CHAPTER 4

BIOTECHNOLOGICAL PRODUCTION OF CAMPTOTHECIN

- A FEASIBLE APPROACH

4.1. INTRODUCTION

The chemodiversity found in plants is a rich source for new economically

important compounds and products. Currently the pharmaceutical companies are

extensively screening plants for new leads for drug development, using high

through put screening, which allows the testing of thousands of samples in a day.

This will certainly result in a series of new plant derived drugs, like previously taxol

and CPT resulted from much sophisticated screenings. Most of these novel drugs

will be derived from wild plants, some d which might be rare, difficult to culture or

with Low levels of the active compound. To ensure production, at least during the

first phase of drug development, plant biotechnology is a promising option to

produce the compounds on kilogram scale. In later stage commercial production

can be envisaged either by cultivated plants, possiblly improved through modem

plant biotechnological approaches, or by means of plant cell cultures (Verpoorte et

al, 1998).

CPT yielded after extraction from plants vary widely, and depend on many

factors difficult to control. In general pests, climate or political instability in the

region where the plants were grown can endanger production in nature. Therefore

the biotechnological production of CPT may be an attractive alternative (Van Hengel

et al, 1992). Of various plant products produced by plant tissue culture,

pharmaceuticals have received maximum attention. This chapter deals with in vitro

production of CPT adopting different culture systems.

4.2. MATERIALS AND METHODS

4.2.1. Source of Explants

The plants of O.rugosa var. decumbens were colleted and maintained in the

green house of Amala Cancer Hospital & Research Centre. To minimize the rate of

contamination in in vztro cultures, prophylactic sprays were given to source plants

and Bavistin and Ekalux 0.1% at weekly intervals. Leaf, stem and petiole explants

were exised from the mother plant.

4.2.2. Surf ace sterilization Surface sterilization of the explants was done as per the procedure described

in materials and methods (Section 2.2.1). Surface sterilized (intermode, 1.5 - 2 cm,

leaf 0.5 - lcm) was inoculated into the media with different concentrations and

combinations of growth hormomes.

4.2.3. Effect of auxins on callusing, shizhogenesis and rhizogenesis

Leaf, stem, internode and petiole were cultured in MS base1 medium

supplimented with auxins such as 2,4-D, NAA and IAA to study their relative

effects on callus induction and growth. Isolation of CPT from callus was

standardized (Fig.4. la)

4.2.4. Effect of Cytokinins and Giberrelic acid on callusing,

shizogenesis and rhizogenesis

Different cytokinins such as BA and KN were incorporated singly or in

combination to different mediums like MS, Bs, Woody plant medium (WPM) and

Whites medium. The responses were close monitered in every 4'h day and different

parameters like, callux index, no. of shoot initiation, length of shoots, number of

roots, root indux, % of root initiation and mean root length and growth index were

recorded.

Growth index = Harvest inoculum/inoculum dry weight

4.2.5. Regulation of in vitro metabolite production

4.2.5.1. Standardization of production medium.

Different explants were incubated on different basal medium and different

64

Fig 4.1 a Camptothecin extraction from callus of 0. rugosa var. decumbens

Callus-freeze dried, ground and sonicated in MeOH

Stirred nv~rnioht

Discarded Evapo ated to dryness f Suspended in distilled water

v Extracted with CHC13 thrice

evaporated to dryness

I f

Dissolyed in MeOH and kept at 40C

Pale yellow crystals

7 Purified by preparative TLC (CHCb : EtOH 24 : 1 v/v)

I

I Residue

f UV, HPLC, IR, ESMS and NMR

hormones and best medium with suitabIe combinations were selected for the *

synthesis of CPT.

4.2.6. Modifying the production Medium

4.2.6.1. Regulation of growth regulator

The production medium was modified by addition; deletion or using various

combinations of growth regulators and CPT production was assessed in weekly

intervals.

4.2.6.2. Modification of carbon source

Production and yield can be increased by manipulation of carbon source. The

carbon source selected in the study was sucrose. Different concentrations of sucrose

like, 10,20,30,40 and 50g 1 .

4.2.6.3. Regulation of photoperiod and pH

The cultures were incubated for 8-16h dark/light photoperiod and monitor

the change in photoperiod affect growth index of the calli. pH values recommended

for plant cell culture media are usually between 5 and 6. The change in pH will

affect the production.

4.2.6.4. Morphological differentiation and production

The formation of CPT in tissue culture was shown inseparably connected

with morphological differentiation of the cells. Morphological differentiation was

induced by changing the concentration of growth regulators singly or in

combinations. The differentiated organoids were extracted with chloroform for

subsequent development into chromatograms for alkaloids screening.

4.2.6.5. Elicitation for CPT .-

The multiple shoot cultures (8 weeks old) were divided into two groups.

Group one was irradiated (10 rads) and group II was given 50 rads using Cobalt-60

Teletherapy unit (Theratron 780, Atomic Enegry, Canada). 24h after irradiation all

65

the cultures were harvested, lyophilized, powdered and extracted with chloroform

and CPT yield was quantified using HPLC.

4.2.7. Induction of hairy roots

Agrobacterium rhizogenes strains like A4,15834 were used for the infection into

stems, leaf explants cultured in agar medium containing 1% sucrose and half

strength rnurashige and skoog salts, subcultured until bacterial infection is stopped.

The hairy roots emerged 80 days after injection, and these were cultured on agar

plates containing 2% sucrose at 25'r.

4.2.8. Establishing suspension cultures

Suspension cultures were established from different explants like leaf, roots

and calli of varying fresh weight (0.5 to 1.0 g) in 50 mI of medium. Erlenmayer

flasks containing media incubated at 120 rpm at 25 * 2 O C with a 16h photoperiod.

Subculturing intervals and cell density was done as described previously in

materials and methods (2.2.7; 2.2.8). In regular intervals crtitical cell density of cell

was monitered (Fig. 4.1), another parameter studied was packed cell volume of the

cells (tiny clumbs) (Fig. 4.2).

4.3. RESULTS AND DISCUSSION

Significantly varying response to basal media employed with respect to

percentage of leaf and stem cultures initiating calli were observed. Highest mean

percentage of cultures initiated calli in different mediums (MS, Bs, and Whites) and

its half strengths were studied. It was well established that MS medium was apt for

callus production along with hormonal combination of auxins (NAA 0.1 to 4) and

cytokinins (BA 0.05 to 0.5). Inhibition of callusing was resulted when activated

charcol was supplemented to medium. It was noted that pH of the medium*plays an

important role in camptothecin production. The production was found maximum at

a pH 4.2 (Fig. 4.3), and it was also interested to note that the amount of dry weight is

directly proportional to CPT yield (Fig. 4.4).

Duration (Days) Fig. 4.1. Standardization of critical cell density and subculture intervals for suspension cultures of 0. rugma vm. &mmhs in Murashige and Skoog medium.

B5 medium c 1 1 I I I I b

0 2 4 6 8 10 12 14 Weeks

Fig. 42. Comparison of psbcked dl volume (pcu) in B5 and MS medium.

600 -

500 - M 6 400 - - -6

-

-

-

I I L I t I

0 2 4 6 8 10 12

weeks Fig. 4.4. A comaparison of increase in dry weight and camptoheck production

7

6

5

Ir . a 3

2

1

7 r 1 0 ' ~ -

Period of incubation (Weeks)

Fig.93 Influence of pH and campbthetin production

6 x 1 0 ~ ~

-

- -

-

5 a 4 x ~ o - 2 - * - 3 x 1 W 2

2 ~ 1 0 - ~

1 x 1 0 - ~

- -

- -

- -

I 1 I I I :

14 12 10 8 6 4 2 0

Shizogenesis: Growth hormones used for shoot multiplication displayed significant

variations in stem derived calli for the percentage of cultures initiating shoot buds,

earliness in shoot induction, number and shoots initiated and length of shoots leaf

explants are found best explant for shoot multiplication. Hormonal combinations of

NAA and BA at 0.05 - 0.01 to 0.5 - 5 mg 1 respectively ranked superior in

initiating shoots in maximum cultures (Table.4.1; 4.2) other ,cytokinins such as KN

was not found so effective. Higher dose of NAA and KN were found deleterious to

the regenerative capacity of in vitro cultures when applied either singly or in

combination (Table. 4.3; Plate No.2).

Rhizogenesis: The regeneration pattern of roots from calli initiated from leaf

explants by effective hormonal combinations are given in the (table. 4.2). The result

of the study reveals various root inducing hormones and differences in their ability

to regenerate roots from leaf explants (Plate No.3). In vitro multiple shoot cultures

and root cultures are the better sources of CPT . Multiple shoot cultures in solid

medium showed a CPT yield of 0.039% in a 45 day old culture with a hormonal

combination of NAA 0.1 mgl-l and BA 4 mgl - and IBA 2 mgl yield a CPT content

of 0.03% in Murashige and Skoog (MS) medium. On the same time shoot suspension

cultures were found as a viable source for CPT. Half strength medium (MS) with a

hormonal combination of BA 5mgl yield 0.085% of CPT on a dry weight basis. But

full strength MS medium with same concentration of BA yield 0.099% of CPT.

4.3.1. Transgenic Roots

Transgenic roots were initiated using (Agrobacferiurn rhizogenes strain) ATCC

15834 from leaf explants were found promising source of CPT. Hairy roots were

initiated at a day of 84. Rapidly growing hairy root lines have selected and

subcultured on to agar medium without any hormones. Hairy roots were confirmed

by morphological evaluation and by TLC with standard agropine. The selected high

yielding clones were cultured in medium for better growth (Plate No.4). The CPT

that accumulated in hairy roots was easily detected and quantified by its

67

Table 4.1. Effect of arowth regulators on multiple shoot production

Values were expressed as mean * SD uf 10 explants

Table 4.2.

Effect of YI & % MS medium witb dlfbrwt hormonal comblrutlohs on mulple shoot devdopment in Ophiorrhiza rum var. ckurnbens

'

Mean No. of muttipte shoots

23 * 1.45

69 5.41

63 * 4.13

08 & 4.66

32 4.14

36 * 1.21

29 4.11

12 i 1.13

39 i 7.61

37 2 4.8

24 2.74

28 3.14

33 * 4-49

17 1.34

Strengthof Ms Medium

'/2

'A

55

%

I !

'A

'42

%

%

%

'!h

Y4

'/r

I/r

GA3

-

-

- -

2

4

5

- - -

2

4

5

W

0.5

0.5

0.5

-

- - -

0.5

0.5

0.5

- - - -

Hwmones

W

2

4

5

5

-

1

-

2

4

5

5

- - -

Table 4.3. E M of NIIA, BA and KN on rooting of the mg~erPted shouts in 60 day OM cuDturs in

full strength MS medium

Values wre expressed as mean * SD uf 10 explants

Growth Regdatm

NAA

0.05

.O.W

0.9

0.1

1.5

2

4

1

2

2

4

5

Frecpnq af

9

13

20

20

40

70

40

40

50

80

60

40

no. mots* SD

8 * 2

7 k 2

I I

13i 2

28 * 4.78

90 * 14.1

29 * 9.8

37 2 8.6

98 * 7.4

1 12.1

78 * 8.5

f l * 0.86

B A ! # N

- -

-

- -

- -

0.01

0.01

0.05

0.05

0.05

~ o o t index

3.6* 0.5

2.44k 0.24

2.88* 0.42

2.88* 0.32

4.8 0.22

Q * 0.96

5.76 * 0.66

6.68 k 0.76

8.4 * 0.5

1 1 . 2 0.93

8.04 * 0.44

7.88* 0.58

LmgVl Of root * SD(m)

3 0.1

I 0.21

1 0.11

8 0.12

1-96 % 0.14

2.2 * 91

2.3 k 1.2

1.89 * 0.24

2.01 * 0.61

2.3* 0.47

2.2 * 0.31

2 . 0.08

- - -

0.01

0.05

0.05

0.05

-

-

- - -

~ r e ~ h W. (g)

0.09* 0.2

0.612 0.1

o.n* 0.05

0.72k 0.2

1 * 0.4

2.28 * 0.3

1.44 * 0.7

1.67 * 0.6

2 0.3

2.8* 0.7

2.01 * 0.5

1 9 0.4

Plate No.2. A. Emergence of multiple shoot from leaf explants (MS + BA 4 + N AA 0.5)

B. Stereomicroscopic view of the multiple shoot

C , D & E- Different stages of growth of multiple shoots with in oitro Flowering.

F- Hardening of the plantlets

G- A comparison of the root system of tissue cultured plant (Right) and field grown plant (Left).

Plate No. 2

Plate No.3. A- In 7~ituo grown callus from 0.rugosa var. decurnbens (MS+ NAA 2 mg 1-1 ).

B & C- Root initiating cell clumbs (Stereomicroscopic view)

D- Suspension root cultures from leaf explants (MS+ NAA2+ BA 0.1)

E- Harvested suspension roots cultures regenerated from roots grown in solid medium

Plate No. 3 7 -c3m

Plate N0.4. - Agrobacerium mediated hairy root induction; A, Control petri dish with our Agrobacterium (Left) ; Transformed culture (Right)

B & C- Tral~sformed roots from 0, rugosa var decumbens.

D,E & F- Selection of different clones for better production.

Plate No. 4

characteristic fluorescence in HPLC analysis. During 5-month culture period the

CPT content was found stable.

Among all auxins used, NAA was found promising in inducing multiple

shoot and roots. It was well documented that morphological differentiation was

found essential for secondary metabolite production. The efforts to determine the

exact mode and the causative factors involved in the synthesis of CPT in in vitro

cultures were determined. From the results it was clearly indicated that CPT was not

synthesized in undifferentiated cultures such as callus and cell suspension cultures.

The accumulation of CPT is increased when the callus differentiated into roots and

shoots. It is also well established that increased production of secondary metabolites

were noticed in stationary phase of the growth, which related to tissue organization

(Thorpe, 1978). From the results it was found that concentration of sucrose also

determine the yield of camptothein (Table. 4.4; Fig. 4.5), and from the results there is

also a relation can be postulated between photoperiod and biomass production

(Table. 4.5).

It was also found that elicitation for CPT is a good strategy for better

production. It was obtained that 10 and 50 rads of y radiation elicit the production of

CPT in 60 days old multiple shoot cultures. The irradiated cultures yield 0.085% CPT

while that of ordinary multiple shoot cultures yield 0.039% of CPT on a dry weight

basis (Table. 4.6). It was well documented that irradiation of cultures is an attractive

strategy for secondary metabolite production (Hirata et al., 1991; 1992).

Table 4.4. Influence ot Carbon Saurce on a1k;rtotd production in Ophionfrha mgosa var.

decumixm (90 day old cuttures)

Wdium used

MS

B 5

W h i

Carbon Source

s u ~ r ~ ~

S u m

SUCTOS~

Sucrose

Sucrose

Sucrose

Sucrose

S u c ~ s e

S m

S m

Sucrose

S ~ c r 8 ~ e

s u w ~ ~ e

Sucrose

Sucrose

carbon used (g~-')

5

I 0

20

30

40

5

10

20

30

4

5

10

20

30

40

Fresh Weight (g)

4.5

7.2

10.9

9.7

6.4

3.9

6.1

Id.7

7. I

4.3

-

2.4

5.7

2- 1

-

Dry uut. (g)

0.29

0.41

1 .I

0.59

0.55

0.1

0.34

0.61

0.37

0.21

-

0.056

0.31

0.112

-

Table 4.5. Influence of photoperiod and biomass production in rnukiple shoot

cultures of 0.rugosa var. decumbms. (90 days culturn psriod)

The values are +SD of 10 replicates; pH of the medium : 5.6 to 5.8; temperature provided : 26 *lOc

Mediumused

MS

MS

MS

MS

B5

E35

B5

B5

Whites

Whites

Whites

Whites

Toetrd 8iorna35 FreshMNt.

3.8 0.61

6 . * 0.44

16.1 * 1.02

5.2 0.34

3.1 0.82

5.61 * 0.59

1 * 0.91

5.98 * 0.92

1.49 0.66

2.2 * 0.91

5.61 i 1.1

1.33 * 0.92

lightexposure(h)

24 h light

20 h light

16 h light

8 h light

24 h light

20 h tight

'l6 h light

8 h light

24 h light

20 h light

16 h light

8 h light

Suaosecon.(gt-1)

20

20

20

20

20

20

20

20

20

20

20

20

Table. 4.6. The O/o of campothccin present in tissue culturcs of 0. nlgosn var . dew rn ben s

Type of tissue

-

Entire Tissue

cultured plant

Callus

Callus

Multiple shoot

Multiple shoot

Multiple shoot

Mu1 tiple shoot (SC)

Multiple shoot (SC)

Multiple shoot (SC)

Root

Root (SC)

Root (SC)

Root (SC)

Hairy roots

Explant used

leaf

leaf

leaf

leaf

leaf

leaf

leaf

petiole

leaf

leaf

leaf

leaf

leaf

leaf

Hormonal

Combinations

NAA 0.5/ BA 2

NAA 1.5/BA 4

NAA 2/ BA 5

BA I/GA 3

BA 1/GA 3

NAA 0.1/ BA 4

BA 5

BA 5

BA 2

NAA 2

NAA 2

NAA 1/ BAl

IAA 2

** Cultured in l/2 MS medium

Sucrose concentration (g/ 1)

I- Full strength I- Half Strength I- 1 / 4th strength

-+-- B5 Medium

-A- Ms Medium

Fig.4.5. Influence of concentration on multiple shoot production in full, half and 1/4th strength MS medium (60 day old culture)