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TRANSCRIPT
Contents
8.1 Introduction
8.2 Materials and methods
8.2.1 Cell cultures
8.2.2 Adventitious root cultures
8.2.3 Permeabilization of cell cultures and root cultures
8.2.3.1 Permeabilization by DMSO
8.2.3.2 Permeabilization by Tween 20
8.2.4 Extraction of Camptothecin after permeabilization
8.2.5 HPLC analysis of camptothecin
8.3 Statistical analysis
8.4 Results
8.4.1. Permeabilization by DMSO
8.4.2. Permeabilization by Tween 20
8.5. Discussion
List of Figures
Fig. 8.1. Release of CPT from cells of O.eriatha by the addition of DMSO
Fig.8.2.Release of CPT from adventitious roots of O.eriatha by the
addition of DMSO
Fig. 8.3. Release of 10-HCPT from cells of O.eriatha by the addition of
DMSO
Fig.8.4. Release of 10-HCPT from adventitious roots of O.eriantha by the
addition of DMSO
Fig. 8. 5. Release of CPT from cell of O.eriantha by the addition of
Tween 20
Fig. 8.6. Release of CPT from adventitious roots of O.eriantha by the
addition of Tween 20
Fig. 8.7. Release of 10-HCPT from cell of O.eriantha by the addition of
Tween 20
Fig. 8.8. Release of 10-HCPT from adventitious roots of O.eriatha by the
addition of Tween 20
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8.1 Introduction
The use of plant cell cultures for the production of commercially useful
compounds from plants is of great interest in biotechnology and
pharmacology (Deus and Zenk 1982). Because secondary plant metabolites
are often stored within the vacuoles of plant cells, which make product
recovery and continuous operation of processes more difficult (Luckner 1990).
In order to release the products from vacuoles of plant cells, two membrane
barriers, the plasma membrane and tonoplast have to be penetrated. Cell
permeabilization depends on the formation of pores in one or more of the
membrane systems of the plant cell, enabling the passage of various
molecules into and out of the cell (Brodelius and Pedersen, 1993). Attempts
have been made to permeabilize the plant cells transiently, to maintain the
cell viability, and to have short time periods of increased mass transfer of
substrate and metabolites to and from the cell (Brodelius, 1988; Brodelius and
Nilsson, 1983; Dornenburge and Knorr, 1992; Felix, 1991; Park and Martinez,
1992; Parr et al., 1984; Sim et al., 1994).
Various substances have been used to initiate product release from
cultured plant cells (Felix et al., 1981; Felix 1982). These methods include
chemical treatments such as with solution of high ionic strength, change of
external pH, permeabilization with dimethylsulfoxide (DMSO) (Delmer 1979),
Tween 20, some mild detergents (Wagner & Matthysse 1992) and physical
treatments including high electric field pulses, ultrasonics, ultra-high pressure
(Dornenburg and Knorr, 1993, Knorr et al., 1985). Another strategy that has
been used to improve productivity is in situ removal of metabolites using
solid adsorbents which can provide a high concentration gradient of
metabolites across the cellular membranes (Asada and Shuler, 1989; Cormier
et al., 1992; Payne et al., 1988; Robin and Rhodes, 1986; Sim et al., 1994).
The objective of the present study is to permeabilize and product
removal to extract a high medicinal value secondary metabolite from plant
tissues. The release of camptothecin (CPT) and 10-hydroxy camptothecin (10-
HCPT), alkaloids with potential anticancer properties, from Ophiorrhiza
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eriantha cell and root cultures was evaluated for the present study.
Dimethylsulfoxide (DMSO) and Tween 20 were used as the permeabilization
agent.
8.2 Materials and methods
8.2.1 Cell cultures
Callus induction in O.eriantha was described in materials and methods section
2.2.5.6. Cell suspension cultures were derived from friable callus and
cultivated in gyratory shaker at 110 rpm and maintained at 250 C with 16-h
photo period.
8.2.2 Adventitious root cultures
Adventitious roots were induced from callus as described in materials and
methods section 2.2.5.10.
8.2.3 Permeabilization of cell cultures and root cultures
Two different permeabilizing agents like Dimethylsulfoxide (DMSO) and
Tween 20 were used for the present study. For the permeabilization
experiments cells in early stationary phase were used. Cells were collected by
filtration, an aliquote of cells (0.5 g fresh weight) were incubated with 10 ml of
medium containing appropriate amount of permeabilizing agent shaken on a
gyratory shaker (80 rpm) in a 25 ml Erlenmeyer flask.
Cells were incubated with different time intervals (15, 30, 45, and 60
min) and the cells were collected by centrifugation (500 xg for 2 min) and
washed with the appropriate medium.
Permeabilization agents were added to 60 day old subcultured
adventitious root cultures in MS basal media. After addition of
permeabilization agent the cultures were incubated for different time
intervals and roots were collected. CPT and 10-HCPT released into the
medium assessed by HPLC.
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8.2.3.1 Permeabilization by DMSO
For studies of permeabilization by DMSO 0.5g cells were incubated with 5 %
and 10% DMSO shaking in 10 ml medium.
8.2.3.2 Permeabilization by Tween 20
Tween 20 was directly added without sterilization with a sterile pipette. The
flask was vigorously shaken to ensure a good mixing of the medium. The
concentrations of Tween 20 were expressed as ml for 100 ml of culture
medium (%). The flask was incubated on a shaker (as described above) until
harvesting was performed. Each experiment was repeated three times.
8.2.4 Extraction of CPT and 10- HCPT after permeabilization
The excreted CPT and 10-HCPT in the medium could be absorbed with XAD-
7 resin and easily purified by elution with methanol. The CPT and 10-HCPT
concentration in the culture medium was measured with HPLC.
8.2.5 HPLC analysis of CPT and 10-HCPT
HPLC analysis of the methanolic extracts was carried out on a Shimadzu
SPD-10AVP HPLC system equipped with a multi solvent delivery system and
an UV-VIS detector. The column was a Purospher star column rp-18, end
capped, 5 μm, 250 x 4.60 mm (Merck, Germany).
The presences of CPT and 10-HCPT in the samples were detected by
comparing with the retention time of the standard (Sigma) samples. The
concentrations of CPT and 10-HCPT in samples were quantified by
integrating peak areas with known concentration of standards (Sigma). The
concentration of CPT and 10-HCPT were expressed in mg/g dry weight. The
analytical method was validated by testing for precision, accuracy, linearity,
limits of detection, limit of quantification using standard methods. For CPT
the mobile phase was composed of acetonitrile and water (60:40) and for 10-
HCPT acetonitrile-water (30:70) with isocratic elution. The flow rate was
1ml/minute nm and sample injection volume was 20l. For CPT detection UV
absorbance was at 256 nm and for 10-HCPT it was 266 nm.
122
8.3 Statistical analysis
All experiments were repeated thrice. The effects of different treatments were
quantified and the data’s were expressed in Mean ± Standard Deviation. The
data were analyzed by one-way ANOVA followed by Dunnet multiple
comparison test. p<0.05 taken as significant. The statistical calculation was
done using InStat GraphPad 3.00 software for Windows, San Diego,
California, USA.
8.4 Results
Various chemicals may be employed to make membranes permeabilization of
plant cell and permeable to different compounds into the extracellular
medium. In order to study the release of CPT and 10-HCPT into the medium,
the cells and adventitious roots of O.eriantha were premeabilized with DMSO
and Tween 20.
8.4.1. Permeabilization by DMSO
Cells permeabilized with 5% DMSO can release up to 0.0066 ± 0.0005 mg/ml
CPT into the medium whereas higher concentration of DMSO (10%) released
the almost same amount of CPT 0.0061 ± 0.008 mg/ml (Fig. 8.1). The release of
CPT from adventious root permeabilization with 5% of DMSO was 0.0284 ±
0.003 mg/ml (Fig. 8.2). The maximum accumulation of CPT was at 30 min
after the treatment with 5% DMSO.
The concentration of excreted 10-HCPT was highest (0.0047 ± 0.0006
mg/ml) in permeabilization with 5% DMSO treatment with cells (Fig. 8.3).
Adventitious roots reated with the same concentration of DMSO also released
maximum 10-HCPT (0.0136 ± 0.0008 mg/ml ) (Fig. 8.4).
8.4.2. Permeabilization by Tween 20
The release profiles of CPT in cells and adventitious roots permeabilized with
2 % of Tween 20 showed that singnificantly improve the release of CPT as
compared with 1% of Tween 20 at 45 min. By the addition of 2% or 1%Tween
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20, the concentration of extracellular CPT from cells increased to 0.00686 ±
0.009 or 0.0049 ± 0.009 mg/ml , respectively (Fig. 8.5). Highest amount of CPT
(0.0289 ± 0.002) released from root permeabilized with 2% Tween 20 (Fig. 8.6).
10-HCPT release from cells and adventitious roots were also studied
with Tween 20. Excreted 10-HCPT from cells were 0.0045 ± 0.0003 mg/ml
and 0.0033 ± 0.0005 by 2% and 1%Tween 20, respectively (Fig. 8.7). Fig. 8.8
demonstrate the release of 10-HCPT from adventitious roots with Tween 20
permeabilization. The result showed that maximum amount of 10-HCPT
(0.0133 ± 0.0007 mg/ml) from roots released with 2% of Tween 20 at 30 min.
After 45 min release of 10-HCPT was decreased.
8.5. Discussion
Plant secondary metabolites are generally located in intracellular
compartments, usually the vacuoles. Therefore, the production of many
metabolites is limited by the capacity of the vacuole(s). To enhance yields of
desired metabolites and reduce total process costs, several approaches for
permeabilizing the cells and thereby releasing products have been applied,
e.g., treatment with organic solvents (dimethyl sulfoxide; Sim et al., 1994) or
surfactants (Boitel-Conti et al,. 1996).
In the present study Dimethyl sulphoxide (DMSO) and Tween 20 has
been used to permeabilize cells and adventitious roots of O.eriantha in
suspension culture and promote the release of intracellular CPT and 10-
HCPT.
Relatively high DMSO concentrations were required to release
intracellularly stored quinoline alkaloids from cells of Cinchona ledgeriana
(Parr et al., 1984). Very limited release of alkaloids from cells of Berberis
stolonifera was observed after DMSO treatment (Rueffer 1985). Brodelius and
Nilsson (1983) reported that treatment of cells with low levels of DMSO may
fulfil the necessary requirement and the repeated cycles of product synthesis
and DMSO permeabilization allows long term harvesting of alkaloids from
Catharanthus roseus. Present study showed that cells and adventitious roots of
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O.eriantha permeabilized with DMSO and the low level of DMSO require for
the release of both CPT and 10-HCPT from roots and cells.
Datura innoxia hairy roots treated with Tween 20 led to the migration of
significant amounts of alkaloids from cells into the culture medium in a study
by Boitel-Conti et al., (1996). Furthermore, the cited authors observed that
total alkaloids production (intracellular plus extracellular) was 3- to 8-fold
higher in treated than in untreated roots. The effects of Tween 20 as
permeabilizing agent on release of CPT and 10-HCPT from O.eriantha cell and
adventitious root cultures have been studied. The 2% Tween 20 triggered
significant enchancement in the amount of CPT and 10-HCPT released into
the medium.
In conclusion, the results presented in this work clearly demonstrate
that it is possible to release CPT and 10-HCPT stored within cells and roots of
O.eriantha by the permeabilization procedure. Permeabilization may in the
future find application in the downstream processing of cultured plant cells.
Fig. 8.1. Release of CPT from cells of O.eriatha by the addition of DMSO
Values are expressed in Mean ± SD, ‘a’, p<0.01 and ‘b’, P<0.05 Fig. 8.2. Release of CPT from adventitious roots of O.eriantha by the addition of DMSO
Values are expressed in Mean ± SD, ‘a’, p<0.01 and ‘b’, P<0.05
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
15 min 30 min 45 min 60 min
5 % of DMSO 10 % of DMSO Control
Incubation time
Rel
ease
of
CP
T (m
g/m
l)
b
a
a
a
a
a
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
15 min 30 min 45 min 60 min
10% DMSO 5% DMS0 Control
Incubation time
Rel
ease
of
CP
T (m
g/m
l)
a
a
a
a
a
a