- Email: [email protected]
Supercritical extraction and HPLC analysis of taxol from Taxus brevifolia using nitrous oxide and nitrous oxide + ethanol mixtures
RUIBPHIB[ EQUIUBRIA ELSEVIER
Fluid Phase Equilibria 116 (1996) 162-169
Supercritical Extraction and H P L C Analysis of Taxol from Taxus brevifolia Using Nitrous Oxide and N i t r o u s Oxide -b Ethanol M i x t u r e s V i s h n u V a n d a n a 1, A m y n S. T e j a a n d L e o n H. Z a l k o w S c h o o l of C h e m i c a l E n g i n e e r i n g ~z School o f C h e m i s t r y a n d B i o c h e m i s t r y G e o r g i a I n s t i t u t e o f T e c h n o l o g y , A t l a n t a , G A 30332-0100
Abstract Taxol is an alkaloid which has been found to be exceptionally promising in the treatment of ovarian cancer. The major current sources of the drug are yew species which are in limited supply. As a result, there is a need for improved separation processes to effectively remove the drug from its natural sources. h this study, the separation of taxol from the bark of Ta~:us brevi/olia has been achieved at 320 K and 331 K and at a pressure range of 10.3 to 38.10 MPa using supercritical nitrous oxide and nitrous oxide + ethanol mixtures. The extracts were quantified by high pressure liquid chromatography with photo diode array detection in the UV region. It was found that supercritical nitrous oxide + ethanol mixtures were able to extract most of the taxol present in the bark and that the extractions were more efficient than those using CO2 + ethanol mixtures.
1
Introduction
Ta.xol is a diterpenoid and was the first compound possessing a taxane ring to exhibit anti-cancer and antileukemic properties. It has been called "the most promising anti-cancer compound to have come along in 15 years" because of its success in the experimental treatment of several forms of cancer-including ovarian, breast and lung cancers. It is currently undergoing phase III clinical trials for the treatment of ovarian cancer and is expected to be approved for clinical use in the near future. The molecule has a highly folded conformation and 11 chiral centers as shown in Figure 1. It was first isolated from the bark of the Pacific yew tree Tazus brevifolia in 1967 and its structure was characterized by Wani et al. in 1971. Since then, isolation of the drug from the bark of other Tazus species has been reported by a number of researchers (McLaughlin et al., 1981; Miller et al., 1981; and Stasko et al., 1989). Synthesis of the drug is obviously of great interest since the natural supply of taxol is limited. Two research groups have been able to achieve total synthesis from simple starting materials (Holton et al., 1994a; Holton et al., 1994b; Nicolaou et al., 1994a; Nicolaou et a l , 1994b). However, the yields obtained in these syntheses are small and it is unlikely that total synthesis will provide the required quantities of taxol in the near future. Semi-synthesis can be expected to provide a more practical solution to the supply problem, but will require more efficient isolation and extraction methods for providing the necessary precursors. Two precursors, baceatin III and 1O-deacetylbaccatin III, are found in the needles of several Tazus species and partial synthesis of taxol from these compounds has been accomplished by Denis et al., 1981. Since the amount of taxol present in the natural sources (yew bark and needles) is small (~ 0.01 wt%), it is important to develop an effective separation process for the extraction and isolation of most of the compound present in the natural product. In this study, a supercritical fluid extraction based separation t Present Address: Advanced SemiconductorTechnologyCenter, IBM Corporation, Route 52, Hopewell Junction, New York 12533.
0378-3812/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI S0378 - 3812(96)02991-3
V. Vandana et al. / Fluid Phase Equilibria 116 (1996) 162-169
163
O
U
CHf'- C - - O
o
....
o
O' I
Ou~C
Figure h Structure of Taxol
o
I
Cffi,ffiO 0t3
process has been developed for this purpose. In our earlier work, we have shown t h a t carbon dioxide and carbon dioxide % ethanol mixtures can be used for the extraction of taxol (Vandana and Teja, 1995a). In this work, another supercritical solvent, nitrous oxide has been used as a solvent with ethanol as a co-solvent. Nitrous oxide was chosen for two reasons. First, it has a moderate critical temperature (To = 35.5 C) so that extractions can generally be carried out at temperatures which minimize thermal degradation of taxol. Second, the polarity of nitrous oxide and the solvent used in our previous studies, carbon dioxide, is very different (CO2 is an apolar solvent, whereas N 2 0 is a polar solvent). Large differences in solubilizing different types of molecules were therefore expected. Also, extractions using nitrous oxide + ethanol mixtures were expected to be more selective towards the mildly polar taxol than extractions using a "powerful" (polar) solvent such as ethanol, which is currently employed in the liquid extraction of taxol.
2 2.1
Experimental Reagents
and Materials
Nitrous oxide (SCF grade) with a m i n i m u m purity 99.995 mole %, acetic acid with a purity of 99.99 mole %, HPLC grade methanol and water and reagent grade toluene and ethanol (99.99 mole %) were used in the experiments. All taxanes (taxol, cephalomannine, 10-deacetylbaccatin III, baccatin III, 7-epi 10-deacetyltaxol and 10-deacetyltaxol) were obtained from the Drug Synthesis and Chemistry Branch, Development Therapeutics Program, Division of Cancer treatment, National Cancer Institute (Bethesda, MD). The Cla reverse phase column was purchased from Alltech Associates Inc. Dry bark of Tazus brevifolia was obtained from Connolly/Eller Cottage Grove, Oregon. The same source of ground tree hark was used in all the experiments. The average particle size of the bark was less than 0.495 m m in diameter. All substances were used without further purification. 2.2
Experimental
Procedure
An a p p a r a t u s consisting of an Isco SFE 2-10 Extractor system was used to perform supercritical extractions of taxol from the bark of Tazus brevifolia. Details of the apparatus are available elsewhere (Vandana a n d Teja, 1995a). Extractions were performed using nitrous oxide as the supercritical fluid with and without ethanol as a co-solvent. During each experiment, two ISCO syringe pumps (model 260 D) were used to p u m p
164
V. Vandana et al. / Fluid Phase Equilibria 116 (1996) 162-169
nitrous oxide and ethanol through static mixers to ensure t h a t the mixtures were homogeneous and at the set b a t h temperature prior to contacting the ground plant material. A known amount (approximately 3.5 g) of ground bark was placed in a high pressure cell (cartridge) of 10 mL internal volume. Filters, of 5 # m diameter, were placed in the cartridge at both the ports to prevent any solid bark from being carried out with the supercritical fluid. The loaded supercritical fluid was depressurized across a heated capillary tube into a collection vessel placed in an ice bath. The depressurized fluid was then passed through a second collection vessel (when co-solvent was present), placed in a dry ice/ethanol bath. The gas volumes of nitrous oxide were measured with a Precision Scientific wet test meter (model 63111, D-3B) which was factory calibrated and certified to be accurate within 4- 0.5 %. Flow rates were kept at approximately 0.3-0.4 m L / m i n to allow sufficient residence time for the solvent to contact the bark. A Heise pressure gauge (model 8400) was used to monitor the system pressure with an uncertainty of 4- 0.046 MPa. A Leeds and Northrup thermistor was used to monitor the system temperature. The thermistor was calibrated using a standard p l a t i n u m resistance thermometer (model Number: R800-3 2.5 ~, Serial Number: Rsg0Y-11)) manufactured by Chino Works America Inc., and traceable to NIST. The uncertainty in the measurement of system temperatures was estimated to be 4- 0.5 K. Blank experiments were performed to calibrate the solvent delivery system for accuracy of the co-solvent composition. The co-solvent composition was found to be accurate within 40.5 mole%. The amount of ethanol and solute (supercritical extract) were determined gravimetrically by weighing the collection vessels before and after the experiment was complete. A Sartorius balance was used for the mass measurements. These measurements were reproducible within 4-0.0001 g and their accuracy was estimated to be within 4-0.0005 g. The extracts were analyzed for taxol content using high pressure liquid chromatography. The amount of taxol present in the bark extract was determined by HPLC analysis of the m e t h a n o l soluble portion of the extract. Dried bark extracts were dissolved in 2 mL of methanol, sonicated and mixed using a vortex mixer, and then filtered using nylon 66 membranes of 0.45 # m pore si~.e. The methanol soluble portion was analyzed using an LDC Analytical HPLC purchased from Thermoseparation Products Inc., equipped with a photo diode array detector. The solution was separated on a 250 m m (]is Alltech reverse phase column using a shallow gradient mobile phase for the analysis. The mobile phase varied from 58.5:41.5 methanol-water to 61.5:1.5 methanol-water for 25 minutes and its flow rate was maintained at 1.45 m l / m i n (Vandana et al., 1994). Taxol and related taxanes (baccatin III, 10-deacetylbaccatin III, cephalomannine, 7-epi 10-deacetyltaxol and 10-deacetyltaxol) absorb light in the ultraviolet (UV) region and exhibit a )~m~® at 227 nm. The photo diode array detector was therefore monitored in the range of 190 - 300 nm. The fingerprint of the absorb£nce of the compounds in the isogram was used to analyze the chromatograms. The absorption at 227 nm was used for quantitation (taxol has a 2 , ~ at this wavelength} and the datafiles containing the chromatograms were stored on disk for analysis. The amount of taxol present in the supercritical extracts was determined by comparing the response of the peak areas of taxol in a standard solution to that in the extracts. The injection volumes were 20 #L for each extract and s t a n d a r d solution. The average deviation in the measurement of peak areas as a function of concentration using 95 % confidence intervals was found to be within 5%.
3
R e s u l t s and D i s c u s s i o n
The results of nitrous oxide and nitrous oxide + ethanol extractions are tabulated in Tables 1,2 respectively. In our previous experiments with carbon dioxide and carbon dioxide + ethanol mixtures, it was observed t h a t the concentration of taxol in the extract decreased with time after attaining a m a x i m u m after approximately 3.0 moles of solvent had passed through the bed (Vandana and Teja, 1995a). Furthermore, the concentration of taxol in the extracts became almost zero after 3.5-4.0 moles of fluid had passed through
V. Vandana et a l . / Fluid Phase Equilibria 116 (1996) 162-169
•
o
o
•
•
.
.
.
.
!
.
.
.
.
.
.
.
.
.
!
=
.
•
•
.
.
.
.
165
.
[] E
"0
[]
[]
[]
[]
0.1
¢,J
M mm
0.01
o x
•
0.001
[ •
0 % Ethanol, 321.4 K
[
I []
11.4 % Ethanol, 319.5 K
I
. . . . . . . . . . . . . . . . . . .
10
.........
'
20
30
40
Pressure (MPa) Figure 2: Mass of taxol extracted per four moles of nitrous oxide as a function of pressure (the results are normalized to show the trends)
. . . . . . . . .
'.
. " .
.....
100
[]
•
[]
[]
[]
[]
10 o e~
•
10
•
|
•
•
.
•
•
[]
CO z - 10.5 % Ethanol
•
N20 - 11.5 % Ethanol
•
I
•
•
.
I
I
•
•
•
•
I
20 30 Pressure (MPa)
.
•
.
.
.
•
,
.
i
40
Figure 3: A comparison of the amounts of taxol extracted by four moles of supercritical nitrous oxide and carbon dioxide at 331 K
166
V. Vandana et al. / Fluid Phase Equilibria 116 (1996) 162-169
19.00
(a)
1 .B8 i
15.7a7.2 l |
|
Zl .~3
|
4.48
(b)
3.1
_.4
19.05
].7.27 A XS.??A /
,NV
A
25.38 27.40 m
29.02 21.05 a2.33
1"
0,00
TJ.~
~5.00
Figure 4: Chromatograms on a CzsReverse Phase Column (a): Standard solution of taxol, retention time = 19.0 rain. (b): Standard solution of a mixture of 10-deacetylbaccatin III (03.8 rain.), baccatin III (04.68 min.), 10-
deacetyltaxol (015.77 min.), cephalomannine (017.27 min.), taxol (019.05 rain.), 7-epi lO-deacetyltaxol (@21.50 min.) (c): Supercritical extract sample: taxol elutes at 19.02 rnin.
167
V. Vandana et al. / Fluid Phase Equilibria 116 (1996) 162-169
Table 1: Extraction of Taxol from Tazns brevifolia Using Supercritical Nitrous Oxide T
P
Mass of Bark
Mass
Moles
Mass
Mass
in the cell
N~O
Taxol in Extract (mg)
Extract
(K)
(MPa)
(g)
Taxol in Bark (mg)
322.1 320.8 322.1 320.6 av: 321.4
13.99 21.08 27.58 34.48
3.4226 3.4646 3.4809 3.4836
0.5476 0.5543 0.5569 0.5574
4.2741 4.1180 3.7528 3.5248
0.0103 0.0479 0.0558 0.0459
0.0395 0.0444 0.0432 0.0841
331.9 331.3 331.7 331.9 av: 331.7
13.75 20.75 27.60 34.76
3.4580 3.6097 3.3946 3.6102
0.5533 0.5776 0.5431 0.5776
6.3876 7.3831 4.0459 3.3745
0.0079 0.0179 0.0190 0.0875
0.0485 0.0595 0.0514 0.0513
(g)
the bed. Therefore, 3.4-7.4 moles of supercritical nitrous oxide and 2.5-4.5 moles nitrous oxide + ethanol mixtures were used in the extractions carried out in this study. Figure 2 shows a plot of the amount (rag) of taxol extracted per approximately 4.0 moles of nitrous oxide passed through the bed as a function of pressure at 320 K. The results were normalized for qualitative comparison of the effect of pressure and cosolvent composition on the amount of taxol extracted. It can be seen t h a t the amount of taxol extracted increases with increase in pressure and also with the the addition of co-solvent. The nitrous oxide + ethanol mixtures proved to be better solvents than nitrous oxide alone. Thus, the selectivity and extractibility of taxol improved with the addition of ethanol. Also, most of the taxol present in the bark was extracted using supercritical nitrous oxide at the highest co-solvent composition of ~ I0 mole % whereas only 15 % was extracted without the use of a co-solvent. All the calculations to determine the taxol content in the extractions were based on total taxol content of 0.016 % in the bark determined by performing an exhaustive hquid ethanol extraction and subsequent HPLC analysis of the extract (Jennings et a l , 1992). A comparison of extraction of taxol using carbon dioxide + ethanol mixtures and nitrous oxide + ethanol mixtures at 331 K is shown in Figure 3 (the tabulated extraction data using carbon dioxide and carbon dioxide ÷ ethanol mixtures are presented in Vandana and Teja, 1995b). Nitrous oxide proved to he a better solvent t h a n carbon dioxide. Also, most of the taxol present in the bark was removed using only 3.0 moles of nitrous oxide + ethanol mixtures at the higher pressures. The HPLC analysis of the supercritical extracts involved a time-optimized method incorporating a gradient elution of the mobile phase. Figure 4a shows the results obtained when a standard solution of pure taxol was injected on the Cls column; taxol is seen to elute at about 19.0 minutes. Figure 4b 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 taxanes, 10deacetylbaccatin III and baccatin III, which are more polar than the other taxanes sluts at the beginning of the run. Cephalomannine, which elutes at ~17.0 minutes, is very similar in structure to ta~ol but does not have any tumor-inhibitory activity. In the method chosen in this study, a shallow gradient ensures clean separation of this compound from taxol as can he seen in the chromatogram. The other two compounds, 10-deacetyltaxol and 7-epi 10-deacetyltaxol, are decomposition products which need to be identified to ensure t h a t taxol has not decomposed during the extraction process. Figure 4c shows a typical chromatogram
V. Vandana et al. / FluM Phase Equilibria 116 (1996) 162-169
168
T a b l e 2: E x t r a c t i o n of Taxol from Tazus brevifolia Using Supercritical N i t r o u s O x i d e + E t h a n o l M i x t u r e s Mass Taxol in Extract
MRss Extract
(ms)
(g)
5.3 6.I 8.8 5.1 5.3 5.I av:5.5
0.0870 0.1257 0.0972 0.0982 0,0956 0.1149
0,0847 0.0981 0.0892 0.0840 0.0658 0.0749
3.2075 4.3010 4.2059 4.0414 2.4543 3.7414
11,0 10.8 10.8 11.3 13.3 11.1 av:11.4
0.1022 0.2155 0.2489 0.3215 0.5697 0.3925
0.0873 0.1764 0.4166 0.3201 0.1751 0.1310
0.5866 0.5793 0.5673 0.5357 0.5642 0.5576
3,3740 3.0687 2.9216 3.3294 3.0023 3.4432
5.8 5.8 5.3 5.8 5.1 6.3 av:5.7
0.0531 0.2379 0.2166 0.3588 0.4866 0.4870
0.0487 0.0594 0.0671 0.0709 0.0806 0.0881
0.5554 0.5482 0.5470
3.8500 2.6953 2.6339
0.5558
2.9302
II.0 13.3 10.9 10.5 av:ll.5
0.1575 0.4470 0.4850 0.5543
0.3877 0.2638 0.2821 0.1155
P
Mass of Bark in the ceU
Mass Taxol in Bark
Moles N20
(K)
(MPa)
(g)
(rag)
320.6 320.3 320.5 320.2 320.3 320.6
10.45 13.92 20.66 27.51 34.37 37.83
3.5651 3.6149 3.4436 3.5632 3.4157 3.5594
0.5704 0.5784 0.5510 0.5701 0.5465 0,5595
2.8350 3.5963 2.5 817 3.6843 2.5965 4.0145
10.54 13.96 20.79 27.62 34.55 38.10
3.0730 3.7120 3.6578 3.5346 3.6274
0.5877 0.5939 0.5853 0.5655 0.5804 0.5942
av:320.4 319.0 319.6 319.0 319.6 319.5
319.3 av:319.5
3.7140
330,6
10.45
3.6660
330.5
13.93
3.6207
330.6 330.5 331.0 331.5 av:330.8
20.73 27.55 34.44 37.91
3.5458 3.3480 3.5250 3.4849
330.6 330,6 331.2 330.8 av:330.8
18.87 20.67 27.51 34.34
3.4710 3.4261 3.4185 3.4739
Mole % Ethanol in SCF
of t h e supercritical extracts. A lot of polar m a t e r i a l elutes at t h e b e g i n n i n g of t h e run, b u t a clear baseline s e p a r a t i o n of t a x o l a n d related t a x a n e s is observed. T h e less polar material, which s t a y s in t h e c o l u m n , is u s u a l l y flushed after t h e r u n with 100 % m e t h a n o l . T h e H P L C m e t h o d developed is simple a n d a c c u r a t e a n d w a s used to a s s a y taxol from t h e rest of t h e p l a n t m a t e r i a l extracted.
4
Conclusions
Supercritical n i t r o u s oxide and nitrous oxide + e t h a n o l m i x t u r e s were used to e x t r a c t t a x o l f r o m t h e b a r k of
Tazu8 brerifolia. T h e m i x t u r e s proved to be better solvents in e x t r a c t i n g t h e d r u g f r o m t h e p l a n t m a t e r i a l . A d d i t i o n of e t h a n o l increases the selectivity a n d extractibility of taxol. T h i s s t u d y s h o w s t h a t supercritical fluid e x t r a c t i o n c a n be used as a valuable a n d b e n i g n s e p a r a t i o n process to e x t r a c t t~xol w i t h o u t t h e u s e o f h a l o g e n a t e d solvents.
V. Vandana et al. /Fluid Phase Equilibria 116 (1996) 162-169
5
169
Acknowledgements
The authors would like to thank Dr. Gordon Cragg and the National Cancer Institute for supplying the bark of Tazus 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.
6
References
Wani, M. C., Taylor, H. L., Wall, M. E., Coggon, P., and McPhail, &. T., 1971. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Ta=us brevi/o]ia. J. Am. Chem. S0c., 93:2325-2327. McLaughlin, J. L., Miller, R. W., Powell, R. G., and Smith, C. R. Jr., 1981. 19-hydroxy baccatin III, 10-deacetyl cephalomannine and 10 - deacetyltaxol: New antitumor taxanes from Tazus waUichiana. J. Nat. Prod., 44:312-319. Miller~ R. W., Powell, R. G., and Smith, C. R. Jr., 1981. Antileukemic alkaloids from Taxus wallichiana Zucc. J. Org. Chem., 46:1469-1474. Stasko, M. W., Witherup, K. M., Ghiorzi, T. J., McCloud, T. G., Look, S. A., Muschik, G. M., and Issaq, H. J., 1989. Multimodal thin layer chromatographic separation of taxol and related compounds from Ta=us brsvifolia. J. Liq. Chrom., 12:2133-2143. Holton, R. A., Somoza, C., Kim, H-B., Liang, F., Biediger, R. J., Boatman, P. D., Shindo, M., Smith, C. C., Kim, S., Na~:lizadeh, H., Suzuki, Y., Tao, C., Vu, P., Tang, S., Zhang, P., Murthi, K. K., Gentile, L. N., and Liu, J. H., 1994a. First total synthesis of taxol 1. Functionalization of the B ring. J. Am. Chem. Sos., 116:1597-1598. Holton, R. A., Kim, H-B., Somoza, C., Liang, F., Biediger, R. J., Boatman, P. D., Shindo, M., Smith, C. C., Kim, S., Na~lizadeh, H., Suzuki, Y., Ta~, C., Vu, P., Tang, S., Zhang, P., Murthi, K. K., Gentile, L. N., and Liu, J. H., 1994b. First total synthesis of taxol 2. Completion of the C and D rings. J. Am. Chem. Soc., 116:1599-1600. Nicola~u, K. C , Yang, Z., Liu, J. J., Ueno, H.j Nantermet, P. G , Guy, R. K., Clairborne, C. F., Renaud I J., Couladourosl E. A., Paulvannan, K., and Sorensen, E. J., 1994a. Total synthesis of taxol. Nature, 367:630-635. Nicolaou, K. C., Clalborne, C. F., Nantermet, P. G., Couladouros, E. A , and Sorensenl E. J., 1994b. Synthesis of novel ta~xoids. J. Am. Chem. S0c., 116:1591-1593. Denis, J. N., Greene, A. E., Guenard, D., Gueritte-Voegelein, F., Mangatlal, L., and Potier, P., 1988. A highly efficient, practical approach to natural taxol. J. Am. Chem. Soc., 110:5917-5919. Vandana, V., and Teja, A. S., 1995a. A novel separation process for isolating taxol, an anti-cancer drug, from Tazus bre~i/olia. Submitted to Bioteeh. ~ Bioengg. Vandana, V., Teja, A. S., Deutsch, H. M, and Zalkow, L. H., 1994. A simple HPLC method for the separation of taxol and related taxanes in supercritical extracts of Tazus brevifolia. Submitted So J. Chrom. Jennings, D. W., Deutzch, H. M., Zalkow, L. H. and Teja, A. S., 1992. Supercritical extraction of taxol from the bark of Tazus bre~i]olia. J. Supercrit. Fluids, 5:1-6. Vandanal V., and Teja, A. S., 1995b. Supercritical extraction of taxol using CO2 and CO2 + ethanol mixtures. Accepted for Publication, Supercritical Science and Technology, ACS Syrup. Set.
Fluid Phase Equilibria 116 (1996) 162-169
Supercritical Extraction and H P L C Analysis of Taxol from Taxus brevifolia Using Nitrous Oxide and N i t r o u s Oxide -b Ethanol M i x t u r e s V i s h n u V a n d a n a 1, A m y n S. T e j a a n d L e o n H. Z a l k o w S c h o o l of C h e m i c a l E n g i n e e r i n g ~z School o f C h e m i s t r y a n d B i o c h e m i s t r y G e o r g i a I n s t i t u t e o f T e c h n o l o g y , A t l a n t a , G A 30332-0100
Abstract Taxol is an alkaloid which has been found to be exceptionally promising in the treatment of ovarian cancer. The major current sources of the drug are yew species which are in limited supply. As a result, there is a need for improved separation processes to effectively remove the drug from its natural sources. h this study, the separation of taxol from the bark of Ta~:us brevi/olia has been achieved at 320 K and 331 K and at a pressure range of 10.3 to 38.10 MPa using supercritical nitrous oxide and nitrous oxide + ethanol mixtures. The extracts were quantified by high pressure liquid chromatography with photo diode array detection in the UV region. It was found that supercritical nitrous oxide + ethanol mixtures were able to extract most of the taxol present in the bark and that the extractions were more efficient than those using CO2 + ethanol mixtures.
1
Introduction
Ta.xol is a diterpenoid and was the first compound possessing a taxane ring to exhibit anti-cancer and antileukemic properties. It has been called "the most promising anti-cancer compound to have come along in 15 years" because of its success in the experimental treatment of several forms of cancer-including ovarian, breast and lung cancers. It is currently undergoing phase III clinical trials for the treatment of ovarian cancer and is expected to be approved for clinical use in the near future. The molecule has a highly folded conformation and 11 chiral centers as shown in Figure 1. It was first isolated from the bark of the Pacific yew tree Tazus brevifolia in 1967 and its structure was characterized by Wani et al. in 1971. Since then, isolation of the drug from the bark of other Tazus species has been reported by a number of researchers (McLaughlin et al., 1981; Miller et al., 1981; and Stasko et al., 1989). Synthesis of the drug is obviously of great interest since the natural supply of taxol is limited. Two research groups have been able to achieve total synthesis from simple starting materials (Holton et al., 1994a; Holton et al., 1994b; Nicolaou et al., 1994a; Nicolaou et a l , 1994b). However, the yields obtained in these syntheses are small and it is unlikely that total synthesis will provide the required quantities of taxol in the near future. Semi-synthesis can be expected to provide a more practical solution to the supply problem, but will require more efficient isolation and extraction methods for providing the necessary precursors. Two precursors, baceatin III and 1O-deacetylbaccatin III, are found in the needles of several Tazus species and partial synthesis of taxol from these compounds has been accomplished by Denis et al., 1981. Since the amount of taxol present in the natural sources (yew bark and needles) is small (~ 0.01 wt%), it is important to develop an effective separation process for the extraction and isolation of most of the compound present in the natural product. In this study, a supercritical fluid extraction based separation t Present Address: Advanced SemiconductorTechnologyCenter, IBM Corporation, Route 52, Hopewell Junction, New York 12533.
0378-3812/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI S0378 - 3812(96)02991-3
V. Vandana et al. / Fluid Phase Equilibria 116 (1996) 162-169
163
O
U
CHf'- C - - O
o
....
o
O' I
Ou~C
Figure h Structure of Taxol
o
I
Cffi,ffiO 0t3
process has been developed for this purpose. In our earlier work, we have shown t h a t carbon dioxide and carbon dioxide % ethanol mixtures can be used for the extraction of taxol (Vandana and Teja, 1995a). In this work, another supercritical solvent, nitrous oxide has been used as a solvent with ethanol as a co-solvent. Nitrous oxide was chosen for two reasons. First, it has a moderate critical temperature (To = 35.5 C) so that extractions can generally be carried out at temperatures which minimize thermal degradation of taxol. Second, the polarity of nitrous oxide and the solvent used in our previous studies, carbon dioxide, is very different (CO2 is an apolar solvent, whereas N 2 0 is a polar solvent). Large differences in solubilizing different types of molecules were therefore expected. Also, extractions using nitrous oxide + ethanol mixtures were expected to be more selective towards the mildly polar taxol than extractions using a "powerful" (polar) solvent such as ethanol, which is currently employed in the liquid extraction of taxol.
2 2.1
Experimental Reagents
and Materials
Nitrous oxide (SCF grade) with a m i n i m u m purity 99.995 mole %, acetic acid with a purity of 99.99 mole %, HPLC grade methanol and water and reagent grade toluene and ethanol (99.99 mole %) were used in the experiments. All taxanes (taxol, cephalomannine, 10-deacetylbaccatin III, baccatin III, 7-epi 10-deacetyltaxol and 10-deacetyltaxol) were obtained from the Drug Synthesis and Chemistry Branch, Development Therapeutics Program, Division of Cancer treatment, National Cancer Institute (Bethesda, MD). The Cla reverse phase column was purchased from Alltech Associates Inc. Dry bark of Tazus brevifolia was obtained from Connolly/Eller Cottage Grove, Oregon. The same source of ground tree hark was used in all the experiments. The average particle size of the bark was less than 0.495 m m in diameter. All substances were used without further purification. 2.2
Experimental
Procedure
An a p p a r a t u s consisting of an Isco SFE 2-10 Extractor system was used to perform supercritical extractions of taxol from the bark of Tazus brevifolia. Details of the apparatus are available elsewhere (Vandana a n d Teja, 1995a). Extractions were performed using nitrous oxide as the supercritical fluid with and without ethanol as a co-solvent. During each experiment, two ISCO syringe pumps (model 260 D) were used to p u m p
164
V. Vandana et al. / Fluid Phase Equilibria 116 (1996) 162-169
nitrous oxide and ethanol through static mixers to ensure t h a t the mixtures were homogeneous and at the set b a t h temperature prior to contacting the ground plant material. A known amount (approximately 3.5 g) of ground bark was placed in a high pressure cell (cartridge) of 10 mL internal volume. Filters, of 5 # m diameter, were placed in the cartridge at both the ports to prevent any solid bark from being carried out with the supercritical fluid. The loaded supercritical fluid was depressurized across a heated capillary tube into a collection vessel placed in an ice bath. The depressurized fluid was then passed through a second collection vessel (when co-solvent was present), placed in a dry ice/ethanol bath. The gas volumes of nitrous oxide were measured with a Precision Scientific wet test meter (model 63111, D-3B) which was factory calibrated and certified to be accurate within 4- 0.5 %. Flow rates were kept at approximately 0.3-0.4 m L / m i n to allow sufficient residence time for the solvent to contact the bark. A Heise pressure gauge (model 8400) was used to monitor the system pressure with an uncertainty of 4- 0.046 MPa. A Leeds and Northrup thermistor was used to monitor the system temperature. The thermistor was calibrated using a standard p l a t i n u m resistance thermometer (model Number: R800-3 2.5 ~, Serial Number: Rsg0Y-11)) manufactured by Chino Works America Inc., and traceable to NIST. The uncertainty in the measurement of system temperatures was estimated to be 4- 0.5 K. Blank experiments were performed to calibrate the solvent delivery system for accuracy of the co-solvent composition. The co-solvent composition was found to be accurate within 40.5 mole%. The amount of ethanol and solute (supercritical extract) were determined gravimetrically by weighing the collection vessels before and after the experiment was complete. A Sartorius balance was used for the mass measurements. These measurements were reproducible within 4-0.0001 g and their accuracy was estimated to be within 4-0.0005 g. The extracts were analyzed for taxol content using high pressure liquid chromatography. The amount of taxol present in the bark extract was determined by HPLC analysis of the m e t h a n o l soluble portion of the extract. Dried bark extracts were dissolved in 2 mL of methanol, sonicated and mixed using a vortex mixer, and then filtered using nylon 66 membranes of 0.45 # m pore si~.e. The methanol soluble portion was analyzed using an LDC Analytical HPLC purchased from Thermoseparation Products Inc., equipped with a photo diode array detector. The solution was separated on a 250 m m (]is Alltech reverse phase column using a shallow gradient mobile phase for the analysis. The mobile phase varied from 58.5:41.5 methanol-water to 61.5:1.5 methanol-water for 25 minutes and its flow rate was maintained at 1.45 m l / m i n (Vandana et al., 1994). Taxol and related taxanes (baccatin III, 10-deacetylbaccatin III, cephalomannine, 7-epi 10-deacetyltaxol and 10-deacetyltaxol) absorb light in the ultraviolet (UV) region and exhibit a )~m~® at 227 nm. The photo diode array detector was therefore monitored in the range of 190 - 300 nm. The fingerprint of the absorb£nce of the compounds in the isogram was used to analyze the chromatograms. The absorption at 227 nm was used for quantitation (taxol has a 2 , ~ at this wavelength} and the datafiles containing the chromatograms were stored on disk for analysis. The amount of taxol present in the supercritical extracts was determined by comparing the response of the peak areas of taxol in a standard solution to that in the extracts. The injection volumes were 20 #L for each extract and s t a n d a r d solution. The average deviation in the measurement of peak areas as a function of concentration using 95 % confidence intervals was found to be within 5%.
3
R e s u l t s and D i s c u s s i o n
The results of nitrous oxide and nitrous oxide + ethanol extractions are tabulated in Tables 1,2 respectively. In our previous experiments with carbon dioxide and carbon dioxide + ethanol mixtures, it was observed t h a t the concentration of taxol in the extract decreased with time after attaining a m a x i m u m after approximately 3.0 moles of solvent had passed through the bed (Vandana and Teja, 1995a). Furthermore, the concentration of taxol in the extracts became almost zero after 3.5-4.0 moles of fluid had passed through
V. Vandana et a l . / Fluid Phase Equilibria 116 (1996) 162-169
•
o
o
•
•
.
.
.
.
!
.
.
.
.
.
.
.
.
.
!
=
.
•
•
.
.
.
.
165
.
[] E
"0
[]
[]
[]
[]
0.1
¢,J
M mm
0.01
o x
•
0.001
[ •
0 % Ethanol, 321.4 K
[
I []
11.4 % Ethanol, 319.5 K
I
. . . . . . . . . . . . . . . . . . .
10
.........
'
20
30
40
Pressure (MPa) Figure 2: Mass of taxol extracted per four moles of nitrous oxide as a function of pressure (the results are normalized to show the trends)
. . . . . . . . .
'.
. " .
.....
100
[]
•
[]
[]
[]
[]
10 o e~
•
10
•
|
•
•
.
•
•
[]
CO z - 10.5 % Ethanol
•
N20 - 11.5 % Ethanol
•
I
•
•
.
I
I
•
•
•
•
I
20 30 Pressure (MPa)
.
•
.
.
.
•
,
.
i
40
Figure 3: A comparison of the amounts of taxol extracted by four moles of supercritical nitrous oxide and carbon dioxide at 331 K
166
V. Vandana et al. / Fluid Phase Equilibria 116 (1996) 162-169
19.00
(a)
1 .B8 i
15.7a7.2 l |
|
Zl .~3
|
4.48
(b)
3.1
_.4
19.05
].7.27 A XS.??A /
,NV
A
25.38 27.40 m
29.02 21.05 a2.33
1"
0,00
TJ.~
~5.00
Figure 4: Chromatograms on a CzsReverse Phase Column (a): Standard solution of taxol, retention time = 19.0 rain. (b): Standard solution of a mixture of 10-deacetylbaccatin III (03.8 rain.), baccatin III (04.68 min.), 10-
deacetyltaxol (015.77 min.), cephalomannine (017.27 min.), taxol (019.05 rain.), 7-epi lO-deacetyltaxol (@21.50 min.) (c): Supercritical extract sample: taxol elutes at 19.02 rnin.
167
V. Vandana et al. / Fluid Phase Equilibria 116 (1996) 162-169
Table 1: Extraction of Taxol from Tazns brevifolia Using Supercritical Nitrous Oxide T
P
Mass of Bark
Mass
Moles
Mass
Mass
in the cell
N~O
Taxol in Extract (mg)
Extract
(K)
(MPa)
(g)
Taxol in Bark (mg)
322.1 320.8 322.1 320.6 av: 321.4
13.99 21.08 27.58 34.48
3.4226 3.4646 3.4809 3.4836
0.5476 0.5543 0.5569 0.5574
4.2741 4.1180 3.7528 3.5248
0.0103 0.0479 0.0558 0.0459
0.0395 0.0444 0.0432 0.0841
331.9 331.3 331.7 331.9 av: 331.7
13.75 20.75 27.60 34.76
3.4580 3.6097 3.3946 3.6102
0.5533 0.5776 0.5431 0.5776
6.3876 7.3831 4.0459 3.3745
0.0079 0.0179 0.0190 0.0875
0.0485 0.0595 0.0514 0.0513
(g)
the bed. Therefore, 3.4-7.4 moles of supercritical nitrous oxide and 2.5-4.5 moles nitrous oxide + ethanol mixtures were used in the extractions carried out in this study. Figure 2 shows a plot of the amount (rag) of taxol extracted per approximately 4.0 moles of nitrous oxide passed through the bed as a function of pressure at 320 K. The results were normalized for qualitative comparison of the effect of pressure and cosolvent composition on the amount of taxol extracted. It can be seen t h a t the amount of taxol extracted increases with increase in pressure and also with the the addition of co-solvent. The nitrous oxide + ethanol mixtures proved to be better solvents than nitrous oxide alone. Thus, the selectivity and extractibility of taxol improved with the addition of ethanol. Also, most of the taxol present in the bark was extracted using supercritical nitrous oxide at the highest co-solvent composition of ~ I0 mole % whereas only 15 % was extracted without the use of a co-solvent. All the calculations to determine the taxol content in the extractions were based on total taxol content of 0.016 % in the bark determined by performing an exhaustive hquid ethanol extraction and subsequent HPLC analysis of the extract (Jennings et a l , 1992). A comparison of extraction of taxol using carbon dioxide + ethanol mixtures and nitrous oxide + ethanol mixtures at 331 K is shown in Figure 3 (the tabulated extraction data using carbon dioxide and carbon dioxide ÷ ethanol mixtures are presented in Vandana and Teja, 1995b). Nitrous oxide proved to he a better solvent t h a n carbon dioxide. Also, most of the taxol present in the bark was removed using only 3.0 moles of nitrous oxide + ethanol mixtures at the higher pressures. The HPLC analysis of the supercritical extracts involved a time-optimized method incorporating a gradient elution of the mobile phase. Figure 4a shows the results obtained when a standard solution of pure taxol was injected on the Cls column; taxol is seen to elute at about 19.0 minutes. Figure 4b 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 taxanes, 10deacetylbaccatin III and baccatin III, which are more polar than the other taxanes sluts at the beginning of the run. Cephalomannine, which elutes at ~17.0 minutes, is very similar in structure to ta~ol but does not have any tumor-inhibitory activity. In the method chosen in this study, a shallow gradient ensures clean separation of this compound from taxol as can he seen in the chromatogram. The other two compounds, 10-deacetyltaxol and 7-epi 10-deacetyltaxol, are decomposition products which need to be identified to ensure t h a t taxol has not decomposed during the extraction process. Figure 4c shows a typical chromatogram
V. Vandana et al. / FluM Phase Equilibria 116 (1996) 162-169
168
T a b l e 2: E x t r a c t i o n of Taxol from Tazus brevifolia Using Supercritical N i t r o u s O x i d e + E t h a n o l M i x t u r e s Mass Taxol in Extract
MRss Extract
(ms)
(g)
5.3 6.I 8.8 5.1 5.3 5.I av:5.5
0.0870 0.1257 0.0972 0.0982 0,0956 0.1149
0,0847 0.0981 0.0892 0.0840 0.0658 0.0749
3.2075 4.3010 4.2059 4.0414 2.4543 3.7414
11,0 10.8 10.8 11.3 13.3 11.1 av:11.4
0.1022 0.2155 0.2489 0.3215 0.5697 0.3925
0.0873 0.1764 0.4166 0.3201 0.1751 0.1310
0.5866 0.5793 0.5673 0.5357 0.5642 0.5576
3,3740 3.0687 2.9216 3.3294 3.0023 3.4432
5.8 5.8 5.3 5.8 5.1 6.3 av:5.7
0.0531 0.2379 0.2166 0.3588 0.4866 0.4870
0.0487 0.0594 0.0671 0.0709 0.0806 0.0881
0.5554 0.5482 0.5470
3.8500 2.6953 2.6339
0.5558
2.9302
II.0 13.3 10.9 10.5 av:ll.5
0.1575 0.4470 0.4850 0.5543
0.3877 0.2638 0.2821 0.1155
P
Mass of Bark in the ceU
Mass Taxol in Bark
Moles N20
(K)
(MPa)
(g)
(rag)
320.6 320.3 320.5 320.2 320.3 320.6
10.45 13.92 20.66 27.51 34.37 37.83
3.5651 3.6149 3.4436 3.5632 3.4157 3.5594
0.5704 0.5784 0.5510 0.5701 0.5465 0,5595
2.8350 3.5963 2.5 817 3.6843 2.5965 4.0145
10.54 13.96 20.79 27.62 34.55 38.10
3.0730 3.7120 3.6578 3.5346 3.6274
0.5877 0.5939 0.5853 0.5655 0.5804 0.5942
av:320.4 319.0 319.6 319.0 319.6 319.5
319.3 av:319.5
3.7140
330,6
10.45
3.6660
330.5
13.93
3.6207
330.6 330.5 331.0 331.5 av:330.8
20.73 27.55 34.44 37.91
3.5458 3.3480 3.5250 3.4849
330.6 330,6 331.2 330.8 av:330.8
18.87 20.67 27.51 34.34
3.4710 3.4261 3.4185 3.4739
Mole % Ethanol in SCF
of t h e supercritical extracts. A lot of polar m a t e r i a l elutes at t h e b e g i n n i n g of t h e run, b u t a clear baseline s e p a r a t i o n of t a x o l a n d related t a x a n e s is observed. T h e less polar material, which s t a y s in t h e c o l u m n , is u s u a l l y flushed after t h e r u n with 100 % m e t h a n o l . T h e H P L C m e t h o d developed is simple a n d a c c u r a t e a n d w a s used to a s s a y taxol from t h e rest of t h e p l a n t m a t e r i a l extracted.
4
Conclusions
Supercritical n i t r o u s oxide and nitrous oxide + e t h a n o l m i x t u r e s were used to e x t r a c t t a x o l f r o m t h e b a r k of
Tazu8 brerifolia. T h e m i x t u r e s proved to be better solvents in e x t r a c t i n g t h e d r u g f r o m t h e p l a n t m a t e r i a l . A d d i t i o n of e t h a n o l increases the selectivity a n d extractibility of taxol. T h i s s t u d y s h o w s t h a t supercritical fluid e x t r a c t i o n c a n be used as a valuable a n d b e n i g n s e p a r a t i o n process to e x t r a c t t~xol w i t h o u t t h e u s e o f h a l o g e n a t e d solvents.
V. Vandana et al. /Fluid Phase Equilibria 116 (1996) 162-169
5
169
Acknowledgements
The authors would like to thank Dr. Gordon Cragg and the National Cancer Institute for supplying the bark of Tazus 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.
6
References
Wani, M. C., Taylor, H. L., Wall, M. E., Coggon, P., and McPhail, &. T., 1971. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Ta=us brevi/o]ia. J. Am. Chem. S0c., 93:2325-2327. McLaughlin, J. L., Miller, R. W., Powell, R. G., and Smith, C. R. Jr., 1981. 19-hydroxy baccatin III, 10-deacetyl cephalomannine and 10 - deacetyltaxol: New antitumor taxanes from Tazus waUichiana. J. Nat. Prod., 44:312-319. Miller~ R. W., Powell, R. G., and Smith, C. R. Jr., 1981. Antileukemic alkaloids from Taxus wallichiana Zucc. J. Org. Chem., 46:1469-1474. Stasko, M. W., Witherup, K. M., Ghiorzi, T. J., McCloud, T. G., Look, S. A., Muschik, G. M., and Issaq, H. J., 1989. Multimodal thin layer chromatographic separation of taxol and related compounds from Ta=us brsvifolia. J. Liq. Chrom., 12:2133-2143. Holton, R. A., Somoza, C., Kim, H-B., Liang, F., Biediger, R. J., Boatman, P. D., Shindo, M., Smith, C. C., Kim, S., Na~:lizadeh, H., Suzuki, Y., Tao, C., Vu, P., Tang, S., Zhang, P., Murthi, K. K., Gentile, L. N., and Liu, J. H., 1994a. First total synthesis of taxol 1. Functionalization of the B ring. J. Am. Chem. Sos., 116:1597-1598. Holton, R. A., Kim, H-B., Somoza, C., Liang, F., Biediger, R. J., Boatman, P. D., Shindo, M., Smith, C. C., Kim, S., Na~lizadeh, H., Suzuki, Y., Ta~, C., Vu, P., Tang, S., Zhang, P., Murthi, K. K., Gentile, L. N., and Liu, J. H., 1994b. First total synthesis of taxol 2. Completion of the C and D rings. J. Am. Chem. Soc., 116:1599-1600. Nicola~u, K. C , Yang, Z., Liu, J. J., Ueno, H.j Nantermet, P. G , Guy, R. K., Clairborne, C. F., Renaud I J., Couladourosl E. A., Paulvannan, K., and Sorensen, E. J., 1994a. Total synthesis of taxol. Nature, 367:630-635. Nicolaou, K. C., Clalborne, C. F., Nantermet, P. G., Couladouros, E. A , and Sorensenl E. J., 1994b. Synthesis of novel ta~xoids. J. Am. Chem. S0c., 116:1591-1593. Denis, J. N., Greene, A. E., Guenard, D., Gueritte-Voegelein, F., Mangatlal, L., and Potier, P., 1988. A highly efficient, practical approach to natural taxol. J. Am. Chem. Soc., 110:5917-5919. Vandana, V., and Teja, A. S., 1995a. A novel separation process for isolating taxol, an anti-cancer drug, from Tazus bre~i/olia. Submitted to Bioteeh. ~ Bioengg. Vandana, V., Teja, A. S., Deutsch, H. M, and Zalkow, L. H., 1994. A simple HPLC method for the separation of taxol and related taxanes in supercritical extracts of Tazus brevifolia. Submitted So J. Chrom. Jennings, D. W., Deutzch, H. M., Zalkow, L. H. and Teja, A. S., 1992. Supercritical extraction of taxol from the bark of Tazus bre~i]olia. J. Supercrit. Fluids, 5:1-6. Vandanal V., and Teja, A. S., 1995b. Supercritical extraction of taxol using CO2 and CO2 + ethanol mixtures. Accepted for Publication, Supercritical Science and Technology, ACS Syrup. Set.