Chapter 8

Permeabilization of camptothecin and 10-hydroxy camptothecin

from Ophiorrhiza eriantha  

 

 

 

 

 

 

 

 

 

 

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.

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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