Download pdf - Separation of taxanes from Taxus canadensis using dynamic pressurized liquid extraction

Biotechnology and Bioprocess Engineering 16: 769-776 (2011)

DOI 10.1007/s12257-010-0330-6

Separation of Taxanes from Taxus canadensis Using Dynamic

Pressurized Liquid Extraction

Yuheng Wang, Joanne Gamage, and Zisheng Zhang

Received: 19 September 2010 / Revised: 3 March 2011 / Accepted: 6 March 2011

© The Korean Society for Biotechnology and Bioengineering and Springer 2011

Abstract An extraction technique, dynamic pressurized

liquid extraction (DPLE), was proposed to extract the

taxanes; including 10-DAB III, Baccatin III, 9-DHB III

and paclitaxel, from powdered Taxus canadensis needles.

A dual-solvent approach was adopted in which the im-

purities were firstly removed by extraction with hexane,

and the taxanes were subsequently extracted with an ap-

propriate solvent. The performance of chloroform, dichloro-

methane, and mixtures of methanol/dichloromethane was

compared for use as the taxane-extracting solvent, and it

was found that solvents containing a higher proportion of

methanol had higher extraction capabilities. The effect of

temperature on DPLE extraction of the taxanes was also

studied, and it was found that higher extraction efficiencies

could be realized with increasing temperature up to a

threshold of 90oC. Based on a progressive conversion

model, a kinetic equation for the extraction process was

proposed. This model successfully confirmed that smaller

needle powder particle sizes would result in higher ex-

traction rates, which is consistent with the data obtained by

experimentation.

Keywords: DPLE (dynamic pressurized liquid extraction),

PLE (pressurized liquid extraction), dual-solvent extraction,

taxanes, Taxus canadensis

1. Introduction

The taxanes are a class of alkaloids which include a

tricyclic ring structure (as shown in Fig. 1). They are able

to stop the proper division of cells through the inhibition of

the microtubule function, and two members of the taxane

class; paclitaxel and docetaxel, are widely recognized as

anticancer drugs. Other taxanes isolated from the biomass

of Taxus species, such as 10-DAB III, baccatin III, and 9-

DHB III, can be used as intermediates for the semisyn-

thesis of paclitaxel and docetaxel since the complexity of

the molecular structures involved make direct chemical

synthesis routes uneconomical. For the production of these

bioactive components, isolation from natural sources often

provides the most economically viable method for produc-

tion [1]. Taxanes isolated from plants are the dominant

source of materials for taxane anticancer drugs, and it is

believed that for the near future, taxanes will continue to be

produced by biological means, namely plantation and

subsequent separation and purification [2]. Research on

paclitaxel production and purification performed using bio-

logical means via cell cultures is currently under investi-

gation [3-7].

However, in the more traditional approach using solvent

extraction methods, an appropriate solvent is mixed with

solid matrices and the concerned chemicals from the solids

are dissolved in the solvent and subsequently separated

through phase separation. Classical or conventional solvent

extraction (CSE) is widely applied in large-scale industrial

production. However, this frequently involves the use of

large amounts of solvent and a highly complicated process.

In a typical CSE for taxane production from Taxus bio-

Yuheng Wang, Joanne Gamage, Zisheng Zhang*

Department of Chemical and Biological Engineering, University ofOttawa, Ontario K1N 6N5, Canada Tel: +1-613-562-5800, Fax: +1-613-562-5172E-mail: [email protected]

RESEARCH PAPER

Fig. 1. The tricyclic ring structure of taxanes.

770 Biotechnology and Bioprocess Engineering 16: 769-776 (2011)

mass, liquid-liquid extraction (LLE) must be capable of

removing the lipids associated with the taxane source, and

this separation involves the handling and use of a large

amount of mixed solvents. There has been much research

attempting to improve CSE or develop new techniques to

improve its operation and decrease its complexity. Within

the literature, supercritical fluid extraction [8,9], microwave

accelerated extraction [10,11], automated Soxhlet extraction

[12], sonication accelerated extraction [13], and pressuriz-

ed liquid extraction (PLE) [14-18], have been reported.

However, at the current state of development, these techno-

logies are unable to surpass and replace CSE in large-scale

production. It should also be noted that, with the exception

of sonication acceleration, the reported extraction techni-

ques all employ the use of elevated operating temperature.

PLE is an extraction technique introduced in 1996 (then-

called accelerated solvent extraction) [14]. In this process,

extraction is operated under elevated temperatures and

pressures, where the elevated pressure is used to prevent

the solvent from boiling. Although PLE performs well at

bench-scale, its operation is batch-wise and limited to

scale-up. To the best of the authors’ knowledge, there is no

report of the use of PLE on an industrial scale to recover

bioactive components from biomass. Additionally, the ex-

traction kinetics of the liquid-solid system is rarely report-

ed, though some researchers have tried to study the process

using the ideal stage calculation [19].

In this work, the PLE technique is improved by continu-

ously pumping the solvent through the extraction cell while

maintaining the elevated temperature, and is called the

dynamic pressurized liquid extraction (DPLE). Since the

fresh solvent continuously flows into the extraction cell,

the concentration gradient of extracted compounds is

increased, enhancing the diffusivity of the extracted com-

pounds. A major advantage of DPLE is that it can be

operated with a continuous flow of solvent, making it very

promising for scale-up and industrial use. Additionally, a

lower pressure is required to maintain the solvents from

boiling within the DPLE than those used within PLE

methods (70 ~ 75 psig for the current work, in comparison

to 1,000 ~ 2,500 psig [14]). This is advantageous from a

scale-up point of view. The extraction of taxanes from

twigs and needles of Taxus canadensis (Canadian yew) is

studied in this DPLE system, and the kinetics of the

extraction is developed and discussed.

2. Materials and Methods

2.1. Material

Fresh twigs and needles of Taxus canadensis were picked

at Hartland and Rexton, New Brunswick, Canada. After

drying for 7 days in the dark at ambient temperature and

humidity, the needles were manually stripped from stems

and ground to powder with the size of < 20 mesh. The

ground needle powder was stored in a freezer below

−10oC. For each experiment, the needle powder was

freshly prepared by grinding the powders of < 20 mesh

using a household coffee mill (Braun, Type 4041, model

KSM2). After grinding, the powder was sieved and dried at

60oC for 4 h in air ventilation dryer with a digital temper-

ature controller (Fisher Scientific, Model 737F). The sieved

needle powder was mixed thoroughly to obtain identical

needle powders for each experiment.

All solvents used were HPLC grade (EM Science,

Gibbstown, NJ). The water was HPLC grade prepared by

a Zenopure® four-cartridge system (Type QUATRA 90LC,

Zenon Environmental Inc., Burlington, ON, Canada). Pacli-

taxel (99%), 10-deacetyl-baccatin III (10-DAB III, 95%),

and baccatin III (90%) were obtained from Fisher Scienti-

Fig. 2. Experimental setup for DPLE.

Separation of Taxanes from Taxus canadensis Using Dynamic Pressurized Liquid Extraction 771

fic (Ontario, Canada; purchased through Acros® Organics).

9-dihydro-13-acetyl-baccatin III (9-DHB III, 95%) was

obtained from NaPro Biotherapeutics, Inc. (Boulder, CO,

USA).

2.2. DPLE Experimental set-up

The process flowchart for DPLE is depicted in Fig. 2. The

solvent feeding pump was a Waters® 501 HPLC pump with

a flow rate of 0.0 ~ 9.9 mL/min. The extraction cell used

was an Omnifit® medium pressure preparative chromato-

graphy column (15 mm inner diameter, 100 mm in length)

made of borosilicate glass with one fixed end-piece and

one adjustable end-piece. The relief valve was Swagelock®,

Type RL3, while the other valves were Omnifit® three-way

valves made of PTFE. The range of the pressure gauges

was 0 ~ 100 psig. All valves were purchased from Ottawa

Fluid System Technologies Inc. (Ottawa, Canada).

2.3. Procedure

Prior to each experiment, the solvent was purged with high

purity helium using the HLPC online degassing system. A

sample of the needle powder (5.000 g) was transferred into

the extraction cell, and the height of the bed was adjusted

to 4.5 cm. The extraction cell was then attached to the ex-

traction system, and the system was purged with nitrogen

gas to remove oxygen. The relief valve was adjusted to

maintain the system pressure within the range of 70 ~ 75

psig to prevent the solvents from boiling. The extraction

cell was immersed into a water bath for 5 min before the

extraction. The solvent was then pumped through the

system at a constant flow rate, and the time recording

started at the point when the first drop of the liquid came

out of the system. The eluate was collected in 10 mL pre-

weighed and labelled test tubes in 5 mL aliquots until the

end of the experiment. After the extraction, the system was

purged with high pressure nitrogen gas (70 ~ 75 psig) to

remove the liquid solvent. The pressure was then reduced

to ambient and purged with low pressure nitrogen (< 10

psig) for 5 min to remove any remaining solvent residues.

2.4. Analysis

The HLPC system comprised of a Millennium™ 2010

Chromatography manager (A NEC™ 486/33i computer with

Millennium™ Software 2.0), a 600E Multisolvent Delivery

System, a Waters™ 717 Autosampler and a 486 Tunable

Absorbance Detector. The HPLC column (Curosil®-PFP,

250 × 4.6 mm) was purchased from Phenomenex USA

(Torrance, CA, USA). The wavelength of the UV detector

was 227 nm, and the injection volume was 10 µL. Com-

mercial taxanes were used as standards for calibration. If a

taxane was not identified clearly by its retention time due

to poor separation with HPLC or the variation of the

retention time, a few drops of acetobitrile solution of the

specific taxane external standard were added to the sample

and then analyzed with HPLC. The specific taxane peak

was then identified as the one with increased height by

comparing the two chromatograms of the sample.

3. Results and Discussion

3.1. Hexane extraction of lipid impurities

Fig. 3 shows a typical HPLC chromatogram obtained from

the 70% acetone/water extract of ground needle powder of

Taxus canadensis in a beaker. Pentafluorophenyl, the

material used for the coating inside the HPLC column,

possesses an aromatic ring with five fluorine atoms. The

large ð bond of the aromatic ring and the even distribution

of fluorine atoms cause the electron cloud to evenly

distribute within the pentafluorophenyl group. Due to this

uniformity, the hydrophobic (non-polar) compounds are

more easily adsorbed onto the column than the hydrophilic

(polar) compounds. As such, in Fig. 3, the compounds with

shorter retention times are the hydrophilic impurities, while

those with longer retention times are the hydrophobic

impurities. In the chromatograms, there are many peaks for

hydrophobic impurities near the paclitaxel peak at 36 min

retention time, which is consistent with is the presence of

much lipid inside the plant. To improve final taxane

recovery and minimize the lipid content of the extract, a

strategy was adopted to remove these lipid impurities prior

to the extraction of the taxanes.

Hexane, due to its low cost and non-polarity, was chosen

to extract the hydrophobic impurities in the needle powder.

The dominant component in the extract is thought to be

lipid, so within this work, the hexane extract is referred to

simply as “lipid”.

In the extraction experiment with hexane in DPLE, the

eluate from DPLE was kept in a fume hood at room

Fig. 3. HPLC chromatogram of the eluate from extraction using70% acetone/water. Extraction conditions: < 100 mesh needlepowder with Celite 545; extraction temperature: 25oC; extractiontime: 10 min; retention time: paclitaxel, 36.0 min; 9-DHB III, 25.9min; Baccatin III, 24.1 min; and 10-DAB III, 16.3 min.


temperature for 12 h. Within this sample, a small amount

of green precipitate appeared on the bottom of the sampl-

ing test tube, and this precipitate was separated by

filtration. The filtrate was collected in Petri dish and kept

in the fume hood for 12 h. The paclitaxel content in both

dried filtrate and precipitate was determined using HLPC.

Among the four taxanes, 10-DAB III, baccatin III, 9-DHB

III, and paclitaxel, only 80 µg of paclitaxel was detected in

the extract (Table 1). This represents 0.0016% of the

needle powder fed into the extraction cell, while the typical

maximum extraction amount of taxanes from the needle

powder using DPLE was about 0.104%. This indicates that

the loss of taxanes within the lipid removal step is very

small, approximately 1.54%.

3.2. Effect of temperature on DPLE

Hexane and dichloromethane extractions were conducted

at several temperatures which were selected based on

considerations including dissolution power of the solvent,

process kinetics, as well as product thermal stability. In the

extraction of taxanes with dichloromethane, all needle

powders were pretreated, i.e. extracted with hexane. It was

observed that the amount of extract increases with increas-

ing temperature (Figs. 4 and 5). A similar trend is notice-

able despite the different particle sizes used, and in the

presence and absence of the hexane pretreatment.

This result is alluded to by the common characteristics

of high temperature shared by the advanced extraction

techniques, as previously mentioned. Though the elevated

pressure assists in the disruption of solute-matrix inter-

actions, its main function in DPLE is to maintain the

solvent in a liquid state when the temperature exceeds its

normal boiling point.

The elevated temperature facilitates extraction by the

following mechanisms [14]: Enhancing mass transfer,

improving the solubility of the solutes in the solvent, and

disrupting the adhesion between the solute and solid

matrices. Additionally, within DPLE, the viscosity of the

extraction solvent is also decreased at elevated temperatures.

This has the effect of lowering the pressure drop through

the bed in the column, which is economically beneficial in

full scale operations. DPLE also offers the distinct advant-

age of a continuous flow of fresh solvent into the column,

increasing the concentration gradient that drives the diffu-

sion of solutes to the solvent.

When the temperature approaches 90oC, the curve tends

to plateau. It is hypothesized that the ability for the solvent

to extract solutes from the solid matrices is nearly saturated

at this temperature. Thus 90oC represents a “threshold”

temperature for this extraction. If the extraction temperature

Table 1. Analysis results of hexane extract after 12 h precipitation at room temperature*

Dried filtrate Precipitate

Net weight (g) 0.28345 0.02425

Percentage of dry needle powder (%) 5.669 0.485

Appearance Dark brown, tar-like semi-solidFine green powder, readily dissolved in dichloromethane or methanol

Taxane content Not detected Paclitaxel only, 80 µg

*DPLE condition: 1.0 mL/min, 90.0°C, 30 min.

Fig. 4. The effect of temperature on DPLE in lipid removal withhexane. Extraction conditions: 5.000 g of 40 ~ 60 mesh needlepowder; flow rate: 1.0 mL/min; extraction time: 60.0 min.

Fig. 5. The effect of temperature on DPLE extraction of taxaneswith dichloromethane. Extraction conditions: 5.000 g of < 100mesh needle powder; flow rate: 1.0 mL/min; extraction time: 60min; biomass in the column pretreated with hexane: 90oC, 1.0 mL/min, and 30 min.


falls below this threshold, the efficiency of DPLE will be

increased by increasing temperature, but above this value

the efficiency will only be slightly improved.

Fig. 6 shows the variation in the amount of paclitaxel in

the first 5.0 mL of hexane extract with temperature. A

linear trend is observed, in which at high temperatures,

there is no evidence that the extracted amount in the first

5.0 mL of eluate decreases when using the hexane solvent.

In the experiments conducted to obtain the data in Fig. 6,

due to the very weak ability of hexane to extract paclitaxel,

a great amount of paclitaxel inside the solid particles has

not been released. In comparison with the results in Fig. 5,

where the experiment was conducted with hexane-pretreat-

ed taxane and dichloromethane solvent, the extraction

ability for chloromethane to extract paclitaxel is nearly

saturated at 90oC. Fig. 7 shows the time course of the

extract weight of lipid in each 5.0 mL eluate sample during

the hexane extraction process. It indicates that within 60

min, lipids have almost been fully extracted. From Figs. 6

and 7, it can be concluded that in Figs. 4 and 5, the plateau

near 90oC is a result of almost full extraction of the lipids

by hexane at this temperature.

The threshold temperature is a meaningful signal indi-

cating that the solutes have been almost fully extracted.

The threshold temperature is dependent on operational

parameters, for example – if the extraction time extends to

120 min, the value of the threshold will be lower than that

in the 60 min extraction. Fig. 7 therefore also indicates that

long operational times may not be economical.

Fig. 8 depicts the weight variation of the extracted

taxanes with temperature, and the plateau in the vicinity of

Fig. 6. Amount of paclitaxel in the first 5.0 mL of hexane extract.Extraction conditions: 5.000 g of < 100 mesh needle powder; flowrate: 1.0 mL/min.

Fig. 7. Profile of extract weight from each 5 mL of extract in lipidremoval by DPLE with hexane. Extraction condition: 5.000 g of40 ~ 60 mesh needle powder; flow rate: 1.0 mL/min; temperature:90oC.

Fig. 8. Temperature effect on the extraction of taxanes withdichloromethane. Extraction conditions: 5.000 g of <100 meshneedle powder; flow rate: 1.0 mL/min; extraction time: 60 min.Biomass in the column pretreated with hexane: 90.0oC, 1.0 mL/min, and 30 min.

Fig. 9. Temperature effect on the concentration of taxanes in theextract. Extraction conditions: 5.000 g of < 100 mesh needlepowder; extraction solvent: dichloromethane; flow rate: 1.0 mL/min; extraction time: 60 min. Biomass in the column pretreatedwith hexane: 90.0oC, 1.0 mL/min, and 30 min.


the 90oC threshold is also realized. However, the concent-

ration of total taxanes, as calculated by the summation of

various taxanes, in the extract decreases with temperature

(Fig. 9). This indicates that higher extraction temperatures

result in a larger ratio of impurities in the extract.

3.3. The effect of particle size on DPLE

Four groups of needle powder with different particle sizes

were compared: 40 ~ 60 mesh, 60 ~ 80 mesh, 80 ~ 100

mesh, and > 100 mesh. Five grams of needle powder was

packed into each extraction column. Although the weight

of the needle powder used was the same in each extraction,

within the same extraction time, the weights of the lipid

extracted from the needle powder increased with decreas-

ing particle size (Fig. 10).

The needle powder of Taxus canadensis is in the form of

loose particles. The solvent can easily enter and react

throughout the particle at all times; which is represented by

the progressive conversion model [20]. Within this model,

the extraction rate is determined by two processes: The

physical/chemical reaction (extracting extract into the solv-

ent), and mass transfer. In the extraction process, a single

particle can be regarded as an entity and the concentration

inside the particle varies according to the first-order rate

law, where:

(1)

The amount that leaves the solid for the solvent can then be

expressed as:

(2)

The amount of the extracted chemical in the eluate is also

influence by the distance that the chemical which is being

extracted travels inside the solid. The average traveling

distance inside one particle is:

(3)

Making the simplification that the traveling distance has a

reversely proportional influence on the extracted amount in

eluate,

(4)

Differentiating with respect to time yields:

(5)

The concentration in the elute (assuming rigorous plug

flow inside the reactor),

(6)

Using the least squares method with Matlab software, the

coefficients were found by regression to be: k = 0.1397/sec

and K = 220 kg/m3. In Fig. 10, the solid lines are results

calculated using the model with regression coefficients,

and are seen to be in good agreement with the experimental

data.

3.4. Effect of flow rate and extraction time on DPLE

The extraction process is comprised of two basic steps: the

disruption of the solute-matrix interactions and mass

transfer (diffusion). A higher flow rate of solvent enables a

dCs

dt-------- = kCs–

Md = GCs0

ρ------------ 1 e

kt––( )

Davg = 3

4πR3

------------

0

R

∫ R r–( )4π r3dr =

R

4---

Me = 4GCs0

k′Rρ--------------- 1 e

kt––( )

dMe

dt---------- =

4kGCs0

k′Rρ------------------e

kt–

Ce = dMe

Qdt---------- =

4kGCs0

Qk′Rρ------------------e

kt– = K

R----e

kt–

Fig. 10. Effect of needle powder particle size on the extraction oflipids. Note that the points refer to experimental data while thelines were obtained by regression of the kinetic model. Extractionconditions: 5.000 g of needle powder; extraction temperature:90.0oC; extraction time: 60 min.

Fig. 11. Effect of flow rate on the extraction of taxanes in DPLEwith dichloromethane. Extraction conditions: 5.000 g of < 100mesh needle powder; extraction temperature: 90.0oC; extractiontime: 60 min. The biomass inside the column was pretreated byDPLE with hexane: 90.0oC, 1.0 mL/min, and 30 min.


more intense mechanical attack on the solute matrix, and is

also beneficial to the diffusion of the chemical in solvent.

As such, a higher flow rate should enhance the extraction

efficiency; and this is observed in the experimental data

(Fig. 11).

The time course for the total amount extracted (Fig. 12)

indicates that long extraction time result in more extracted

lipid, however the extraction rate (extracted amount per

unit time), decreases. In addition, the eluate color of the

hexane extraction changed from dark green at the beginn-

ing to light white after 25 min in the extraction experiment

at 90oC. This implies that besides lipid, another impurity,

chlorophyll, appears to be fully removed within 25 min.

3.5. Effects of solvent selection on DPLE

Based on a preliminary screening, extraction experiments

were conducted with chloroform, dichloromethane, and a

dichloromethane-methanol mixture as the taxane-extracting

solvents. In each experiment, the extraction column was

packed with 5.000 g of < 100 mesh needle powder and

pretreated with hexane using the following conditions:

Flow rate of 1.0 mL/min; pressure of 0.070 ~ 0.075 psig;

extraction temperature of 90oC, and extraction time of 30

min.

The extraction capabilities of the three solvents were

found to be (in decreasing order): methanol-dichloromethane

with the highest percentage of methanol, dichloromethane,

and chloroform (Fig. 13). Inside di- or trichloroformethane

molecules, the chlorine atom with strong negativity attracts

the electron cloud, and a dipole pointed to the carbon atom

is formed. Obviously, trichloromethane (chloroform) has a

higher polarity than dichloroethane due to the additional

chlorine atom. The polarizability of chloroform is 8.865;

whereas that of dichloromethane is 7.205 [21]. This indi-

cates that even after hexane extraction, there are still more

extracts which prefer slightly lower polarity than those that

prefer high polarity.

In the purging stage of DPLE, the flow rates of the

residue solvents decreased dramatically with the concent-

ration of methanol in mixing solvent. When purging with

0.015 psig nitrogen gas after the extraction, the purging

time varied from 20 sec to more than 10 min when the

methanol content increased from 0 to 4%. This indicates

that higher methanol concentrations could possibly result

in an unacceptably long time required to rinse the ex-

traction columns.

4. Conclusion

The proposed extraction technique, DPLE, is an effective

approach to extract taxanes from powdered Taxus canadensis

needles. The main advantage of this method is its con-

tinuous operation, which makes it very promising for

application in large scale production. Additionally, the

pressure requirements are lower than in previously reported

pressurized liquid extraction methods. A threshold temper-

ature of approximately 90oC is realized for an efficient

extraction process. Below this temperature, there is an

increase in efficiency with increasing temperature, while

above this temperature, efficiency only slightly increases

with increasing temperature. The extraction process follows

the progressive conversion model in which the solvent

permeates into the whole particle solid and the extraction

rate is dependent on the rate that the chemical leaves the

solid for the solvent and the mass transfer resistance of the

solid matrices. Using dichloromethane as the solvent, the

Fig. 12. Cumulative extract weight profile of DPLE with hexane.Extraction conditions: 5.000 g of 40 ~ 60 mesh needle powder;flow rate: 1.0 mL/min.

Fig. 13. Extract weight profiles of various solvents in DPLE.Extraction conditions: 5.000 g of < 100 mesh needle powder; flowrate: 1.0 mL/min; extraction temperature: 90.0oC. The needlepowder inside the extraction column was pretreated with hexane:90.0oC, 1.0 mL/min, and 30 min.


addition of a small amount of methanol adjusts the polarity

of solvent to enhance the process of taxanes in the solid

matrix dissolving into the solvent. Future work should be

completed to optimize processing temperature and process-

ing time.

Nomenclature

C : Concentration of chemicals which are being

extracted (kg/m3)

D : Traveling distance inside one particle (m)

G : Total mass of the solid fed into the extraction cell

(kg)

k : Reaction rate coefficient (1/sec)

k' : Resistance coefficient of mass transfer (1/m)

K : Overall coefficient (kg/cm3)

M : Mass (kg)

Q : Flow rate (kg/m3)

R : Particle radius (m)

r : Distance between any point and the centre of the

particle sphere (m)

t : Time(sec)

ρ : Density of the solid particles (kg/m3)

avg : Average

d : Solute leaving the solid for the solvent

s : Chemical concentration being extracted

o : Original concentration inside solid

e : Extracted chemical in the elute

References

1. Cragg, G. M., M. R. Boyd, R. Khanna, D. J. Newman, and E. A.Sausville (1999) Natural product drug discovery and develop-ment: The United States National Cancer Institute Role. Proceed-ings of the 38th Annual Meeting at the Phytochemical Society ofNorth America on Phytochemicals in Human Health Protection,Nutrition, and Plant Defense. July 26-31. Pullman, USA.

2. Walker, K. and R. Croteau (2001) Taxol biosynthetic genes.Phytochem. 58: 1-7.

3. Pyo, S. -H., H. -B. Park, B. -K. Song, B. -H. Han, and J. -H. Kim(2004) A large-scale purification of paclitaxel from plant cell cul-tures of Taxus chinensis. Proc. Biochem. 39: 1985-1991.

4. Pyo, S. -H., M. -S. Kim, J. -S. Cho, B. -K. Song, B. -H. Han, andH. -J. Choi (2004) Efficient purification and morphology char-acterization of paclitaxel from cell cultures of Taxus chinensis. J.

Chem. Technol. Biotechnol. 79: 1162-1168. 5. Jeon, K. -Y. and J. -H. Kim (2007) Optimization of micellar

extraction for the pre-purification of paclitaxel from Taxus chin-ensis. Biotechnol. Bioproc. Eng. 12: 354-358.

6. Jeon, K. -Y. and J. -H. Kim (2009) Improvement of fractionalprecipitation process for pre-purification of paclitaxel. Proc. Bio-chem. 44: 736-741.

7. Han, M. -G., K. -Y. Jeon, S. Mun, and J. -H. Kim (2010) Devel-opment of a micelle-fractional precipitation hybrid process forthe pre-purification of paclitaxel from plant cell cultures. Proc.Biochem. 45: 1368-1374.

8. Oostdyk, T. S., R. L. Grob, J. L. Snyder, and M. E. McNally(1993) Study of sonication and supercritical fluid extraction ofprimary aromatic amines. Anal. Chem. 65: 596-600.

9. Kim, J. W., Y. H. Choi, K. P. Yoo, M. J. Noh, and J. H. Han(2003) Method and apparatus for preparing taxol using supercrit-ical fluid from source materials. US Patent 6,503,396.

10. Abu-Samra, A., J. S. Morris, and S. R. Koirtyohann (1975) Wetashing of some biological samples in a microwave oven. Anal.Chem. 47: 1475-1477.

11. Hao, J. Y., W. Han, S. D. Huang, B.Y. Xue, and X. Deng (2002)Microwave-assisted extraction of artemisini from Artemisia. Sep.Purif. Technol. 28: 191-196.

12. USEPA: United States Environmental Protection Agency (1995)Test methods for evaluating solid waste, method 3541. USEPASW-846, 3rd ed., Update III; U.S. GPO: Washington DC.

13. Golden, G. and E. Sawicki (1978) Determination of benzo(a)-pyrene and other polynuclear aromatic-hydrocarbons in airborneparticulate material by ultrasonic extraction and reverse phasehigh-pressure liquid-chromatography. Anal. Lett. 11: 1051-106.

14. Richter, B. E., B. A. Jones, J. L. Ezzell, N. L. Porter, N. Avdal-ovic, and C. Pohi (1996) Accelerated solvent extraction: A tech-nique for sample preparation. Anal. Chem. 68: 1033-1039.

15. Benthin, B., H. Danz, and M. Hamburger (1999) Pressurizedliquid extraction of medicinal plants. J. Chromatogr. A 837: 211-219.

16. Alonso-Salces, R. M., E. Korta, A. Barranco, L. A. Berrueta, B.Gallo, and F. Vicente (2001) Pressurized liquid extraction for thedetermination of polyphenols in apple. J. Chromatogr. A 933: 37-43.

17. Lee, H. K., H. L. Koh, E. S. Ong, and S. O. Woo (2002) Deter-mination of ginsenosides in medicinal plants and health supple-ments by pressurized liquid extraction with reversed phase highperformance liquid chromatography. J. Sep. Sci. 25: 160-166.

18. Choi, M. P. K., K. K. C. Chan, H. W. Leung, and C. W. Huie(2003) Pressurized liquid extraction of active ingredients frommedicinal plants using non-ionic surfactant solutions. J. Chro-matogr. A 983: 153-162.

19. Kawasaki, J., H. Kosuge, H. Habaki, and Y. Morita (2006) Solid-liquid extraction of taxane compounds from yew needle. Sep. Sci.Technol. 41: 1077-1097.

20. Levenspiel, O. (1972) Chemical Reaction Engineering. p. 360.John Wiley & Sons, Inc., NY-London-Sydney-Toronto.

21. Lide, D. R. (2006-2007) In CRC Handbook of Chemistry andPhysics. 85th ed., CRC Press, Boca-Roton-London-NY-Washing-ton D. C., USA.