JOURNAI.

OF BIOVXNC~ AND BIO~NGIN~ERINC

Vol. 93, No. 2, 2 15-220. 2002

Production of Podophyllotoxin by Plant Cell Cultures

of Podophyllum hexandrum in Bioreactor
SAURABH CHATTOPADHYAY,’ ASHOK K. SRIVASTAVA,’
SANT S. BHOJWANI,’ AND VIRENDRA S. BISARIA’*

Department of Biochemical Engineering & Biotechnolog)i Indian institute qf Technology-Delhi, Hauz Khas
New Delhi I10016, India’ and Department of Botany. University qf Delhi, Delhi 110003. India’

Received 4 October 200liAccepted 28 November 2001

Submerged cultivation of Podophyllum hexandrum for the production of podophyllotoxin was

carried out in a 3 I stirred tank bioreactor fitted with a low-shear Setric impeller. The specific re-
quirements of the medium, such as carbon source (sugar) and light, were established for the
growth of and podophyllotoxin production by I? hexandrum in suspension cultures. Substitution
of sucrose by glucose resulted in higher growth and podophyllotoxin production. The biosynthesis
of podophyllotoxin was favored when plant cells were cultivated in the dark. An agitation speed of
100 rpm was sufficient to mix the culture broth in the bioreactor without causing any significant
cell damage. Biomass and podophyllotoxin accumulation in 3 1 bioreactor under batch growth
conditions were 6.5 g/l and 4.26 mgll, respectively, in 22 d. This resulted in an overall podophyllo-
toxin productivity of 0.19mgl(l.d), which represented an increase of 27% in comparison to its
productivity in a shake flask. Podophyllotoxin production was found to be a combined growth-as-
sociated and non-growth associated process.

[Key words: podophyllotoxin, Podophyltum hexandrum, plant cell culture, bioreactor, shear stress,
anticancer drugs]

Plants have provided us with food, fuel and fiber since
prehistoric times. They have been an inexhaustible source
of a diverse array of chemicals such as flavors, fragrances,
natural pigments, pesticides and pharmaceuticals. This
seemingly unrelated collection of chemicals can be grouped
together under a broad category of plant secondary metabo-
lites. Currently, these compounds are produced by cultiva-
tion of the plants followed by extraction of the desired prod-
uct. This conventional process presents a number of disad-
vantages such as non-availability of the secondary metabo-
lite from the plant throughout the year and inconsistent
product yield due to climatic variations. Plant cell culture
technology provides a viable alternative to whole plant cul-
tivation for the production of secondary metabolites. By
proper manipulation of culture conditions, use of precur-
sors/elicitors and cultivation in low-shear bioreactors, it is
possible to achieve high product concentration and produc-
tivity (l-8).
Podophyllum hexandrum Royle (May Apple), belonging
to the Berberidaceae family, is an Indian medicinal herb that
grows in the northern Himalayan region at an altitude of
3500-4000 meters. It is an erect herbaceous plant about 15-
30 cm in height with a perennial root system and annual
parts that appear around the middle of March after dor-
mancy during winter (9). The rhizomes of this plant species
are well known in medicine as a source of podophyllin
resin. The resin consists of various lignans that are pharma-
* Corresponding author. e-mail: vbisaria@dbeb.iitd.ernet.in
phone: +91-l I-6591002 fax: +91-l I-6868521
cologically active plant-based compounds. Podophyllotoxin
is the major lignan present in the resin and is a dimerized
product of the intermediates of the phenylpropanoid path-
way (10). The cytotoxic activity of podophyllotoxin is based
on its ability to inhibit the microtubule assembly during cell
division. It is used as a starting material for the chemical
synthesis of anticancer drugs, etoposide (VP- 16-2 13) and
teniposide (VM-26), which have fewer side effects (11).
These drugs act as inhibitors of topoisomerase II and are
widely used in the treatment of lung and testicular cancers
( 12). Podophyllotoxin is also produced in relatively minute
quantities by other plant species, viz., P peltatum, P ver-
sipelle, LinumJlavum, L. album and Juniperous chine&s.
The Indian Podophyllum hexandrum is superior to its
American counterpart, I? peltatum, in terms of its higher
podophyllotoxin content (4% in the dried roots in compari-
son to only 0.25% for J! peltatum) ( 10, 13).
Podophyllotoxin is presently being isolated by solvent
extraction from the rhizomes of II hexandrum. Due to indis-
criminate collection and unorganized cultivation, L! hexan-
drum from the Himalayan region has been declared as an
endangered species. The risk of extinction of this economi-
cally valuable plant can be overcome by mass cultivation of
the plant cells under in vitro suspension culture conditions.
Kadkade developed the callus culture and was the first to
report the production of podophyllotoxin by tissue culture
of 19 peltatum (14). The production of podophyllotoxin by
cell cultures of F! hexandrum has thereafter received great
attention from different investigators as being a promising
alternative source (13, 15). Studies on the suspension cul-

215
216 CHATTOPADHYAY ET AL.
ture of P hexandrum for improving productivity have so far
concentrated mainly on precursor feeding strategies and
immobilization techniques (16). The development of the
culture and operating conditions for suspension culture of i?
hexandrum will pave the way for the large-scale production
of podophyllotoxin.
Major culture conditions such as medium carbon source
(type of sugar), light and hydrodynamic stress play a signif-
icant role in product synthesis. Accordingly, the effects of
suitable carbon source, light and shear stress have been
studied for the growth of and podophyllotoxin production
by I? hexandrum plant cells under in vitro conditions. Using
the optimum conditions of the shake flask system, the
growth and product formation kinetics was investigated in a
3 1 bioreactor.
MATERIALS AND METHODS
Culture conditions The methodology used by the authors for
the initiation of callus from the seeds of p hexandrum and its
maintenance has been described elsewhere (17). The suspension
culture of R hexandrum was initiated from its callus in Murashige
and Skoog (MS) medium (18) supplemented with glucose (30 g/l)
and indole-3-acetic acid (IAA, 11.4umol/l) in the presence of pec-
tinase (1.5 mgll) and polyvinylpyrrollidone (PVP, 10 g/r). An inoc-
ulum of 2 g/l [dry cell weight (DCW) basis] was used. The suspen-
sion-grown cells were maintained by subculturing in fresh medium
containing ‘MS+glucose (30 gIl)+PVP (5 g/Q)’ every three weeks.
Pectinase and IAA were withdrawn from the medium during sub-
cultures. The cultures were incubated at 125 rpm on a gyratory
shaker at 20°C in the dark.
Effect of sugar on the growth of and podophyllotoxin pro-
duction by I? hexandrum in shake flask The cells of l? hexan-
drum (2 g/l, DCW basis) were inoculated into 50 ml hormone-free
MS medium containing either sucrose or glucose as carbon source
at 30 g/l in 250 ml Erlenmeyer flasks. The cultures were incubated
at 125 rpm on a gyratory shaker at 20°C in the dark. Samples (15
ml) were withdrawn every 4 d to determine DCW as well as resid-
ual sugar and podophyllotoxin concentrations.
Effect of light on the growth of and podophyllotoxin pro-
duction by R hexandrum in shake flask The cells of p hex-
andrum (2 g/l, DCW basis) were inoculated into 50 ml hormone-
free MS medium supplemented with 30 g/l glucose. The cultures
were incubated at 125 rpm on gyratory shaker at 20°C under 16/8 h
light/dark regime and in the dark. Samples (15 ml) were withdrawn
every 4 d to determine DCW as well as residual glucose and podo-
phyllotoxin concentrations.
Effect of hydrodynamic shear stress on L hexandrum cells
cultivated in shake flask I? hexundrum cells were grown in
250 ml Erlenmeyer flasks containing 50 ml medium [MS+glucose
(30 gIZ)+PVP (IO g/l)]. The cultures were incubated on a gyratory
shaker at different rotational speeds (125, 150, 200, 250 and 300
rpm) at 20°C in the dark. Samples (5 ml) were collected every 4 d
and analyzed for cell viability. The triphenyltetrazolium chloride
(TTC) method (19) was adopted for the accurate and quantitative
analysis of cell viability.
Cultivation of l? hexandrum in 3 I bioreactor The cells of
I? hexandrum were cultivated in a 3 1 laboratory-scale bioreactor
using a three-week dark-grown culture as inoculum (2 g/l, DCW
basis). The medium used was ‘MS +glucose (30 g/l) +PVP (10
g/c)’ and the bioreactor was operated at 100 rpm at 20°C in the
dark. The pH of the culture medium was adjusted to 5.8 before
autoclaving and controlled at 5.8 by the automatic addition of
aq. NaOH (0.5 molil) and aq. HCI (0.5 molil) during cultivation.
J. BIOSU. BIOENG.,
Temperature was controlled by passing chilled water into the cool-
ing finger of the bioreactor. All parameters were controlled by a
bio-controller (ADI 1030, Applikon Dependable instruments, The
Netherlands). A specially designed low-shear impeller (Setric im-
peller) was used for gentle agitation to avoid damage to the cells
caused by impeller rotation. An air flow rate of 0.5 vvm was main-
tained only in the head space of the bioreactor to avoid extensive
foaming of the culture broth and to prevent cell damage. Samples
(I 5 ml) were collected in duplicate every 2 d to determine DCW as
well as residual glucose and podophyllotoxin concentrations.
Measurement of DCW and sugars The samples (15 ml)
were centrifuged at 3000 rpm for 15 min, after which the cells
were washed with distilled water, centrifuged again and then
weighed to obtain the fresh weight. The cells were then dried at
60°C for I6 h to determine DCW. The reducing sugars were esti-
mated as glucose by the dinitrosalicylic acid method (20) and total
sugars by phenol-sulphuric acid method (21).
Extraction and detection of podophyllotoxin Podophyllo-
toxin was extracted by heating the dried cell mass in ethanol at
60°C for 30 min with constant stirring using a magnetic stirrer, fol-
lowed by sonication (Soniprep, USA) for I5 min. Constant stirring
promoted the leaching out of the lignans from the cells. The super-
natant obtained after centrifugation at 12,000 rpm for 10 min was
evaporated to dryness under vacuum. The dry mass was dissolved
in analytical-grade ethanol. HPLC analysis of the extract was
carried out using a NovaPak C,, (Waters, USA) column (250x4.6
mm) in an HPLC system (Waters) connected to a UV detector
(Waters, Model 440 Absorbance Detector). Acetonitrile-water-
methanol (37 : 58 : 5) was used as the mobile phase at a flow rate of
1.5 ml/min. The extract (20 pl), after passing through a 0.45 urn
filter, was injected into the column. Podophyllotoxin was detected
at 280 nm. Commercially available podophyllotoxin (0.1 mg/ml,
Sigma, USA) was used as the standard for the calculation of podo-
phyllotoxin content in the samples based on the total area of the
peak obtained from an integrator (Waters 746 Data Module).
Quantification of relevant growth and production parame-
ters A few selected growth and production parameters have been
commonly employed for the submerged culture of plant cells in a
shake flask and in a bioreactor (22). These parameters were used
for analyzing the efficiency of growth and podophyllotoxin pro-
duction in suspension cultures.
Growth yield (Y,):
[Maximum cell mass - Initial cell mass]
/[Sugar consumed] (g/g)
Growth index (GI):
[Maximum cell density - Initial cell density]
/[Initial cell density]
Average growth rate (Q,):
[Maximum cell density - Initial cell density]
/[(Initial cell density)(Culture time)] (d-‘)
Volumetric productivity (Qp):
[Amount of product]
/[(Culture volume)(Culture time)] [mgi(l.d)]
Specific productivity (qp):
[Amount of product]
/[(Cell growth)(Culture time)] [mg/(g.d)]
RESULTS AND DISCUSSION
Effect of sugar on the growth of and podophyllotoxin
production by l? hexandrum in shake flask Glucose and
sucrose at 30 gll were studied for the growth of and product
formation by P hexandrum. The profiles of growth and
podophyllotoxin production (Fig. 1) show that glucose was
a better carbon source than sucrose. A maximum biomass of
VOL. 93,2002 PRODUCTION OF PODOPHYLLOTOXIN IN BIOREACTOR 217

IO

Tim:(d)
0
IL
::
0 IO 20 30 40 (b) ,^
Tie(d)
4 4o[
FIG. I. Growth and product (podophyllotoxin) profiles of P: hex-
andrum in MS media containing sucrose and glucose. DCW in glucose
medium (open circles); DCW in sucrose medium (solid circles); podo-
phyllotoxin in glucose medium (open squares); podophyllotoxin in su-
crose medium (solid squares).

8.3 gll (DCW basis) and podophyllotoxin of 4.9 mgll were

produced after 28 and 32 d, respectively, when glucose was
used as carbon source in the culture medium, whereas a IO 30 40

maximum biomass of 6.8 g/l (DCW basis) and podophyllo- Tim:(d)

toxin of 3.5 mgll were produced after 32 d on sucrose, the
most commonly used carbon source for plant cells. The
growth and production parameters Y,, Gl, Q,, Qp, and q,, are
listed in Table 1. The higher values of the above parameters
for glucose indicate that the cultivation of rl hexandrum in
MS medium containing glucose produced both significantly
higher biomass and podophyllotoxin. The volumetric and
specific productivities were enhanced by 36% and 19%,
respectively, when I? hexandrum was cultivated in glucose
medium. The profile of the consumption of glucose and su-
crose during the growth ofl? hexandrum is presented in Fig.
2. Apparently, sucrose, after being taken up from the culture
media, was hydrolyzed to glucose and fructose by cell wall
bound invertase of plant cells (23). The slow consumption
of sucrose might be due to low activity of the invertase.
The selection of carbon source (glucose or sucrose) has
long been recognized as an important factor in the produc-
tion of secondary metabolites by plant cell cultures (24).
Production of ajmalicine was induced in Cutharanthus ro-
sew in the presence of glucose only (25). In contrast, glu-
cose was reported to be less effective than sucrose for shiko-
FIG. 2. (a) Consumption of glucose during growth of F1 hex-
andrum. (b) Consumption of sugars during growth of /? hexandrum in
medium containing sucrose as carbon source. Total sugar (solid trian-
gles); reducing sugar (open triangles); sucrose (open circles).
nin production by Lithospermum erythrorhizon (26).
Effect of light on the growth of and podophyllotoxin
production by I? hexandrum in shake flask It was ob-
served that the callus of p hexandrum was more friable
when it was grown in the dark compared to those grown
under 16/S h light/dark regime. The effect of light on the
growth and product formation was studied in I? hexandrum
suspension cultures. Figure 3 presents the profile of growth
and podophyllotoxin production during the cultivation under
both dark and light/dark conditions. Although the growth
and production rates were quite similar under both con-
ditions, the overall growth and production were relatively
higher in the dark (Table 2).
Light provided to naturally growing plants is required for
photosynthesis. However, the light provided to the in vitro
grown cultures is utilized for certain complex photochemi-

TABLE I. Comparative values of growth and production parameters in I? hexundrum suspension cultures with sucrose and glucose

DCW Podophyllotoxin GI
Carbon source
(g/l) (mdl) (&) ____
Sucrose 6.8 3.5 0.24 2.4 0.075 0.1 I 0.016
Glucose 8.3 4.9 0.31 3.15 0.1 I 0.15 0.019

TABLE 2. Comparative values of growth and production parameters in suspension cultures

of p hexundrrumunder 1618 h light/dark (L/D) regime and under darkness
-_-
DCW Podophyllotoxin Cl P,
Light
(gll) (mgll) &) (f:) Imsi(ld)l [m&L,-d)l
~_~__~~___
L/D regime 7.0 3.8 0.22 2.5 0.08 0.12 0.017
Dark 8.3 4.9 0.31 3.15 0.1 I 0.15 0.019
218 CHATTOPADHYAY ET AL.
0 IO 20 30
Time (d)
FIG. 3. Growth and podophyllotoxin profiles during the
tion of !! hexandrum under dark and 1618h light/dark (L/D)
DCW in the dark (open circles); DCW in L/D regime (solid
podophyllotoxin in the dark (open squares); podophyllotoxin
regime (solid squares).
cal metabolic reactions, as the sugar is already supplied
along with other nutrients in the culture media. Light has
been an important factor in secondary metabolism both in
intact plants and in suspension cultures. In non-photosyn-
thetic cultures, light intensity and duration can induce the
production of enzymes required for secondary metabolism.
The effect of light is also associated with medium composi-
tion. The effect of light on the regulation of shikonin pro-
duction by L. erythvorhizon cell cultures has been studied,
where the dark-grown cultures were reported to accumulate
an elevated level of acetylshikonin (27). The pigment con-
tent of L. erythrorhizon also increased linearly with time
after a lag phase when callus tissues were grown in the dark,
whereas it markedly decreased when irradiated with blue
light (28). Enhanced catharanthine (29) and taxol (30) pro-
duction during the cultivation of C. roseus and Taxus cuspi-
data in the dark was reported. In the suspension culture of
C. roseus grown in the dark, only ajmalicine synthesis was
induced whereas the syntheses of serpentine, phenolics and
anthocyanins were repressed (31). Products normally syn-
thesized in photosynthetic cells because of more favorable
thermodynamic conditions for synthesis are expected to be
absent in non-photosynthetic cells lacking these conditions.
This point may be elucidated by the apparent inability of
heterotrophic cells of C. roseus to accumulate substantial
amounts of two major leaf alkaloids, vindoline and catha-
ranthine, while the alkaloids found in the roots of the plant
(serpentine and ajmalicine) accumulated to very high con-
centrations in heterotrophic cell cultures (32).
The production of podophyllotoxin by I? hexundrum in
the present study was found to be inhibited by light. A 29%
increase in podophyllotoxin production was observed when
P hexandrum cells were cultivated in the dark. Both volu-
metric productivity and specific productivity were also en-
hanced by 25% and 12% respectively in the dark (Table 2).
Effect of hydrodynamic shear stress on I? hexandrum
cells cultivated in shake flask P hexandrum cells, like
many other plant species, were found to be sensitive to
hydrodynamic stress when cultivated in a shake flask on a
J. BIOKI. BIOENF.,
5 10 15 20 25 30
Time (d)
40
FIG. 4. Viability profile of P: hamdrum cells when cultivated in
shake flask at different rotational speeds. I25 rpm (open circles);
cultiva- I50 rpm (open squares); 200 rpm (open triangles); 250 rpm (solid cir-
regime. cles); 300 rpm (solid squares).
circles);
in LID
gyratory shaker. Hydrodynamic stress was generated by in-
creasing the rotational speed. The immediate consequence
of exposing the plant cells to higher rotational speeds was
the reduction of cell viability, which was quantitatively esti-
mated by the triphenyltetrazolium chloride method (19).
The viability profile of l? hexandrum cells cultivated on a
gyrator-y shaker at different rotational speeds is shown in
Fig. 4. It is clear that cell viability decreased with increas-
ing rotational speed. The cells grown at 125 and 150 rpm
showed comparatively less damage caused by the shear
stress. When the rotational speeds were increased to 200
rpm or more, the cell viability decreased significantly with
cultivation time.
The shear stress generated by the rotational speed on the
cells when cultivated in shake flasks could be quantified by
an empirical formula proposed by Sumino et al. (33). The
specific power input, P/V (kW/m’) can be expressed as
P/V=l.O9x 104V,-o.‘5xN2.8’ (1)
where VL is the volume of the liquid and N is the rotational
speed (rpm).
The energy dissipation rate, E, which can be used as a cor-
relating parameter for the system response, is related to the
specific power input (34) by the following relationship:
E= (P/q/p (2)
where p is the density of the liquid.
It was noted from Fig. 4 that not more than a maximum
hydrodynamic stress of 0.52 kWJkg (corresponding to 150
rpm on gyratory shaker) should be applied as the survival of
P hexandrum cells decreased significantly at higher stresses
in suspension cultures.
This concept was extrapolated to determine the most fa-
vorable impeller speed necessary to operate the bioreactor
for the cultivation of P hexandrum. A specially designed
low-shear impeller (Setric impeller) was used for the culti-
vation of F hexandrum in the bioreactor to reduce the beat-
ing effect to levels lower than those of other agitators. The
data available for a marine impeller were used due to the
non-availability of the correlation between Reynolds num-
ber and Power number for a Setric impeller. The Reynolds
vol.. 93, 2002 PRODUCTION OF PODOPHYLLOTOXlN IN BIOREACTOR 219

TABLE 3. Comparative values ofgrowth and production parameters in suspension cultures

of P: hexundrum in shake tlask and in bioreactor
DCW Podophyllotoxin Gil
Cultivation
(g/0 (m@) (gk) (31 [nl~~dil [m&&-d)1
Shake flask 8.3 4.9 0.3 I 3.15 0.11 0.019
Bioreactor 6.5 4.26 0.15 2.25 0.1 0.19 0.03

number (N,c=NiD,‘p/p) was calculated to be in the range of
5200 to 7900, which corresponded to rotational speeds of 80
to 120 rpm. The corresponding values of Power number
(N,,=PlpNi’D,‘) were found to be in the range of 0.55-0.6
from the correlation between N,, and NKe (35). The energy
dissipation rate was calculated as:
E=PI~V=N,N,~D,‘IV
where N, is the rotational speed (s-l), D, is impeller diameter
(m), N,, is the Power number for marine impeller, and V is
the working volume (1).
The suitable impeller speed for the cultivation of p hex-
andrum in the bioreactor calculated by equating 1 and 3 was
found to be in the range of 64-l 27 rpm (which corresponds
to the shear stress of 0.32-0.52 kW/kg). An impeller speed
of 100 rpm was therefore selected for adequate mixing and
aeration of the culture broth. This impeller speed was fur-
ther experimentally verified to ensure the maximum viabil-
ity of p hexandrum cells.
Cultivation of R hexandrum in 3 1 bioreactor After
the requirements of sugar, light and shear sensitivity were
established, P. hexundrztm was cultivated in a 3 I stirred tank
bioreactor. The time courses of growth, podophyllotoxin
production and consumption of sugar are shown in Fig 5.
The maximum biomass of 6.5 g/l (DCW basis) was ob-
tained on day 22 with an average growth rate of 0.1 d ’ and
the highest concentration of podophyllotoxin obtained was
4.26 mgil. The growth yield was 0.15 gig. It was also ob-
served that the production of podophyllotoxin followed a
combined growth-associated and non-growth associated ki-
netics during the cultivation of J! hexandrum in the bioreac-
tor. This was significant as a large number of plant second-
ary metabolites follow non-growth associated kinetics (30).
The results of a comparative study of the different pa-
rameters for growth and product formation of p hexandrum
in shake flask and bioreactor are presented in Table 3. It
was found that maximum biomass and podophyllotoxin ac-
cumulation were higher in shake flask than in bioreactor.
However, since the cultivation time of p hexandrum was
(3) significantly reduced from 32 d to 22 d in bioreactor, both
volumetric and specific productivities of podophyllotoxin,
which are the parameters of interest, were enhanced by 27%
and 58%, respectively, compared to those of the shake flask
cultivation. Moreover, in contrast to shake flask cultivation,
the bioreactor has the ability to process large volumes of
culture. During scale-up of several plant cell cultures, a
somewhat reduced product formation has often been re-
ported compared to that of shake flask cultivation (36, 37).
Reduced levels of ajmalicine and catharanthine were re-
ported during the cultivation of C. roseus in bioreactor, al-
though the volumetric productivity was significantly higher
(29).
Conclusion The growth of and podophyllotoxin pro-
duction by F! hexandvum were found to be better with glu-
cose than with sucrose as the medium carbon source. Both
growth and podophyllotoxin production were found to be
light-independent during the cultivation of p hexandrum in
submerged culture. At agitation speeds of up to 150 t-pm, the
viability of the cells was higher than 85% in shake flask cul-
tivation. An agitation speed of 100 rpm was found to be
sufficient to tnix the culture broth in the bioreactor without
causing any significant cell damage. The cultivation of I?
hexundrum in a 3 1 stirred tank bioreactor resulted in bio-
mass accumulation of 6.5 g/l (DCW basis) and podophyllo-
toxin formation of 4.26 mgll, which represented a 27% in-
crease in volumetric productivity compared to that of shake
flask cultivation. Contrary to many secondary metabolites,
podophyllotoxin production was a combined growth-associ-
ated and non-growth associated process in I? hexandrum
suspension cultures.

REFERENCES
Sajc, L., Grbisic, D., and Novakovic, G. V.: Bioreactors for
plant engineering: an outlook for further research. Biochem.
Eng. J., 4, 89999 (2000).
Panda, A. K., Mishra, S., Bisaria, V. S., and Bhojwani,
S. S.: Plant cell reactors - a perspective. Enz. Microbial
0 Technol., II, 386-397 (1989).
0 0 I? IS 24 30 Panda, A. K., Mishra, S., and Bisaria, Y S.: Alkaloid pro-
Time (d) duction by plant cell suspension cultures of Holarrhena anti-
dysentericu: I. Effect of major nutrients. Biotechnol. Bioeng.,
FIG. 5. Growth, podophyllotoxin production and glucose con- 39, 1043-1051 (1992).
sumption during cultivation of’ P hrxunu’rum in 3 1 bioreactor. DCW Panda, A. K., Bisaria, V. S., and Mishra S.: Alkaloid pro-
(open circles); podophyllotoxin (open squares); residual glucose (open duction by plant cell cultures of Holarrhena antidysenterica:
triangles). II. Effect of precursor feeding and cultivation on stirred tank
220 CHATTOPADHYAY ET AL.
bioreactor. Biotechnol. Bioeng., 39, 1052-1057 (1992).
5. Endo, T., Goodbody, A., and Misawa, M.: Alkaloid produc-
tion in root and shoot cultures of Cathavanthus rosez~s. Planta
Med., 53,479-482 (1987).
6. Son, S. H., Choi, S. M., Lee, Y. H., Choi, K. B., Yun, S. R.,
Kim, J. K., Park, H. J., Kwon, 0. W., Noh, E. W., Seon,
J. H., and Park, Y. J.: Large-scale growth and taxane pro-
duction in cell cultures of Taxus cuspid&~ (Japanese Yew)
using a novel bioreactor. Plant Cell Rep., 19,628-638 (2000).
7. Fujita, Y., Hara, Y., Ogino, T., and Suga, C.: Production of
shikonin derivatives by cell suspension cultures of Litho-
spermum erythrorhizon. I. Effects of nitrogen sources on the
production of shikonin derivatives. Plant Cell Rep., 1, 59-60
(1981).
8. Furuya, T., Yoshikawa, T., Orihara, Y., and Oda, H.:
Studies of the culture conditions for Panux ginseng cells in jar
fermentors. J. Nat. Prod., 47, 70-75 (1984).
9. Gupta, R. and Sethi, K. L.: Conservation of medicinal plant
resources in the Himalayan region, p. 101-107. In Jain, S. K.
and Mehra, K. L. (ed.), Conservation of tropical plant re-
sources. Botanical Survey of India, Howrah (1983).
10. Jackson, D. E. and Dewick, P.M.: Arltetralin lignans from
Podophyllum hexandrum and Podophyllum peltatum. Phyto-
them., 23, 1147-l 152 (1984).
11. Canel, C., Moraes, R. M., Dayan, F. E., and Ferreira, D.:
Molecules of interest podophyllotoxin. Phytochem., 54, 115-
120 (2000).
12. Holthuis, J. J. M.: Etoposide and teniposide. Bioanalysis,
metabolism and clinical pharmacokinetics. Pharm. Weekbl.
Sci., 10, 101-116 (1988).
13. van Uden, W., Pras, N., Visser, J. F., and Malingre, T. M.:
Detection and identification of podophyllotoxin production
by cell cultures derived from Podophyllum hexandrum Royle.
Plant Cell Rep., 8, 165-l 68 (I 989).
14. Kadkade, P. G.: Growth and podophyllotoxin production in
callus tissues of Podophyllum peltatum. Plant Sci. Lett., 25,
107-115 (1982).
15. Heyenga, A. G., Lucas, J. A., and Dewick, P. M.: Produc-
tion of tumour-inhibitory lignans by callus cultures of Podo-
phyllum hexandrum. Plant Cell Rep., 9, 382-385 (1990).
16. van Uden, W., Pras, N., and Malingre, T. M.: On the im-
provement of the podophyllotoxin production by phenylpro-
panoid precursor feeding to the cell cultures of Podophyllum
hexandrum Royle. Plant Cell Tissue Organ Cult., 23, 217-
224 (1990).
17. Chattopadhyay, S., Srivastava, A. K., Bhojwani, S. S., and
Bisaria, V. S.: Development of suspension culture of Podo-
phyllum hexandrum for the production of podophyllotoxin.
Biotechnol. Lett., 23,2063-2066 (2001).
18. Murashige, T. and Skoog, F.: A revised medium for rapid
growth and bioassay with tobacco tissue cultures. Physiol.
Plant, 15,473-497 (I 962).
19. Towill, L. and Mazur, P.: Studies on the reduction of 2,3,5-
triphenyltetrazolium chloride as a viability assay for plant tis-
sue cultures. Can. J. Bot., 53, 1097-l 102 (1975).
20. Miller, G. M.: Use of dinitrosalicylic acid reagent for de-
termination of reducing sugar. Anal. Chem., 31, 426-428
(1959).
21. Dubois, M., Gilles, K. A., Hamilton, J. K., Roberts, P. A.,
J. BIOSCI. BIOENG.,
and Smith, F.: Calorimetric method for determination of
sugars and related substances. Anal. Chem., 28, 350-356
(1956).
22. Scragg, A. H.: The problems associated with high biomass
levels in plant cell suspensions. Plant Cell Tissue Organ Cult.,
43, 163-170 (1995).
23. Martinez, B.C. and Park, C. H.: Characteristics of batch
suspension cultures of preconditioned Coleus blumei cells:
sucrose effect. Biotechnol. Prog., 9,97-100 (1993).
24. Knobloch, K. H. and Berlin, J.: Influence of medium com-
position on the formation of secondary metabolites in cell
suspension cultures of Cutharanthus roseus (L.) G. Don. Z.
Naturforsch., 35C, 551-556 (1980).
25. Schlatmann, J.E., Koolhaas, C.M. A., Vinke, J. L., ten
Hoopen, H. J. G., and Heijnen, J. J.: The role of glucose in
ajmalicine production by Catharunthus roseus cell cultures.
Biotechnol. Bioeng., 47, 525-534 (1995).
26. Mizukami, H., Konoshima, M., and Tabata, M.: Effect of
nutritional factors on shikonin derivative formation in Litho-
spermum callus cultures. Phytochem., 16, 1183-l 186 (1977).
27. Gaisser, S. and Heide, L.: Inhibition and regulation of
shikonin biosynthesis in suspension cultures of Lithosper-
mum. Phytochem., 41, 1065-1072 (1996).
28. Tabata, M., Mizukami, M., Hiraoka, N., and Konoshima,
M.: Pigment formation in callus cultures of Lithospermum
erythrorhizon. Phytochem., 13,927-932 (1974).
29. Zhao, J., Zhu, W. H., and Hu, Q.: Enhanced catharanthine
production in Catharanthus roseus cell cultures by combined
treatment in shake flasks and bioreactors. Enz. Microbial
Technol., 28,673-681 (2001).
30. Pestchanker, L. J., Roberts, SC., and Shuler, M.L.:
Kinestics of taxol production and nutrient use in suspension
cultures of Taxus cuspidata in shake flasks and a Wilson-type
bioreactor. Em Microbial Technol., 19, 256-260 (1996).
31. Knobloch, K. H., Bast, G., and Berlin, J.: Medium- and
light-induced formation of serpentine and anthocyanins in
cell suspension cultures of Cutharunthus roseus. Phytochem.,
21,591-594 (1982).
32. Stafford, A., Morris, P., and Fowler, M. W.: Plant cell bio-
technology: a perspective. Enz. Microbial Technol., 8, 578-
586 (1986).
33. Sumino, Y., Akiyama, S., and Fukuda, H.: Performance of
the shaking flask (I) power consumption. J. Ferment. Tech-
nol., 50,203-208 (I 972).
34. Kieran, P.M., Malone, D.M., and MacLoughlin, P.F.:
Effect of hydrodynamic and interfacial forces on plant cell
systems, p. 139-177. In Scheper, T., Schugerl, K., and
Kretzmer, G. (ed.), Adv. Biochem. Eng./ Biotechnol., vol. 67.
Springer-Verlag, Berlin (2000).
35. Doran, P. M.: Bioprocess Engineering Principles, p. 150.
Academic Press, London (1995).
36. Schlatmann, J. E., Nuutila, A.M., van Gulik, W. M., ten
Hoopen, H. J. G., Verpoorte, R., and Heijnen, J. J.: Scale-
up of ajmalicine production by plant cell cultures of Catha-
ranthus roseus. Biotechnol. Bioeng., 41,253-262 (1993).
37. Scragg, A. H., Morris, P., Allan, E. J., Bond, P., and
Fowier, M. W.: Effect of scale-up on serpentine formation
by Cutharanthus roseus suspension cultures. Enz. Microbial
Technol., 9, 619-624 (1987).