Supercritical Extraction of Paclitaxel Using CO2 and CO

Chapter 29

Supercritical Extraction of Paclitaxel Using CO and CO -Ethanol Mixtures 2

2

1

Vishnu Vandana and Amyn S. Teja

Downloaded by UNIV OF CINCINNATI on May 20, 2016 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0608.ch029

School of Chemical Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0100 Taxol is an exceptionally promising cancer therapeutic agent with an unusually broad spectrum of antileukemic and tumor inhibiting activity. To date, slow growing yew trees are the major source of this compound. New separation methods are therefore of interest for its extraction from the plant material. The extraction of taxol from the bark of Taxus brevifolia using supercritical carbon dioxide with and without ethanol as a co-solvent was studied in this work. The extractions were performed at 319 Κ and 331 Κ and at pressures ranging from 10 to 38 MPa. The addition of co-solvent significantly increased the rate of extraction of taxol. Furthermore, supercritical extraction was found to be more selective than conventional liquid ethanol extraction and could remove a significant amount of the taxol present in the bark.

Taxol is an alkaloid currently in phase III clinical trials for the treatment of ovarian cancer. It is found in trees of the Taxus species and was first isolated from the bark of Taxus brevifolia by Wani et al. in 1971 [1]. Its chemical structure shown in Figure 1. Isolation of the drug from the bark of several Taxus species has been reported by a number of researchers [2, 3, 4], although there is some concern about the natural supply of taxol. Total and partial synthesis of the drug are being studied in several lab oratories [5, 6, 7, 8, 9, 10]. Total synthesis of taxol has been achieved from simple starting materials by Holton et al. and Nicolaou et al. [11, 12, 13, 14]. However, the yields obtained in these syntheses are small and total synthesis 1

Corresponding author

NOTE: Paclitaxel is the generic name for Taxol, which is now a registered trademark.

0097-6156/95/0608-0429$12.00/0 © 1995 American Chemical Society

Hutchenson and Foster; Innovations in Supercritical Fluids ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

INNOVATIONS IN SUPERCRITICAL FLUIDS


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Figure 1. Structure of Taxol.


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is unlikely to provide the required quantity of taxol in the near future. Partial synthesis from precursors such as baccatin III and 10-deacetylbaccatin III is a more practical solution to the supply problem, and has been achieved by Dennis et. al [15]. However, this approach will require more efficient isolation and extraction methods for the precursors. Production from tissue culture is another alternative that is being pursued [16]. However, it is difficult to establish long term micro cultures of conifers due to the difficult juvenile tissue, production of detrimental secondary metabo Downloaded by UNIV OF CINCINNATI on May 20, 2016 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0608.ch029

lites and recalcitrance of this group of plants to form in-vitro cultures. Though shoot cultures are difficult to establish, long term maintenance of callus and cell cultures is being established and effects of various parameters such as plant growth regulators, vitamins and minerals on growth rate, production of secondary metabolites, rate of production of taxol, and formation of other taxanes, are being studied. Genetic engineering of taxol is also being inves tigated. Transferring the enzyme system to a faster growing species or to a yeast or a bacterium is being considered [17]. However, the apparent difficulty in executing this method is the bioactivity of the product towards eukaryotic tubulin. The other obstacle is the absence of information on the biosynthetic pathway. One of the major sources of taxol is material from the trees of the Taxus species. Since the amount of taxol present in the trees is small (~ 0.01 %), it is important to develop an effective separation process for its extraction. Current production methods are estimated to extract only half the amount of taxol that is present in the tree. In this study, a new supercritical based separation process has been developed to extract and isolate taxol and related taxanes from the bark and needles of the Taxus brevifolia. Supercritical extraction offers a number of advantages over more conven tional separation techniques in biochemical and biotechnology applications. The solvents (e.g., carbon dioxide) used are physiologically inert thus avoiding toxicity and environmental problems. Since the the density of a supercritical fluid is a strong function of temperature and pressure, separation of the solute and solvent can be achieved with small changes in temperature or pressure. Also, addition of a small amount of co-solvent generally increases the solvent power and selectivity of the solute in the supercritical phase. Finally, super critical extraction can be combined with other post extraction processes to yield hybrid separation processes. Because of these advantages, the use of


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supercritical fluids in the recovery of taxol and precursors was studied in this work.

Experimental Reagents and Materials.

Coleman grade carbon dioxide of minimum pu

rity 99.99 % was obtained from Matheson Gas Products. HPLC grade Methanol and Water and reagent grade (99.99 %) Toluene and Ethanol were purchased from J. T. Baker Incorporated. Acetic acid with a purity of 99.99 % was Downloaded by UNIV OF CINCINNATI on May 20, 2016 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0608.ch029

obtained from Fischer Scientific Incorporated. The taxane standard kit con sisting of taxol, cephalomannine, 10-deacetylbaccatin III, baccatin III, 7-epi 10-deacetyltaxol, and 10-deacetyltaxol was obtained from the Drug Synthesis and Chemistry Branch, Development Therapeutics Program, Division of Can cer treatment, National Cancer Institute (Bethesda, MD). The Cis reverse phase column was purchased from Alltech Associates Inc. Supercritical ex tracts were filtered using nylon 66 membranes 13 mm in diameter and 0.45 micron pore size, purchased from Alltech Associates Inc. Dry bark of Taxus brevifolia from Connolly/Eller Cottage Grove, Oregon. The bark was ground before use and the same source of ground tree bark was used in all the exper iments. All substances were used without further purification. Apparatus.

An ISCO SFE 2-10 Extractor system was used to perform su

percritical extractions of taxol from the bark of Taxus brevifolia. The system employed two ISCO syringe pumps (Model 260 D) to pump liquid carbon diox ide and the co-solvent (ethanol) into the extractor. A Buchler micro-rotary evaporator from Fischer Scientific Inc. was used to evaporate the solvents from the supercritical extracts. The extracts were analyzed using an LDC Analyti cal HPLC system. The HPLC system was equipped with a constaMetric 4100 quaternary solvent delivery pump, a membrane degasser and a spectroMonitor 5000 photo-diode array detector. The HPLC system was also equipped with a 486DX IBM PC/AT computer containing the LCTalk 2-channel analog data acquisition system for automated analysis. A vortex mixer purchased from Thermolyne Corporation (model M16715) was used to mix the extracts beforefiltering.A wet test meter purchased from Precision Scientific Inc. (model 63111, D-3B) was used to measure the gas volumes of carbon dioxide. The system pressure and temperature were moni tored by a pressure gauge purchased from Heise (model 8400) and a Leeds and Northrup thermistor respectively. A standard platinum resistance thermome ter (SPRT) was used to calibrate the thermistor.


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Experimental Procedure.

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A schematic diagram of the apparatus used in

this work is shown in Figure 2. Liquid carbon dioxide from a cylinder equipped with an eductor tube was pumped using an ISCO syringe pump while a second ISCO syringe pump was used to pump ethanol when needed. The syringe pump used to pump carbon dioxide was equipped with a cooling jacket to ensure that carbon dioxide remained in the liquid phase while being pumped into the extractor. Absolute ethanol was stored in a reservoir connected to the other syringe pump. A common pump controller unit was used to set the Downloaded by UNIV OF CINCINNATI on May 20, 2016 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0608.ch029

ethanol concentration and the system pressure. The supercriticalfluidmixture then passed through static mixers into the extractor which was maintained at a constant temperature. A thermistor was used to record the temperature of the experiments. Extractions of taxol form the bark were performed using carbon dioxide as the supercritical fluid with and without ethanol as a co-solvent. During each experiment, carbon dioxide and ethanol were pumped through a coil and static mixers placed to ensure that the CO2 + ethanol mixtures were homoge neous and at bath temperature prior to contacting the ground Taxus brevifolia bark. Approximately 3.5 grams of ground bark was placed in a high pressure cell (cartridge) of 10 mL internal volume. Filters, 5 microns in diameter, were placed in the cartridge at the exit ports to prevent any bark from being carried out with the supercritical fluid. The loaded supercritical fluid was depressurized across a heated capillary tubing into a collection vessel placed in an ice bath. The depressurized fluid was then passed through a second collection ves sel (in experiments involving a co-solvent) placed in a dry ice/ethanol bath. The gaseous carbon dioxide was then passed through the wet test meter (Fis cher Scientific Inc., model 63111,D-3B) for flow totalization. Flowrates were kept at approximately 0.3-0.4 mL/min to allow sufficient residence time for the solvent to contact the bark. The soluble bark extract, as well as some of the ethanol (if present), were precipitated and collected in the first collection vessel. The collection vessel incorporated a coldfingerwith the exit arm of the trap packed with glass wool to prevent the precipitated material from being carried out of the trap. A Utube packed with glass wool served as an additional trap for any remaining ethanol, leaving practically pure carbon dioxide to pass through the wet test meter.




434

3

S-H

0>

a; OH


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The pressure was controlled by the pump controller unit and measured with a Heise pressure gauge (model 8400). The Heise gauge was calibrated against a Budenberg dead weight tester. The uncertainty in the measurement of system pressure was found to be ± 0.046 MPa. The temperature was monitored by a Leeds and Northrup thermistor in the extractor, and a thermocouple inserted inside the extractor was used to control the temperature of the extractor. The thermistor was calibrated against a standard platinum resistance thermometer (model number: R800-3 2.5 Ω, Serial Number: RS90Y-11) traceable to NIST. Downloaded by UNIV OF CINCINNATI on May 20, 2016 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0608.ch029

The uncertainty in the measurement of system temperature was estimated to be ± 0.5 K. The volume of carbon dioxide was determined using the wet test meter which was factory calibrated with a stated accuracy of ± 0.5 %. Blank experiments were performed to calibrate the solvent delivery system and the co-solvent composition was found to be accurate within db 0.5 mole %. The amount of ethanol was determined gravimetrically by weighing the collection vessels before and after the experiment. A Sartorius balance was used for the gravimetric analysis and had an accuracy of ± 0.0005 g. Since the total weight of bark extract was small, it was neglected in determining the weight of ethanol collected. The amount of dried bark extract was also determined gravimetrically as follows. At the end of the extraction run, the contents of the first collection vessel, extracted bark material, flush solvent, and co-solvent (if used), were transferred to a distillation flask. 5-10 mL of toluene were then used to flush the collection vessel and the liquid was also transferred to the flask. The flush solvent and co-solvent ethanol were then evaporated under reduced pressure in a rotary evaporator. The bark extract was dissolved in approximately 5 mL of toluene and transferred into a preweighed vial; and the toluene was again evaporated by rotary evaporator. A slow stream of nitrogen was passed over the bark extract to ensure that all the toluene was removed. The vial was then weighed to determine the total weight of bark extract. The amount of taxol present in the bark extract was determined by HPLC analysis of the methanol soluble portion of the extract. The HPLC samples were prepared and analyzed in the following manner. A known amount of methanol was first added to the dried bark extract. The solution was soni cated and mixed in a vortex mixer to ensure that the soluble components had dissolved. The solution was then filtered and the methanol soluble portion was analyzed by HPLC. The solution was separated on a 250 mm Cis Alltech


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Table I: Extraction of Taxol from Taxus brevifolia Using Supercritical Carbon Dioxide and Carbon Dioxide + Ethanol Mixtures


Τ

Ρ

Mass Taxol in Bark

(g) 3.4085 3.5968 3.5746 3.5773

(mg) 0.5454 0.5755 0.5719 0.5724

6.9619 7.1960 6.0154

(Κ)

(MPa)

319.7 318.5 318.9 319.3 av:319.1

13.67 20.67 27.59 34.39

320.4 320.2 320.0 319.9 319.9 319.8 av:320.0

10.29 13.75 20.63 27.37 34.18 38.20

3.4460 3.5923 3.6762

0.5514 0.5748 0.5882

3.4936 3.6516 3.7699

319.7 319.8 319.9 319.7 319.6 319.3 av:319.7

10.33 13.78 20.37 27.12 34.34 37.74

330.6 330.5 330.5 331.0 331.6 av:330.8 330.8 330.7 330.7 331.2 330.8 330.8 av:330.8

Moles

Mass Bark in Cell

C0

2

Mole % Ethanol

Mass Taxol in Extract

Mass Extract

(mg)

(g) 0.0100 0.0055 0.0021

0 0 0 0

0.0021 0.0025 0.0078 0.0021

0.5590 0.5843 0.6032

3.6253 3.3908 3.9933 3.4673 3.1254 3.3769

6.1 5.0 5.5 5.1 5.6 4.9 av:5.4

0.0665 0.0377 0.0721 0.0746 0.1410 0.1670

0.1122 0.0673 0.0705 0.0915 0.1408 0.1285

3.5876 3.4301 3.1699 3.6723 3.5411 3.4988

0.5740 0.5488 0.5072 0.5876 0.5666 0.5598

3.3465 4.9237 3.7093 3.6687 4.9789 3.5089

11.0 9.8 10.0 11.1 10.0 11.1 av:10.5

0.1210 0.0784 0.1210 0.2200 0.3020 0.2590

0.0586 0.4083 0.0726 0.1318 0.4865 0.1427

13.71 20.74 27.64 34.30 38.10

3.8346 3.7293 3.5530 3.9139 3.6255

0.6135 0.5967 0.5685 0.6262 0.5801

3.2005 4.4236 3.3603 3.7439 3.7015

4.6 5.1 4.5 4.9 4.7 av:4.9

0.0693 0.0509 0.0951 0.0858 0.1150

0.0822 0.1157 0.2279 0.1269 0.0965

10.65 14.07 20.80 27.62 34.28 37.95

3.8197 3.7333 3.7819 3.5230 3.7584 3.7523

0.6112 0.5973 0.6051 0.5637 0.6013 0.6004

3.2593 5.5343 3.8924 4.4458 3.3728 3.7390

10.4 11.0 9.8 11.1

0.1470 0.1300 0.1570 0.1620 0.2440 0.2470

0.2669 0.0506 0.0681 0.0629

6.3740

11.0 9.4 av:10.5

0.0289

0.0648 0.0584


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reverse phase column using a shallow gradient of mobile phase which varied from 58.5:41.5 methanol-water to 61.5:38.5 methanol-water for 25 minutes. The mobile phaseflowratewas maintained at ~ 1.45 ml/min. The absorp tion at 227 nm was monitored and the datafiles containing the chromatograms were stored on disk for future reference. The amount of taxol present in the bark was quantified by comparing the response of the peak area of taxol in the extract sample to the peak area of taxol from a standard solution of taxol in methanol. The injection volumes were 20 μ!< for each extract and standard Downloaded by UNIV OF CINCINNATI on May 20, 2016 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0608.ch029

solution.

Results and Discussion Table I shows the results of extractions using supercritical carbon dioxide and carbon dioxide + ethanol mixtures at 319 Κ and 331 K. All the experi ments were performed with ground bark of Taxus brevifolia, with an average particle size less than 0.495 mm in diameter. As expected, the amount of taxol extracted is small and increased with an increase in pressure. Addition of ethanol as a co-solvent led to an increase in the amount of taxol present in the extracts as shown in Table I. In our previous studies [18], it was observed that the concentration of taxol in the extract decreased with extraction time and became quite small after 3.5 - 4.0 moles of carbon dioxide had passed through the bed. Therefore, 3.2 - 5.5 moles of carbon dioxide -f ethanol mix tures were used in the extractions carried out in this study. Figure 3 shows a plot of moles of taxol extracted by 4 moles of carbon dioxide as a function of pressure at 318 K. It can be seen that the amount of taxol extracted increases with an increase in pressure and with the the addition of co-solvent. The selectivity and extractability of taxol improved with the addition of ethanol. A plot of solubility enhancement (E) = y ternary/Y

binary

is shown in Figure 4

which shows solubility enhancements of 5-20 fold. The supercritical extracts obtained using carbon dioxide contained small amounts of taxol and were spiked with the taxol standard to quantify the total amount of taxol extracted. However, with the addition of ethanol, samples could be directly injected on to the HPLC columns. A comparison of the amount of taxol extracted shows that almost 50 % of taxol present in the bark was extracted using approximately 4 moles of supercritical carbon dioxide at the highest co-solvent composition of ~ 10.5 mole % ethanol, whereas only 9 % was extracted without the use of a co-solvent. The composition of taxol in the supercritical extracts using carbon dioxide -f ethanol mixtures varied from 0.05 to 0.5 mass %, an increase of approximately 5-50 fold from that


438


10

••

ο

ι ι ι ι ι ι ι ιι

5.4 % Ethanol 10.5% Ethanol

• •

• "ο

•


u Q. Ο

•

Η

β

I IIIIII I

I I II I I II

ο 10

40 30 Ρ (MPa) Figure 3. Moles of Taxol Extracted per 4 Moles of Carbon Dioxide as a Function of Pressure (the results are normalized to show the trends). 20

20

1

ι

1

Ο 5.4 % Ethanol •

15

10.5 % Ethanol

10

Ο Ο ι I ι ι ι ι I ι ι ι ι

0 10

15

20

25

30

35

Ρ (MPa) Figure 4. Solubility Enhancement.


29. VANDANA & TEJÀ

Supercritical Extraction

ofPaclitaxel

439

seen in the bark. All the calculations to determine the taxol content in the extracts were based on total taxol content of 0.016 % in the bark obtained by an exhaustive liquid ethanol extraction of the bark [19]. The HPLC analysis of the supercritical extracts involved a time-optimized method incorporating a gradient elution of the mobile phase. Figure 5 shows the results obtained when a standard solution of pure taxol was injected on the Cis column; taxol is shown to elute at about 19.0 minutes. Figure 6


shows the results when a standard solution containing taxol and related taxanes was injected on the column using the same mobile phase; there is clear baseline separation of all the taxanes. Two of the more polar taxanes, 10deacetylbaccatin III and baccatin III elute at the beginning of the run. These two compounds are found in the leaves of the Taxus species and have been used as starting materials to partially synthesize taxol. This represents an excel lent solution to the supply problem as the leaves are renewable and taxol can be obtained without depleting the yew population. Cephalomannine, which elutes at 17.27 minutes is very similar in structure to taxol but does not have any tumor-inhibitory activity. Since it elutes very closely to taxol, it gener ally creates a problem in the separation of taxol. With the method chosen in this study, we have been able to eliminate the problem as can be seen clearly in the chromatogram. The other two compounds, 10-deacetyltaxol and 7-epi 10-deacetyltaxol, are decomposition products which need to be identified to ensure that taxol has not decomposed during the extraction process. Figure 7 shows a typical chromatogram of the supercritical extracts. A lot of polar material elutes at the beginning of the run, but a clear baseline separation of taxol and related taxanes is observed. Addition of ethanol as a co-solvent alters the components present in the supercritical extracts. When supercritical extractions were performed using carbon dioxide, the extracts consisted of predominantly non-polar components. With the addition of ethanol, the extracts included both non-polar and some polar components. Liquid ethanol extracts of the plant material consisted predominantly of polar material. This can be seen clearly in the HPLC analysis of the extracts using a mobile phase starting with 50:50 methanol-water for the first five minutes and a gradient to 100 % methanol in the next five minutes, followed by 100 % methanol in the last 10 minutes. Figure 8 illustrates the composition of the supercritical extracts (with and without a co-solvent) and the liquid ethanol extracts on a Cis reverse phase column. The more polar


440


4>

u Ο


en •fi

Τ "

5

10

τ* 15

20

25

Time (minutes) Figure 5. Chromatogram of a Standard of Pure taxol on a C i Reverse 8

Phase Column.

Π

I »' »»ι 0 5

1 1 1 1

10

ι

1 1 1 1

15

ι

1 1 1 1

20

I 25

Time (minutes) Figure 6. Chromatogram of a Mixture of 10-deacetylbaccatin III (I), baccatin III (II), 10-deacetyltaxol (III), cephalomannine (IV), taxol (V), and 7-epi-10-deacetyltaxol (VI) on a Cis Reverse Phase Column.



29.

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VANDANA & TEJA

1 • ' ' '

0

I . . , » ! » .

5

» • I . . .

10 15 20 Time (minutes)

» I

25

Figure 7. Chromatogram of a Supercritical Extract on a Cis Reverse Phase Column. Taxol Peak is Labelled as (V).

(a)

\ Taxol

Taxol

^

_

L

0.0

20.0

Time (minutes)

Figure 8. Comparison of the Composition of the Extracts Depicted in the Chromatograms on a Cis Reverse phase Column a: Supercritical Extract Using C 0 Without Co-solvent, b: Supercritical Extract Using C 0 With 2

2

Ethanol as a Co-solvent, c: Liquid Ethanol Extract.



442


material elutes at the beginning of the run, followed by the less polar material when 100 % methanol is used as the mobile phase towards the end of the run. Taxol elutes at 12.8 minutes in this case. Conclusions Supercritical fluid carbon dioxide and carbon dioxide -f ethanol mixtures were used to extract taxol from the bark of Taxus brevifolia. The carbon dioxide 4- ethanol mixtures proved to be better solvents in extracting the drug from the plant material than carbon dioxide alone. Addition of ethanol led to an increase in selectivity and extractability of taxol. This study shows that supercritical fluid extraction can be used as a valuable step in the extraction of taxol. Acknowledgments The authors would like to thank Dr. Gordon Cragg and the National Cancer Institute for supplying the bark of Taxus brevifolia; Dr. K. Snader and the Drug Synthesis and Chemistry Branch, Development Therapeutics Program, Division of Cancer treatment, National Cancer Institute (Bethesda, MD) for supplying the taxanes standards kit; and Dr. H. M. Deutsch for his help and suggestions in the HPLC method development. Literature Cited [1] Wani, M.C.;Taylor, H. L.; Wall, M. E.; Coggon, P.; and McPhail, A. T. J. Am. Chem.Soc.,1971, 93, 2325. [2] McLaughlin, J. L.; Miller, R. W.; Powell, R. G.; and Smith, Jr., C. R. J. Nat. Prod., 1981 44, 312. [3] Miller, R. W.; Powell, R. G.; and Smith, C. R. J. Org. Chem., 1981, 46, 1469. [4] Stasko, M. W.; Witherup, K. M.; Ghiorzi, T. J.; McCloud, T. G.; Look, S. Α.; Muschik, G. M.; and Issaq, H. J. J. Liq. Chrom., 1989, 12, 2133. [5] Wender, P. Α.; and Muccario, T. P. J. Am. Chem.Soc.,1992, 114,5878. [6] Deng, L. and Jacobsen, Ε. N. J. Org. Chem., 1992, 57, 4320. [7] Queneau, Y.; Krol, W. J.; Bornmann, W. G.; and S. J. Danishefsky, S. J. J. Org. Chem., 1992, 57, 4043. [8] Magee, T. V.; Bornmann, W. G.; Richard, C. Α.; and Danishefsky, S. J. J. Org. Chem., 1992, 57, 3274. [9] Benchikh Le-Hocine, M.; Do Khac Duc; Fetizon, M.; Guir, F.; Guo, Y.; and Prange, T. Tetrahedron Lett., 1992, 33, 1443.


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[10] Denis, J. N.; Correa, Α.; and Greene, A. E. J. Org. Chem., 1991, 56, 6939. [11] Holton, R. Α.; Somoza, C.; Kim, H-B.; Liang, F.; Biediger, R. J.; Boat man, P. D.; Shindo, M.; Smith, C. C.; Kim, S.; Nadizadeh, H.; Suzuki, Y.; Tao, C.; Vu, P.; Tang, S.; Zhang, P.; Murthi, Κ. K.; Gentile, L. N.; and Liu, J. H . J. Am. Chem.Soc.,1994, 116, 1597. [12] Holton, R. Α.; Kim, H-B.; Somoza, C.; Liang, F.; Biediger, R. J.; Boat man, P. D.; Shindo, M.; Smith, C. C.; Kim, S.; Nadizadeh, H.; Suzuki, Downloaded by UNIV OF CINCINNATI on May 20, 2016 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0608.ch029

Y.; Tao, C.; Vu, P.; Tang, S.; Zhang, P.; Murthi, Κ. K.; Gentile, L. N.; and Liu, J. H. J. Am. Chem.Soc.,1994, 116, 1599. [13] Nicolaou,K. C.; Yang, Z.; Liu, J. J.; Ueno, H.; Nantermet, P. G.; Guy, R. K.; Clairborne, C. F.; Renaud, J.; Couladouros, Ε. Α.; Paulvannan, K.; and Sorensen, E. J. Nature, 1994, 367, 630. [14] Nicolaou,K. C; Clairborne, C. F.; Nantermet, P. G.; Couladouros, Ε. Α.; and Sorensen, E. J. J. Am. Chem.Soc.,1994, 116, 1591. [15] Denis, J. N.; Greene, A. E.; Guenard, D.; Gueritte-Voegelein, F.; Mangatlal, L.; and Potier, P. J. Am. Chem.Soc.,1988, 110, 5917. [16] Christen, Α. Α.; Bland, J.; and Gibson, D. M. Proc. Am. Assoc. Cancer Res., 1989, 30, 2252. [17] Towers, G. Η. N. Workshop on Taxol and Taxus: Current and Future Perspectives, 1990, June. [18] Vandana, V.; and Teja, A. S. Submitted to Biotech. & Bioeng., 1994. [19] Jennings, D. W.; Deutsch, H. M.; Zalkow, L. H.; and Teja, A. S. J. Supercrit. Fluids., 1992, 5, 1. RECEIVED July 2 4 , 1 9 9 5


Supercritical Extraction of Paclitaxel Using CO2 and CO

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