Biotechnology Letters 22: 16571660, 2000. 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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Single step Al2 O3 chromatography for improved separation and recovery of Taxol
Zhiqiang Zhang & Zhiguo Su
State Key Laboratory of Biochemical Engineering, Institute of Chemical Metallurgy, Chinese Academy of Science, Beijing 100080, P.R. China Author for correspondence and present address: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Science, Beijing 100080, P.R. China (Fax: +86 10 62560912; E-mail: zqzhang@263.net)
Received after revisions 24 August 2000; Accepted 1 September 2000

Key words: alumina chromatography, taxanes, Taxol

Abstract An improved method that uses a single alkaline Al2 O3 chromatography step was developed both for separating Taxol from the extracts of Taxus cuspidate callus cultures, and meanwhile converting other taxanes to Taxol catalyzed by the Al2 O3 adsorbent. Under the optimized operating conditions, a Taxol recovery of 170% was obtained. The nal Taxol content was 29% starting from less than 1% in the initial extracts.

Introduction Taxol, a diterpene obtained from Taxus spp., has signicant anti-cancer activity. Currently, Taxus spp. are the only commercial sources of Taxol. With the growing demand for Taxol and its limited supply, various methods to increase the yield of Taxol from extracts of Taxus have been developed. Taxol separation and purication with a single reversed-phase HPLC step was devised by Wu et al. (1995). However, some contaminants, such as waxes, chlorophylls and non-polar components are still present in the crude samples, which foul the high-resolution column and consequently shorten its life. In addition, in the preparative purication of Taxol, the low solubility of Taxol components always limits the loading of a reverse phase column with a low organic solvent concentration. Practically, a two-step process may be more suitable than a one-step for Taxol separation. In the rst step, a somewhat inexpensive treatment to clarify the crude extracts should be used. This step may not give a very high Taxol purity but will result in a very high recovery. In the preparative isolation of a natural product, adsorption on to silica gel or Al2 O3 is often used as an initial clean-up. Silica gel and Al2 O3

are relatively inexpensive materials, but have high capacities of adsorption. They have also catalytic activity for isomerization and dehydration. In extracts of Taxus, there are several taxanes including 7-epi-Taxol and sugar-bound Taxol, such as 7xylosyl Taxol (Senilh et al. 1984, Huang et al. 1986). Durzan & Ventimiglia (1994) have used xylanase to free Taxol from these taxanes, but this operation is too expensive and difcult to scale up. Carver et al. (1994) also indicated an increase in the Taxol recovery from extracts of Taxus brevifolia by using ion-exchange media. In this paper, we have developed an improved method using an alkaline Al2 O3 chromatography eluted in an isocratic mode for separating Taxol from extracts of Taxus cuspidate callus cultures, and meanwhile converting other taxanes to Taxol to increase the total Taxol recovery. Materials and methods Materials Taxol standard was purchased from Sigma (USA). Alkaline, neutral and acidic Al2 O3 chromatographic supports (5070 m) were supplied by Shanghai Chem-

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Table 1. The inuence of chromatographic medium on isolation and recovery of Taxol from 10 g crude sample. Adsorbent matrix Silica gel Alkaline Al2 O3 Neutral Al2 O3 Acidic Al2 O3 Purication factor 22 45 26 23 Taxol recovery (%) 99 170 101 96

(25:35:40, by vol.) at 1.0 ml min1 . The sample and the mobile phase were ltered through 0.2 m PVDF lter before entering the column. Taxol was quantied by comparing the average peak area of the sample with that of the Taxol standard.

Results and discussion The recovery of Taxol with silica-gel and Al2 O3 chromatography

ical Works (Shanghai, China). Silica gel (5070 m) was supplied by Qindao Ocean Chemical Works (Qindao, China). The HPLC system was Beckman FL750. A C18 column (4.5 mm 250 mm) was also provided by Beckman Instruments Inc. (California, USA). Molecular sieves (Type A4) were purchased from Beijing Chemical Reagent Company. Sample preparation Taxus cuspidate callus cultures were established as described by Xu et al. (1998). The 68 weeks old, slowgrowing callus samples were harvested and freezedried. Calluses were extracted with methanol at ambient temperature for 2 days. The resulting solution was ltered through two lter papers. The methanol was evaporated with a rotary vacuum evaporator at 4050 C. The residue was redissolved in chloroform for the subsequent separation. Alkaline Al2 O3 adsorption chromatography The Al2 O3 powders were packed in a 15 mm 250 mm column and equilibrated with chloroform/methanol (98.5:1.5 v/v). The sample was then loaded onto the column at ow rate 2.0 ml min1 , followed by chloroform/methanol (99:1, v/v) wash to remove non-adsorbed impurities. Then, isocratic elution was performed with chloroform/methanol (95:5, v/v) at 3.5 ml min1 . The eluent was detected at 227 nm with a Pharmacia UV monitor. The collected eluent was evaporated with a rotary vacuum evaporator at 3040 C. The residue was resuspended in methanol for HPLC assay. HPLC analysis Taxol was assayed by HPLC using a C18 -silica column (4.5 mm 250 mm). A 20 l sample was injected each time and detected at 227 nm. The mobile phase for isocratic elution was methanol/acetonitrile/water

The results of Taxol separation on silica gel, alkaline Al2 O3 , neutral Al2 O3 and acidic Al2 O3 adsorption columns are presented in Table 1. The purication factor was dened as the ratio of Taxol content in the sample after chromatography to that in the initial extracts. The alkaline Al2 O3 chromatography was the most efcient to give high recovery and good separation. The alkaline Al2 O3 possibly catalyzed the conversion of other taxanes to Taxol so that the total amount of Taxol was increased as shown by the recovery of 170% after the step of Al2 O3 chromatography, with a purication factor of 45. In contrast, the silica chromatography only gave 99% recovery with a purication factor of 22, the neutral Al2 O3 chromatography gave 101% recovery with a purication factor of 26, and the acidic Al2 O3 chromatography gave 96% recovery with a purication factor of 23. The result of recovery 170% was reproducible and could not be interpreted as an analytical error since the same analysis was used for all chromatography processes. The recoveries of other methods were within the range of literature. The only explanation is that the process of alkaline Al2 O3 chromatography increased the total amount of Taxol in the production stream so that the calculated recovery based on the Taxol before chromatography was more than 100%. In our previous experiments, a chromatographic protocol using C18 silica gel phase and Al2 O3 phase to research the increasing amount of Taxol from the crude methanol extracts of Taxus cuspidate callus cultures had developed. The study indicated that the main source of Taxol increasing was the isomerization of 7-epi-Taxol in the crude extracts to Taxol. The inuence of water on the separation and recovery of Taxol During the alkaline Al2 O3 chromatography, the activity of medium is a very important factor that affects the separation of Taxol and the conversion of taxanes

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Fig. 1. The inuence of water on isolation and recovery process. (A) Both the Al2 O3 and the solvent were dehydrated. (B) The Al2 O3 was dehydrated but the solvent was humidied. (C) Both the solvent and Al2 O3 were humidied. Chromatographic conditions: the solvent for sample loading/washing was chloroform; an alkaline Al2 O3 column of 15 mm 250 mm was loaded with 200 mg sample and isocratically eluted at 3.5 ml min1 with chloroform/methanol (95:5, v/v). The value 100% means all of the Taxol in the initial extracts was recovered. The purication factor was dened as the ratio of Taxol content in the sample after chromatography to that in the initial extract.

Fig. 2. The inuence of the methanol content in the loading/washing buffer on the separation and recovery of Taxol. Chromatographic conditions: an alkaline Al2 O3 column of 15 mm 250 mm was loaded with 200 mg sample and eluted at 3.5 ml min1 with chloroform/methanol (95:5, v/v). Recovery ( ), purication factor ( ). The value 100% of recovery means all of the Taxol in the initial extracts was recovered. The purication factor was dened as the ratio of Taxol content in the sample after chromatography to that in the initial extract.

to free Taxol. Figure 1 shows the inuence of water on the processes. The highest recovery of Taxol was reached when Al2 O3 was dehydrated at 600 C for 2 h and the solvent was dried with molecular sieves. In contrast, if Al2 O3 was dehydrated but the solvent was humidied to contain 0.07% (w/v) water, the purication factor increased, but the recovery of Taxol decreased. If both the solvent and Al2 O3 were humidied, then both the Taxol recovery and the purication factor decreased. According to Snyder & Kirland (1979), the presence of water could improve the separation of adsorption chromatography because water would remove the strongest adsorption cores on the surface of the medium. However, it was possible that the presence of water would also deactivate some reaction cores in the medium, which would reduce the conversion reaction from other taxanes to free Taxol. As a result, the recovery of Taxol decreased. The inuence of methanol on the separation and recovery of Taxol The inuence of the methanol content of the loading/washing buffer on the Taxol recovery and purication factor is shown in Figure 2. Usually, the purpose of column washing after sample loading is to remove unadsorbed impurities on the column. However, with the addition of methanol in the loading/washing buffer in this work, both the Taxol recovery and the purica-

Fig. 3. The inuence of the retention time on the recovery of Taxol. Chromatographic conditions: the loading/washing buffer was chloroform/methanol (98.5:1.5, v/v). An alkaline Al2 O3 column of 15 mm 250 mm was loaded with 200 mg sample and eluted at 3.5 ml min1 with chloroform/methanol (95:5, v/v). The value 100% means all of the Taxol in the initial extracts was recovered.

tion factor were increased. The two values reached the maximum at 1.5% (v/v) of methanol. Further increasing in methanol concentration to 5% caused a decrease in these two values. However, the Taxol recovery was still higher than that without methanol supplement. It was possible that the presence of methanol enhanced the conversion reaction of other taxanes to Taxol in the alkaline Al2 O3 chromatography.

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Table 2. Results of Taxol separation and recovery from 10 g crude sample with a single alkaline Al2 O3 chromatography step. Step Crude sample Al2 O3 chromatography Taxol (mg) 65 111 Purity of Taxol (%) 0.6 29 Recovery (%) 100 170

Conclusion The procedure reported here is a reproducible, simple and inexpensive method that combined the separation of Taxol from the callus cultures extracts and conversion of other taxanes to Taxol in a single step. The optimized operating conditions were selected as follows: use alkaline Al2 O3 as medium; both the medium and solvent were dehydrated; the retention time was set at 30 min; the loading/washing buffer was chloroform/methanol (98.5:1.5, v/v) and eluent was chloroform/methanol (95:5, v/v). Under these conditions a Taxol content of 29% was obtained from the initial callus extracts with less than 1% Taxol and the Taxol recovery exceeded 170%.

Fig. 4. The prole of alkaline Al2 O3 chromatography. Chromatographic conditions: both the medium and the solvent were dehydrated. The loading/washing buffer was chloroform/methanol (98.5:1.5, v/v1 ). A 200 mg sample was loaded and eluted at 3.5 ml min1 with chloroform/methanol (95:5, v/v). The eluent was detected at 227 nm. Peak 1 represented Taxol.

References
Carver DR, Prout TR, Workman CT (1994) Method of using ion exchange media to increase taxane yields. United States Patent 5281727. Durzan DJ, Ventimiglia F (1994) Texanes and the release of bound compounds by xylanse in female haploid-derived cell suspension culture. In Vitro Cell Dev. Biol. Plant. 30: 219227. Huang CHO, Kingston DGI, Magri NF, Samaranayake G, Boettner FE (1986) New taxanes from Taxus brevifolia. J Nat. Prod. 49: 665669. Senilh VS, Blechert MC, Guenard D, Picot F, Potier P, Varenne P (1984) Mise en vidence de nouveaux analogues du Taxol extracts de Taxus baccata. J Nat. Prod. 47: 131137. Snyder LR, Kirland JJ (1979) Introduction to Modern Liquid Chromatography. New York: Inc. New York Chester Brisbane Toronto. Wu DR, Lohse K, Greenblatt HC (1995) Preparative separation of taxol in normal- and reversed-phase operations. J. Chromatogr. A. 702: 233241. Xu J, Yin P, Wei X, Su Z (1998) Self-immobilized aggregate culture of Taxus cuspidata for improved Taxol production. Biotechnol. Tech. 12: 241244.

The suitable retention time of samples in the column Figure 3 demonstrates the inuence of the retention time on the recovery of Taxol. The Taxol recovery reached a maximum at the retention time of 30 min. Further increasing the retention time resulted in a decrease in the Taxol recovery. This indicated that on alkaline Al2 O3 column, while other taxanes could convert to free Taxol, Taxol could decompose to other moleculars. In order to obtain the maximum Taxol recovery, a retention time of 30 min should be selected. The total results of Taxol separation and recovery The results of Taxol separation and recovery with inexpensive alkaline Al2 O3 chromatography in a single step are shown in Table 2. The content of Taxol reached 29% from the initial callus extracts (<1% Taxol) with a Taxol recovery of 170%. The prole of alkaline Al2 O3 chromatography is shown in Figure 4.