method is suitable for large-scale purification of paclitaxel from plant cell cultures and yew tree for the purposes of clinical use.
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S.-H. Pyo et al. / Process Biochemistry 40 (2005) 1113–1117
2. Materials and methods 2.1. Plant materials and culture conditions Suspension cells originating from T. chinensis, were maintained in darkness at 24 ◦ C with shaking at 150 rpm. Suspension cells were cultured in a modified Gamborg’s B5 medium supplemented with 30 g/1 sucrose, 10 M of NAA (naphthalene acetic acid), 0.2 M of BA (6-benzylamino purine), 1 g/1 of casein hydrolysate, and 1 g/1 of 2-(Nmorpholino) ethanesulphonic acid (MES). Cell cultures were transferred to a fresh medium every 2 weeks. In a prolonged culture for production, 1 and 2% maltose was added to the culture medium on day 7 and day 21, respectively, and 4 M of AgNO3 was added on the initiation of culture as elicitor [20]. After the culture, biomass was recovered with a decanter (Westfalia, CA150 Clarifying Decanter) and high-speed centrifuge (␣-Laval, BTOX 205GD-35CDEFP). Paclitaxel in solution was recovered by absorption with resin. 2.2. Analysis of paclitaxel Quantitative analysis for intermediate or finished products was performed with a Hewlett-Packard 1090 HPLC system with a Curosil PFP column (Phenomenex, 4.6 mm×250 mm, dp = 5 m). Elution was performed with a gradient water and acetonitrile from 65:35 to 35:65 within 30 min (flow rate = 1.0 ml/min),The eluent was monitored at 227 nm (paclitaxel) or 255 nm (internal standard) with a photo diode-array detector. Purity determinations of paclitaxel were made using an internal standard assay to compare the paclitaxel content of the test material to the paclitaxel content of the reference paclitaxel. Purity values were calculated by comparing the response ratio determined for the test sample to that obtained for the reference paclitaxel and concentrations of paclitaxel were estimated by reference to the paclitaxel peak area. The dried residue was redissolved in methanol and used for the quantitative analysis of paclitaxel. Authentic paclitaxel and internal standard (n-propyl paraben), were purchased from Sigma and used for standard. 2.3. Preparation of the crude extract by extraction from suspension cultured cells and yew tree The solvent extractions were carried out employing methanol and dichloromethane. The cells recovered from culture were added to methanol, stirred at room temperature for 1 h and filtered to give a methanol extract, where the biomass was preferably added to methanol at a ratio of 100%. Extraction was repeated at least four times and the methanol extracts obtained from each extraction, were collected, and concentrated at a temperature of 20 to 40 ◦ C under a reduced pressure, to reduce the volume of the methanol extract to 20 to 30% of original. To eliminate polar impurities, liquid–liquid (2nd) extractions were carried out
with dichloromethane. The concentrated methanol extract was added to organic solvents (dichloromethane) at a volume ratio of 28%, stirred at room temperature for 30 min. The extraction was repeated at least three times and the crude extracts thus obtained, were pooled and dried at room temperature under reduced pressure. The recovery yield and purity of dried extract were 98 and 6.5%, respectively (Fig 3A). Extraction from yew tree was performed with the same method with the exception of chlorophylls removal by extraction using hexane from methanol concentrate. The purity of dried extract was 0.62% (Table 2, Fig. 4). 2.4. Adsorbent treatment of the crude extract The dried crude extract obtained from the extraction step was dissolved in dichloromethane (CH2 Cl2 ) at a ratio of 5.5 of absorbent (v/w) and followed by the addition of the synthetic adsorbents, such as active clay F-l (Mizukalife Chemical Co., Japan), sylopute (Fuji Silysia Chemical Ltd., Japan), activated carbon A Supra Euro (NORIT, Netherlands) at a ratio of 200% from 50% (w/w) of dried crude extract, stirred at room temperature for 30 min and filtered with the synthetic adsorbent to obtain the filtration solution. The absorbent cake thus obtained, was washed several times with dichloromethane and washings were combined with the filtration solution. The solution was then concentrated at 30 ◦ C under reduced pressure to the level equivalent to 1000% of the crude extract prior to adsorbent treatment. To analysis materials absorbed in absorbents, treated absorbents were washed with methanol and analyzed by HPLC. 2.5. 1st and 2nd precipitation of the crude paclitaxel for further purification For further purification of crude paclitaxel, the solution concentrated in adsorbent treatment step was added to 1000% volume of n-hexane to obtain the precipitate, and filtrated to give crude paclitaxel whose paclitaxel content was over 15%. The crude paclitaxel was dissolved in methanol and dropped the distilled water until 61.5% (v/v) methanol and left to stand at 4 ◦ C for 3 days to obtain paclitaxel precipitate. The paclitaxel content in 61.5% (v/v) methanol solution was 0.5% in pure paclitaxel basis (w/v). After precipitation, the paclitaxel precipitate was filtrated and dried at 30 ◦ C under reduced pressure. Highly purified paclitaxel over (99.5%) was obtained by ODS-HPLC, followed by Silica-HPLC with high yield.
3. Results and discussion 3.1. The effect of absorbent treatment in the purification process of paclitaxel from plant cell cultures For further removal of impurities, which appeared to have a deep color, tar, and insoluble material dissolved in
S.-H. Pyo et al. / Process Biochemistry 40 (2005) 1113–1117
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Table 1 Comparison of efficacy of each absorbent in the 1st and 2nd precipitation Step
Absorptiona and 1st precipitation Purity (%)
Yield (%)
Purity (%)
Yield (%)
Activated carbon Active clay (F-l) Sylopute
14.5 18.5 26.7
88.3 89.5 94.2
40.0 57.7 63.0
45.0 75.6 85.1
a
2nd Precipitation
Yield (overall %)
39.7 67.7 80.2
The purity and amount of starting material, which is crude extract from liquid–liquid extraction, are 6.8% and 5 g, respectively.
dichloromethane, the crude extract was treated with synthetic adsorbent, such as active clay F-l, sylopute, activated carbon. The result on efficiency of each absorbent treatment is summarized in Table 1. In this process, a large amount of impurities were able to be removed simply by treatment and filtration. Furthermore, the efficacy of these absorbents in the purification steps from crude extract to 2nd precipitate are also evaluated with yield and purity in Table 1. There are differences of purity and overall yield until the 2nd precipitation step in each case. On one side, synthetic absorbents, such as sylopute and active clay showed better efficacy in purity and yield than activated carbon. With 50% (w/w) excess of absorbent amounts, the purity of crude paclitaxel after treatment using, activated carbon, active clay, and sylopute followed by 1st and 2nd precipitation were 40, 57.7, and 63%, respectively (Table 1). The overall yield from crude extract for activated carbon, active clay and sylopute were, 39.7, 67.7, and 80.2%, respectively (Table 1). Based on these results the best of removal of impurities was obtained from the combination of sylopute treatment followed by 1st , 2nd precipitation. The higher the purity of the 1st precipitate after absorbent treatment obtained, the higher the purity of crude paclitaxel can be obtained in the 2nd precipitation step. Moreover the efficacy as well as overall yields can be enhanced using sylopute. The quantity of sylopute was monitored to optimize the amount to crude extract. The purity of crude paclitaxel in the 2nd precipitation step showed a tendency to improve in purity as an increasing of treated amounts of sylopute (Fig. 1). The optimum amount of sylopute was 150% (w/w) of dried crude extract. However, the yield starts drop when 200% (w/w) of sylopute was applied. Absorption of paclitaxel with absorbent and lack of washing for treated absorbent also lead to a decrease in yield. In the case of active clay (Fig. 1), a similar tendency was shown in yield and purity, but the yield and purity were lower than those of sylopute, while the lowest yield was obtained for activated carbon purity (data not shown).
Fig. 1. Effect of the ratio of sylopute to liquid–liquid extract (w/w) on overall yield and purity: overall yield for sylopute (A); purity for sylopute (B); overall yield for active clay (C); purity for active clay (D).
absorbent were recovered and analyzed (Fig. 2). In amount of the material absorbed, elute from sylopute was the best amount compare to activated carbon. On the other hand, the loss of product abounds in case of active clay. The result of this comparison with removal efficacy of impurities and loss of product was demonstrated the superiority of selectivity on sylopute. Fig. 3C showed the HPLC profile of impurities removal by absorption. Meanwhile, the average particle size of each absorbents is 10–50 m. In contrast with activated carbon, main component of sylopute is mainly, SiO2 (99.8%) and active clay are the mixture SiO2 (60%), MgO (28%), and other metal oxides (12%), respectively. Consequently, the efficacy of absorbents can be arisen from the difference of the interaction between absorbents and absorbed materials.
3.2. The comparison of adsorption ability and selectivity by analysis of material eluted from each absorbent To compare the absorption ability and selectivity of each absorbent, applied absorbents were treated with 100% MeOH and the materials eluted with methanol from each
Fig. 2. Comparison of materials and paclitaxel desorbed from absorbents: the amounts of applied crude extract and absorbent are 5 and 7.5 g, respectively.
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Fig. 4. Chromatograms of purification steps from yew tree analyzed by RP-HPLC: liquid–liquid extract (A); 1st precipitation (B), respectively. The arrow indicates the peak of paclitaxel.
Fig. 3. Chromatograms of purification steps from plant cell cultures analyzed by RP-HPLC: liquid–liquid extract (A); 1st precipitation (B); materials desorbed from sylopute (C); and 2nd precipitation (D), respectively. The arrow indicates the peak of paclitaxel.
3.3. The effect of absorbent treatment in purification of paclitaxel from yew tree The purity of crude extract was very low (0.62%) while the purity of crude extract from plant cell cultures was 6.8%. Fortunately, the removal effect of impurities showed the excellent result by means of reduction by half of the weight of dried crude paclitaxel with high yield. The purity after the 1st precipitation using the same methods was improved more than four times to 5.43% from 1.38%, with high yield (Table 2) as shown in HPLC chromatograms of Fig. 4A and B. However, the purity is still lower than that from the plant cell cultures and is not suited for the 2nd precipitation Table 2 Effect of absorbent treatment and 1st precipitation in the purification of paclitaxel from yew tree
Dried weight (g) Purity (%) Overall yield (%)
Liquid–liquid extraction
Sylopute treatment 1st
1st Precipitation
3.27 0.62 –
1.40 1.38 95.3
0.33 5.43 88.4
process because, too low purity of 1st precipitate could not be precipitated in 2nd precipitation step. However, after performing low-resolution column chromatography with silica gel, the resulting crude paclitaxel is sufficiently pure HPLC (data not shown). Based on these results, this technique can be applied for partial purification of paclitaxel or natural products. Another obvious advantage over conventional methods, is the removal of waxy compounds and impurities. Crude extract, was pre-purified adequately to perform HPLC through a combination with sylopute treatment and precipitation. The chromatograms of each step are shown in Fig. 3. To increase efficacy of the HPLC process, which costs a great deal, higher purity of samples was required, because purification of samples with low purity requires a large number of HPLC runs. Therefore, the development of effective and convenient pretreatment process is necessary. The Pre-purification, such as absorbent treatment, low-resolution chromatography, precipitation and solvent extraction have been used to achieve these purposes [16,17]. Absorbents, such as, Amberlite, XAD, Silica, ODS (octadecylsilylated), provide applied adsorption processes as well as chromatography methods, but selective removal of derivatives, which have similar properties to the product, are never achieved using simple filtration with organic solvent with low cost. However, sylopute was the best choice of absorbent, because sylopute not only removed color but also better efficacy than activated carbon for the removal of other impurities in the crude product. Another advantage of rising sylopute is the fast filtration rate. Therefore, the abilities of selective impurity removal of sylopute can be applied for other purification processes of many natural source compounds. References [1] Wani MC, Taylor HL, Wall ME, Coggon P, McPhail AT. Plant antitumor agents. VI. The isolation and structure of taxol. J Am Chem Soc 1971;93:2325–7.
S.-H. Pyo et al. / Process Biochemistry 40 (2005) 1113–1117 [2] Arbuck SQ, Christian MC, Fisherman JS, Cazenave LA, Sarosy Q, Suffhess M, Adams J, Canetta R, Cole KE, Friedman MA. Clinical development of taxol. J Natl Cancer Inst Monogr 1993;15:11–24. [3] McGuire WP, Rowinsky EK, Rosenshein NB, Grumbine FC, Ettinger DS, Armstrong DK, Donehower RC. Taxol: a unique antineoplastic agent with significant activity in advanced ovarian epithelial neoplasms. Ann Intern Med 1989;111:273–9. [4] Rowinsky EK, Cazenave LA, Donehower RC. Taxol: a novel investigational antimicrotubule agent. J Natl Cancer Inst 1990;82:1247–59. [5] Witherup KM, Look SA, Stasko MW, Ghiorzi TJ, Muschik GM, Cragg GM. Taxus spp. needles contain amounts of taxol comparable to the bark of Taxus brevifolia: analysis and isolation. J Nat Prod 1990;53:1249–55. [6] Rao KV, Hanuman JB, Alvarez C, Stoy M, Juchum J, Davies RM, Baxley R. A new large-scale process for taxol and related taxanes from Taxus brevifolia. Pharm Res 1995;12:1003–10. [7] ElSohly HN, Croom EM, Kopycki WJ, Joshi AS, McChesney JD. Diurnal and seasonal effects on the taxane content of the clippings of certain Taxus cultivars. Phytochem Anal 1997;8:124. [8] van Rozendaal ELM, Lelyveld GP, van Beek TA. Screening of the needles of different yew species and cultivars for paclitaxel and related taxoids. Phytochemistry 2000;53:383–9. [9] Vance NC, Kelsey RQ, Sabin TE. Seasonal and tissue variation in taxane concentrations of Taxus brevifolia. Phytochemistry 1994;36:1241–4. [10] Baloglu E, Kingston DG. A new semisynthesis of paclitaxel from baccatin III. J Nat Prod 1999;62:1068–71. [11] Nicolaou KC, Yang Z, Liu JJ, Ueno H, Nantermet PQ, Guy RK, Claiborne CF, Renaud J, Couladouros EA, Paulvannan K. Total synthesis of taxol. Nature 1994;6464:630–4.
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[12] Choi HK, Kim S, Son JS, Hong SS, Lee HS. Enhancement of paclitaxel paclitaxel production by temperature shift in suspension culture of Taxus chinensis. Enzyme Microb Technol 2000;27:593– 8. [13] Kim JH, Yun JH, Hwang YS, Byun SY, Kim DI. Production of taxol and related taxanes in Taxus brevifolia cell cutures: effect of sugar. Biotechnol Lett 1995;17:101–6. [14] Pare G, Canaguier A, Landr P, Hocquemiller R, Chriqui D, Meyer M. Production of taxoids with biological activity by plant and callus culture from selected Taxus genotypes. Phytochemistry 2002;59:725– 30. [15] Hong SS, Song BK, Kim JH, Lim CB, Lee HS, Kim KW, Kang IS, Park HB. Method for purifying taxol from Taxus biomass US Patent 1999;5:900–79. [16] Kim JH, Kang IS, Choi HK, Hong SS, Lee HS. A novel prepurification for paclitaxel from plant cell cultures. Process Biochem 2002;37:679–6828. [17] Pyo SH, Park HB, Song BK, Han BH, Kim JH. A large-scale purification of paclitaxel from cell cultures of Taxus chinensis. Process Biochem 2004 [in press]. [18] Dabrowski A. Adsorption-from theory to practise. Adv Colloid Interface Sci 2001;93:135–224. [19] Diez MC, Mora ML, Videla S. Adsorption of phenolic compounds and color from bleached Kraft mill effluent using allophonic compounds. Water Res 1999;33:125–30. [20] Choi HK, Adams TL, Stahlhut W, Kim SI, Yun JH, Song BK, Kim JH, Song JS, Hong SS, Lee HS. Method for mass production of taxol by semi-continuous culture with Taxus chinensis cell culture US Patent 1999;5:871.
Effects of absorbent treatment on the purification of paclitaxel from cell cultures of Taxus chinensis and yew tree. Sang-Hyun Pyo a , Bong-Kyu Song a , Chang-Hun Ju a , Byung-Hee Han b , Ho-Joon Choi a,∗ b
a Samyang Genex Biotech Research Institute, 63-2 Hwaam-Dong, Yuseong-Gu, Daejeon, 305-348, South Korea Department of Chemistry, Chungnam National University, 220 Gung-Dong, Yuseong-Gu, Daejeon, 305-764, South Korea
Received 1 February 2004; accepted 27 March 2004
Abstract A new synthetic absorbent, sylopute was applied for the purification of paclitaxel from plant cell cultures and yew tree. There was selective absorption of impurities in the treatment of sylopute. Therefore, higher purity paclitaxel could be obtained simply in the following process with a higher yield than with active clay or activated carbon. The purity of crude paclitaxel after sylopute treatment followed by precipitation was increased up to 63%, while the purities for active clay and activated carbon were 57.7 and 40%, respectively. The overall yields from sylopute, active clay, and activated carbon were 80.2, 67.7, and 35.5%, respectively. The best result in removing impurities was obtained from the combination of sylopute treatment followed by 1st and 2nd precipitation processes. A similar result was observed in the case of absorbent treatment of crude extract from yew tree. Consequently, sylopute can be applied for production scale purification of other natural products as an alternative to synthetic absorbents, activated carbon, and other alternative processes. © 2004 Elsevier Ltd. All rights reserved. Keywords: Sylopute; Paclitaxel; Isolation; Purification; Absorbent; Adsorption
1. Introduction Paclitaxel, first reported in 1971 [1], is one of the most important anticancer agents in the treatment of refractory ovarian, breast, and other cancers [2–4]. Supplies of paclitaxel are potentially limited and not affordable from the environmental point of view, because the original and major source material of the drug is the bark of Taxus brevifolia [5]. The yield of purification of paclitaxel from T. brevifolia is about 0.01% of the dry weight of bark [6]. It is known that there are significant differences in the contents according to location, season, and tissue variations [7–9]. In addition, chemical semi or total synthesis of paclitaxel [10,11] is very complex with a very low yield and may not be feasible commercially on an industrial scale. Paclitaxel production in plant cell and tissue cultures has the potential to provide sufficient quantities of paclitaxel for chemotherapeutic use
∗
Correspondence author. Fax: +82 42 865 8398. E-mail address: [email protected] (H.-J. Choi).
0032-9592/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.03.004
[12–14]. Meanwhile, cell culture offers the potential advantage of availability and reliable production of using a renewable resource whereas bark-stripping methods [5,6] lead to the destruction of scarce plant material. Nevertheless, the development of a more effective process for separation and purification are important subject. An efficient procedure for large-scale productionof paclitaxel from plant cell cultures has been developed [15–17]. To develop more efficient production methods on a commercial scale, absorption selectivity using several absorbents including, active clay, sylopute, and activated carbon were studied. Generally, activated carbon is usually used as absorbent and has been used broadly to remove color for decolorization of natural product and water as well as the removal of impurities in the crude product [16–19]. However, some synthetic absorbents show better efficiency in purity and yield than activated carbon. In this investigation, we have developed a large-scale and efficient method for removal of impurities in crude extracts using sylopute followed by precipitation. This method is suitable for large-scale purification of paclitaxel from plant cell cultures and yew tree for the purposes of clinical use.
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2. Materials and methods 2.1. Plant materials and culture conditions Suspension cells originating from T. chinensis, were maintained in darkness at 24 ◦ C with shaking at 150 rpm. Suspension cells were cultured in a modified Gamborg’s B5 medium supplemented with 30 g/1 sucrose, 10 M of NAA (naphthalene acetic acid), 0.2 M of BA (6-benzylamino purine), 1 g/1 of casein hydrolysate, and 1 g/1 of 2-(Nmorpholino) ethanesulphonic acid (MES). Cell cultures were transferred to a fresh medium every 2 weeks. In a prolonged culture for production, 1 and 2% maltose was added to the culture medium on day 7 and day 21, respectively, and 4 M of AgNO3 was added on the initiation of culture as elicitor [20]. After the culture, biomass was recovered with a decanter (Westfalia, CA150 Clarifying Decanter) and high-speed centrifuge (␣-Laval, BTOX 205GD-35CDEFP). Paclitaxel in solution was recovered by absorption with resin. 2.2. Analysis of paclitaxel Quantitative analysis for intermediate or finished products was performed with a Hewlett-Packard 1090 HPLC system with a Curosil PFP column (Phenomenex, 4.6 mm×250 mm, dp = 5 m). Elution was performed with a gradient water and acetonitrile from 65:35 to 35:65 within 30 min (flow rate = 1.0 ml/min),The eluent was monitored at 227 nm (paclitaxel) or 255 nm (internal standard) with a photo diode-array detector. Purity determinations of paclitaxel were made using an internal standard assay to compare the paclitaxel content of the test material to the paclitaxel content of the reference paclitaxel. Purity values were calculated by comparing the response ratio determined for the test sample to that obtained for the reference paclitaxel and concentrations of paclitaxel were estimated by reference to the paclitaxel peak area. The dried residue was redissolved in methanol and used for the quantitative analysis of paclitaxel. Authentic paclitaxel and internal standard (n-propyl paraben), were purchased from Sigma and used for standard. 2.3. Preparation of the crude extract by extraction from suspension cultured cells and yew tree The solvent extractions were carried out employing methanol and dichloromethane. The cells recovered from culture were added to methanol, stirred at room temperature for 1 h and filtered to give a methanol extract, where the biomass was preferably added to methanol at a ratio of 100%. Extraction was repeated at least four times and the methanol extracts obtained from each extraction, were collected, and concentrated at a temperature of 20 to 40 ◦ C under a reduced pressure, to reduce the volume of the methanol extract to 20 to 30% of original. To eliminate polar impurities, liquid–liquid (2nd) extractions were carried out
with dichloromethane. The concentrated methanol extract was added to organic solvents (dichloromethane) at a volume ratio of 28%, stirred at room temperature for 30 min. The extraction was repeated at least three times and the crude extracts thus obtained, were pooled and dried at room temperature under reduced pressure. The recovery yield and purity of dried extract were 98 and 6.5%, respectively (Fig 3A). Extraction from yew tree was performed with the same method with the exception of chlorophylls removal by extraction using hexane from methanol concentrate. The purity of dried extract was 0.62% (Table 2, Fig. 4). 2.4. Adsorbent treatment of the crude extract The dried crude extract obtained from the extraction step was dissolved in dichloromethane (CH2 Cl2 ) at a ratio of 5.5 of absorbent (v/w) and followed by the addition of the synthetic adsorbents, such as active clay F-l (Mizukalife Chemical Co., Japan), sylopute (Fuji Silysia Chemical Ltd., Japan), activated carbon A Supra Euro (NORIT, Netherlands) at a ratio of 200% from 50% (w/w) of dried crude extract, stirred at room temperature for 30 min and filtered with the synthetic adsorbent to obtain the filtration solution. The absorbent cake thus obtained, was washed several times with dichloromethane and washings were combined with the filtration solution. The solution was then concentrated at 30 ◦ C under reduced pressure to the level equivalent to 1000% of the crude extract prior to adsorbent treatment. To analysis materials absorbed in absorbents, treated absorbents were washed with methanol and analyzed by HPLC. 2.5. 1st and 2nd precipitation of the crude paclitaxel for further purification For further purification of crude paclitaxel, the solution concentrated in adsorbent treatment step was added to 1000% volume of n-hexane to obtain the precipitate, and filtrated to give crude paclitaxel whose paclitaxel content was over 15%. The crude paclitaxel was dissolved in methanol and dropped the distilled water until 61.5% (v/v) methanol and left to stand at 4 ◦ C for 3 days to obtain paclitaxel precipitate. The paclitaxel content in 61.5% (v/v) methanol solution was 0.5% in pure paclitaxel basis (w/v). After precipitation, the paclitaxel precipitate was filtrated and dried at 30 ◦ C under reduced pressure. Highly purified paclitaxel over (99.5%) was obtained by ODS-HPLC, followed by Silica-HPLC with high yield.
3. Results and discussion 3.1. The effect of absorbent treatment in the purification process of paclitaxel from plant cell cultures For further removal of impurities, which appeared to have a deep color, tar, and insoluble material dissolved in
S.-H. Pyo et al. / Process Biochemistry 40 (2005) 1113–1117
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Table 1 Comparison of efficacy of each absorbent in the 1st and 2nd precipitation Step
Absorptiona and 1st precipitation Purity (%)
Yield (%)
Purity (%)
Yield (%)
Activated carbon Active clay (F-l) Sylopute
14.5 18.5 26.7
88.3 89.5 94.2
40.0 57.7 63.0
45.0 75.6 85.1
a
2nd Precipitation
Yield (overall %)
39.7 67.7 80.2
The purity and amount of starting material, which is crude extract from liquid–liquid extraction, are 6.8% and 5 g, respectively.
dichloromethane, the crude extract was treated with synthetic adsorbent, such as active clay F-l, sylopute, activated carbon. The result on efficiency of each absorbent treatment is summarized in Table 1. In this process, a large amount of impurities were able to be removed simply by treatment and filtration. Furthermore, the efficacy of these absorbents in the purification steps from crude extract to 2nd precipitate are also evaluated with yield and purity in Table 1. There are differences of purity and overall yield until the 2nd precipitation step in each case. On one side, synthetic absorbents, such as sylopute and active clay showed better efficacy in purity and yield than activated carbon. With 50% (w/w) excess of absorbent amounts, the purity of crude paclitaxel after treatment using, activated carbon, active clay, and sylopute followed by 1st and 2nd precipitation were 40, 57.7, and 63%, respectively (Table 1). The overall yield from crude extract for activated carbon, active clay and sylopute were, 39.7, 67.7, and 80.2%, respectively (Table 1). Based on these results the best of removal of impurities was obtained from the combination of sylopute treatment followed by 1st , 2nd precipitation. The higher the purity of the 1st precipitate after absorbent treatment obtained, the higher the purity of crude paclitaxel can be obtained in the 2nd precipitation step. Moreover the efficacy as well as overall yields can be enhanced using sylopute. The quantity of sylopute was monitored to optimize the amount to crude extract. The purity of crude paclitaxel in the 2nd precipitation step showed a tendency to improve in purity as an increasing of treated amounts of sylopute (Fig. 1). The optimum amount of sylopute was 150% (w/w) of dried crude extract. However, the yield starts drop when 200% (w/w) of sylopute was applied. Absorption of paclitaxel with absorbent and lack of washing for treated absorbent also lead to a decrease in yield. In the case of active clay (Fig. 1), a similar tendency was shown in yield and purity, but the yield and purity were lower than those of sylopute, while the lowest yield was obtained for activated carbon purity (data not shown).
Fig. 1. Effect of the ratio of sylopute to liquid–liquid extract (w/w) on overall yield and purity: overall yield for sylopute (A); purity for sylopute (B); overall yield for active clay (C); purity for active clay (D).
absorbent were recovered and analyzed (Fig. 2). In amount of the material absorbed, elute from sylopute was the best amount compare to activated carbon. On the other hand, the loss of product abounds in case of active clay. The result of this comparison with removal efficacy of impurities and loss of product was demonstrated the superiority of selectivity on sylopute. Fig. 3C showed the HPLC profile of impurities removal by absorption. Meanwhile, the average particle size of each absorbents is 10–50 m. In contrast with activated carbon, main component of sylopute is mainly, SiO2 (99.8%) and active clay are the mixture SiO2 (60%), MgO (28%), and other metal oxides (12%), respectively. Consequently, the efficacy of absorbents can be arisen from the difference of the interaction between absorbents and absorbed materials.
3.2. The comparison of adsorption ability and selectivity by analysis of material eluted from each absorbent To compare the absorption ability and selectivity of each absorbent, applied absorbents were treated with 100% MeOH and the materials eluted with methanol from each
Fig. 2. Comparison of materials and paclitaxel desorbed from absorbents: the amounts of applied crude extract and absorbent are 5 and 7.5 g, respectively.
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Fig. 4. Chromatograms of purification steps from yew tree analyzed by RP-HPLC: liquid–liquid extract (A); 1st precipitation (B), respectively. The arrow indicates the peak of paclitaxel.
Fig. 3. Chromatograms of purification steps from plant cell cultures analyzed by RP-HPLC: liquid–liquid extract (A); 1st precipitation (B); materials desorbed from sylopute (C); and 2nd precipitation (D), respectively. The arrow indicates the peak of paclitaxel.
3.3. The effect of absorbent treatment in purification of paclitaxel from yew tree The purity of crude extract was very low (0.62%) while the purity of crude extract from plant cell cultures was 6.8%. Fortunately, the removal effect of impurities showed the excellent result by means of reduction by half of the weight of dried crude paclitaxel with high yield. The purity after the 1st precipitation using the same methods was improved more than four times to 5.43% from 1.38%, with high yield (Table 2) as shown in HPLC chromatograms of Fig. 4A and B. However, the purity is still lower than that from the plant cell cultures and is not suited for the 2nd precipitation Table 2 Effect of absorbent treatment and 1st precipitation in the purification of paclitaxel from yew tree
Dried weight (g) Purity (%) Overall yield (%)
Liquid–liquid extraction
Sylopute treatment 1st
1st Precipitation
3.27 0.62 –
1.40 1.38 95.3
0.33 5.43 88.4
process because, too low purity of 1st precipitate could not be precipitated in 2nd precipitation step. However, after performing low-resolution column chromatography with silica gel, the resulting crude paclitaxel is sufficiently pure HPLC (data not shown). Based on these results, this technique can be applied for partial purification of paclitaxel or natural products. Another obvious advantage over conventional methods, is the removal of waxy compounds and impurities. Crude extract, was pre-purified adequately to perform HPLC through a combination with sylopute treatment and precipitation. The chromatograms of each step are shown in Fig. 3. To increase efficacy of the HPLC process, which costs a great deal, higher purity of samples was required, because purification of samples with low purity requires a large number of HPLC runs. Therefore, the development of effective and convenient pretreatment process is necessary. The Pre-purification, such as absorbent treatment, low-resolution chromatography, precipitation and solvent extraction have been used to achieve these purposes [16,17]. Absorbents, such as, Amberlite, XAD, Silica, ODS (octadecylsilylated), provide applied adsorption processes as well as chromatography methods, but selective removal of derivatives, which have similar properties to the product, are never achieved using simple filtration with organic solvent with low cost. However, sylopute was the best choice of absorbent, because sylopute not only removed color but also better efficacy than activated carbon for the removal of other impurities in the crude product. Another advantage of rising sylopute is the fast filtration rate. Therefore, the abilities of selective impurity removal of sylopute can be applied for other purification processes of many natural source compounds. References [1] Wani MC, Taylor HL, Wall ME, Coggon P, McPhail AT. Plant antitumor agents. VI. The isolation and structure of taxol. J Am Chem Soc 1971;93:2325–7.
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