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Chapter 10 Identification of camptothecin
and 10-hydroxy camptothecin in Ophiorrhiza incarnata wild
plant and tissue cultures
Contents
11.1. Introduction
11.2. Materials and methods
11.3. Statistical analysis
11.4. Results
11.5. Discussion
List of tables
Table: 10.1.Amount of camptothecin and –Hydroxy camptothecin in O. incarnata
Table: 10.2. Effect of basal mediums on callus formation from leaf segments of O.
incarnata after 8 weeks of cultures and the amount of camptothecin and
10-Hydroxy camptothecin present in them
Table 10.3. Effect of plant growth regulators (NAA and BA) on callus formation
from leaf segments of O. incarnata after 8 weeks of cultures and the
amount of camptothecin and 10-Hydroxy camptothecin present in them.
Table 10.4. Effect of growth-regulators on shoot differentiation from leaf derived
callus of O. incarnata after 12 weeks of cultures and the amount of
camptothecin and 10-Hydroxy camptothecin present in them.
Table: 10.5. Influence of different auxins on rooting of in vitro formed shoots of
O.incarnata after 8 weeks culture and the amount of camptothecin and
10-Hydroxy camptothecin present in the rooted plant let.
List of Figures
Figure:10.1. Tissue cultures of O. incarnata
Figure:10.2. Chromatogram of standard Camptothecin (Sigma).
Figure:10.3. Chromatogram of camptothecin from whole plant extract
Figure:10.4. Chromatogram of camptothecin from leaf extract
Figure:10.5. Chromatogram of camptothecin from stem extract
Figure:10.6. Chromatogram of camptothecin from root extract
Figure:10.7. Chromatogram of camptothecin from NAA 4.0/ BA 0.5 mg/Lcallus
cultures
Figure:10.8. Chromatogram of camptothecin from BA 4.0 mg/L shoot cultures
Figure:10.9. Chromatogram of camptothecin from NAA 4.0 mg/L rooted plantlet
cultures
Figure:10.10. Chromatogram of camptothecin from IBA 4.0 mg/L rooted plantlet
cultures
Figure: 10.11. Chromatogram of standard 10-Hydroxy camptothecin (Sigma).
Figure: 10.12. Chromatogram of 10-Hydroxy camptothecin from whole plant extract
Figure: 10.13. Chromatogram of 10-Hydroxy camptothecin from leaf extract
Figure: 10.14. Chromatogram of 10-Hydroxy camptothecin from stem extract
Figure: 10.15. Chromatogram of 10-Hydroxy camptothecin from root extract
Figure: 10.16. Chromatogram of 10-Hydroxy camptothecin from NAA 4.0/ BA 0.5
mg/L callus cultures
Figure: 10.17. Chromatogram of 10-Hydroxy camptothecin from BA 5.0 mg/L shoot
cultures
Figure: 10.18. Chromatogram of 10-Hydroxy camptothecin from IBA 3.0 mg/L
rooted plantlet cultures
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10.1. Introduction
Ophiorrhiza incarnata (Family: Rubeaceae) is herbaceous plant distributed in
southern Western Ghats, in kerala. The genus Ophiorrhiza has got several species of
which O. rugosa (Vineesh et al,, 2007), O. eriantha (Jaimsha et al,, 2010), O. mungos
(Tafur et al,, 1976) has been reported for the presence of an anticancer drug
camptothecin (Wall and Wani, 1998) and various other secondary metabolites with
wide pharmacological activities. The identification of camptothecin and their
derivatives has not yet been done in O. incarnata. Presently the identification of
camptothecin and 10-hydroxy camptothecin is done by HPLC. For analytical purpose
the HPLC is being the most widely used. HPLC results are considered to be accurate
and can be used for quantitative determination of substituents in complex mixtures.
The secondary metabolites are present in very low quatinty in plants and also O.
incarnata is endemic to Western Ghats. If this plant is ulillized for the production of
camptothein can cause the elimination of this native plant from biodiversity. The plant
tissue culture technique plays an important role in the preservation and
micropropagation of germplasm that is endangered or on the brink of extinction and
also for commercial propagation. And also a major tool in the production of high
value secondary metabolites. In vitro regenerated cultures and plants have been used
successfully for mass propagation (Bouman and De Klerk, 2001) and high quality
plant based medicines (Murch et al., 2000).
The present study is aimed to establish tissue cultures of O. incarnate, and
also to identify and quantitate camptothecin and 10-hydroxy camptothecin in O.
incarnata wild and tissue cultures using HPLC system.
10.2. Materials and Methods
10.2.1. Plant material
O. incarnata is herbaceous plant collected from Wayanad district, kerala.
10.2.2. Quantification of Camptothecin and 10-hydroxy camptothecin in O.
incarnata plant parts
The collected plants were washed and separated in to leaves, stem and roots. These
parts were dried and extracted with chloroform. The extract was evaporated and dried
extract is used for HPLC analysis (explained in 2.2.25 of chapter 2).
157
10.2.3. Establishment of tissue cultures in O. incarnata
10.2.3.1. Establishment of callus cultures
Leaf explants were collected (explained in of chapter 2) and cultured in MS medium
supplemented with various hormones. The cultures were kept in dark and were
checked on every day for 1 month. Those cultures having contamination are removed
and the callus generated explants were sub cultured into fresh medium with same
hormonal combination.
10.2.3.2. Establishment of shoot cultures
To induce shoot regeneration, well-established compact calluses (~500 mg fresh
weight) were transferred to MS basal medium supplemented with different
combinations of plant growth regulators BA (0.5 – 5 mg/l) alone or in combination
with NAA (3 – 4 mg/l). The number of shoot-buds induced on 500 mg of calluses was
counted after 12 weeks. The percentage of callus induction was calculated using the
formula
Induction % = (No. of calli producing adventitious buds/No. of calli inoculated)
x100%.
10.2.3.3. Establishment of root cultures
For root induction in vitro differentiated elongated shoots were excised from culture
grown on MS medium supplemented with BA 5 mg/l. The excised shoots were
cultured on four concentrations of NAA (1 - 5 mg/l), IBA (1 - 5 mg/l) and IAA (1 - 5
mg/l). Twelve shoots were used per treatment with three replications. Data were
recorded on percentage of rooting and root number after 8 weeks on rooting media.
The percentage of root induction was calculated using the formula
Induction % = (No. of root produced/No. of shoots inoculated) x100%.
10.2.4. Quantification of camptothecin and 10-hydroxy camptothecin in O.
incarnata tissue cultures.
The lyophilized in vitro cultured plantlets and calluses were powdered, and were
subjected to extraction with methanol. The methanol extract was used for HPLC
analysis (explained in 2.2.24 of chapter 2).
158
10.4. Results
10.4.1. The HPLC analysis
The camptothecin content analysed by HPLC in O. incarnata revealed 0.154 ± 0.001
mg/g dry weight in whole plant (Figure 10.3), 0.050 ± 0.002 mg/g dry weight in
leaves (Figure 10.4), 0.007 ± 0.001mg/g dry weight in stem (Figure 10.5) and 0.287
± 0.003 mg/g dry weight in roots (Figure 10.6). The HPLC spectrum at 256 nm
produced peak for camptothecin with a retention time of 3.5 for both the standard and
plant samples. On the other hand the 10-hydroxy camptothecin was found to be 0.015
± 0.002 mg/g dry weight in whole plant (Figure 10.12), 0.003 ± 0.002 mg/g dry
weight in leaves (Figure 10.13), 0.001 ± 0.001 mg/g dry weight in stem (Figure
10.14) and 0.018 ± 0.001 mg/g dry weight in roots (Figure 10.15). The HPLC
spectrum at 266 nm produced peak for 10-hydroxy camptothecin with a retention time
of 5.9 minute for both the standard and plant samples (Table 10.1).
10.4.2. Callus induction
In O. incarnata, callus formation varied significantly depending on the basal medium
medium and hormones supplied. MS basal medium supplemented with NAA 4.0/BA
1.0 mg/L showed the earliest signs of callus formation after 3 weeks of culture, but
explants cultured in MS medium supplemented with other hormones in combination
or alone started to initiate callus after 4 - 5 weeks of culture. When cultured in MS,
Whites and Gamborg’s basal medium without hormones, the highest callus induction
(27.77%) was achieved in MS medium was used in comparison with on 11.11 %
Gamborg’s medium, and no callus induction with Whites medium (Table 10.2). With
NAA4.0/BA1.0 mg/l showed 100 % callus formation after 8 weeks. However, the
other hormonal combinations exhibited callus formation which was significantly
lower than NAA4.0/BA1.0 mg/l (Table 10.3, Figure 10.1)
10.4.3. Shoot regeneration
Proliferated compact calli were transferred to MS medium supplemented with
different BA and NAA concentrations under light conditions to investigate their
potential for shoot regeneration. After 5 weeks of culture, most of the calli started to
turn to light green, and they gradually became dark green in the following week of
culture. BA alone could induce shoot regeneration at the rate of 22.32 ± 3.58 with
159
100 % when cultured on medium with 4.0 mg/l BA; but the highest BA concentration
at 5.0 mg/l appeared to show a suppressive effect on shoot differentiation and
produced only 19.35 ± 3.27 shoot. Shoot regeneration was found in combinations of
BA and NAA compared with the use of BA alone but it was lower than that of BA
alone. The shoot differentiation rate was also obtained on the medium containing BA
and NAA that produced 17.65 ± 4.25 shoots per callus with 83.33% of shoot
formation. The addition of kinetin, the callus generated shoots but with low shoot
formation frequency (Table 10.4, Figure 10.1).
10.4.4. Root formation
Root formation was achieved by culturing with different auxins (NAA, IBA and IAA)
at concentrations ranging from 0.5 - 5 mg/L. Root formation increased with higher
IBA concentrations (3.0 mg/L) with 8.69 ± 2.64 roots per shoot. Although with
NAA and IAA root formation was observed but this was with low frequency (Table
10.5, Figure 10.1).
10.4.5. Camptothecin (CPT) and 10-hydroxy camptothecin (HCPT) content in O.
incarnata callus cultures
To evaluate the amount of CPT in callus cultures the cultures thus obtained in
different hormonal combination and basal medias were subjected for HPLC analysis.
The callus obtained in hormonal combination of NAA (4.0 mg/l) and BA (0.5 mg/l)
showed more amount of CPT (Figure 10.7) than in NAA and BA alone treated and
their other combinations (Table 10.3). Although all other cultures contained some
amount of CPT that was lower than cultures obtained in NAA 4.0 and BA 0.5 mg/l. In
the case of HCPT, the highest amount found was 0.009 ± 0.001 mg/g dry weight with
NAA 4.0 and BA 0.5 mg/l (Figure 10.16).
10.4.6. Camptothecin (CPT) and 10-hydroxy camptothecin (HCPT) content in O.
incarnata shoot cultures
For shoot regeneration the callus cultures obtained were cultured in basal media
containing different concentration of cytokinins i.e, BA and KIN alone or in
combination with NAA. The shoots thus obtained were evaluated for the presence of
CPT by HPLC. The results from HPLC analysis showed high content of CPT with
BA 4.0 mg/l (0.187 ± 0.001 mg/g dry weight) treated cultures (Figure 10.8). The
160
cultures treated with KIN also produced CPT, but this was lower than that of in BA
4.0 mg/l, the KIN produced only 0.157 ± 0.003 mg/g dry weight of CPT with KIN 3.0
mg/l. The highest concentration of HCPT obtained was in BA 5.0 mg/l (0.025 ± 0.002
mg/g dry weight) (Table 10.4, Figure 10.17).
10.4.7. Camptothecin (CPT) and 10-hydroxy camptothecin (HCPT) content in O.
incarnata rooted plantlets
The amount of CPT in rooted plantlets obtained in different concentration of auxins
(NAA, IAA and IBA) was calculated (Table 10.5). The cultures containing NAA 3.0
mg/l produced higher amount of CPT (0.137 ± 0.05 mg/g dry weight) (Figure 10.9).
The cultures treated with IBA also produced cultures with CPT content of about 0.133
± 0.005 mg/g dry weight (Figure 10.10). While the rooted plantlets cultured in IAA
also showed the presence CPT, but that was lower than that of other two auxins. The
highest amount of HCPT was found in cultures treated with IBA 3.0 and 4.0 mg/l
(both produced 0.015 mg/g dry weight) (Table 10.5, Figure 10.18).
10.5. Discussion
In the present study the presence of antitumour compounds camptothecin and 10-
Hydroxy camptothecin were identified using HPLC system and also established
successful callus induction, shoot and root production in O. incarnata. The HPLC
analysis showed the presence of CPT and HCPT in this plant with a highest amount of
accumulation in root tissues than the leaves and stem.
To achieve tissue cultures, the leaf explants of O. incarnata were cultured in
different hormone combinations that could induce callus, shoot buds and roots on
culture media. The callus was with the supplementation of various phytohormones to
cultures of this the combination of NAA (4.0 mg/l) and BA (0.5 mg/l) produced the
maximum biomass. However, the explants cultured in basal mediums alone, MS
medium only showed the production of callus but with least frequency. This may be
due to the fact that, for optimum callus production the presence of phytohormones is
necessary. Since shoot generation were found to be associated with cytokinins, the
BA and KIN were added to cultures for shoot formation. Off these BA produced more
shoots than KIN. There are reports that the role of BA in shoots induction from callus
(Ayabe et al., 1995; Guo et al., 2005; Xu et al., 2008; Barandiaran et al., 1999 and
Luciani et al., 2006). The rooting was established with the culturing of shoots in
161
different auxins. The cultures supplemented with IBA 3.0 mg/L produced more
number of roots than those cultures treated with NAA and IAA.
The amount of camptothecin in callus, shoot and rooted plantlets were also
analysed by HPLC. Some of the cultures produced these compounds near and higher
than that of wild whole plant. However, this amount was lower than that obtained
from HPLC analysis of wild plants root.
Over 25% of the new drugs approved in the last 30 years are based on a
molecule of plant origin, and about 50% of the top selling chemicals derive from
knowledge on plant secondary metabolism (Terryn et al., 2006). For the production of
secondary metabolites, most medicinal plants are not cultivated; rather they are
collected from the wild. In the past, quantities needed to meet demand were relatively
low; however, increasing commercial demand is fast outpacing supply. Currently
between 4,000 and 10,000 medicinal plants are on the endangered species list and this
number is expected to increase (Canter et al., 2005). To counter over-exploitation of
natural resources and consequent threats to biodiversity, sustainable practices have
been recommended and several worldwide organizations have established guidelines
for collection and sustainable cultivation of medicinal plants (Klingenstein et al.,
2006). Cultivation of medicinal plants has conservation advantages; however, costs
are frequently prohibitive because of their slow growth rate and the fact that many
tropical plants are very difficult to cultivate in a commercial setting. Despite such
difficulties, the production of useful and valuable secondary metabolites from plant
tissue and cell cultures is an attractive proposal. Since the plant cells have the ability
to regenerate an entire plant form each cell, a phenomenon known as totipotency.
Thus cultured plant cells can synthesize, accumulate and sometimes exude many
classes of metabolites as like their mother plant.
The results indicate that CPT and HCPT were found in O. incarnata. And the
tissue cultures of this plant also contained CPT.
162
Table: 10.1. Amount of camptothecin and –Hydroxy camptothecin in O. incarnata
Whole plant and
parts
Camptothecin content
(mg/g dry weight)
10-Hydroxycamptothecin content
(mg/g dry weight)
Whole plant
Leaves
Stem
Root
0.154 ± 0.001a
0.050 ± 0.002b,a
0.007 ± 0.001c
0.287 ± 0.003d,b
0.015 ± 0.002a
0.003 ± 0.002b
0.001 ± 0.001c
0.018 ± 0.001d
Values represent the mean ±S.D. The experiment was conducted in triplicates. Letters represent significant differences in comparisons, p<0.01. Same alphabet in the column defines non-significance, p>0.05.
Table: 10.2. Effect of basal mediums on callus formation from leaf segments of O. incarnata after 8 weeks of cultures and the amount of
camptothecin and 10-Hydroxy camptothecin present in them
Basal Medium Mean fresh weight of callus ( in grams)
Percentage of callus induction
Camptothecin content(mg/g dry weight)
10-Hydroxy camptothecin content
(mg/g dry weight) Murashique and Skoog (MS)
Gamborg
Whites
0.124 ± 0.010
0.054 ± 0.001
-
27.77
11.11
0
Trace amount
Trace amount
-
Trace amount
Trace amount
-
Values represent the mean ±S.D. The experiment was conducted in triplicates
163
Table 10.3. Effect of plant growth regulators (NAA and BA) on callus formation from leaf segments of O. incarnata after 8
weeks of cultures and the amount of camptothecin and 10-Hydroxy camptothecin present in them. Growth regulators (mg/L) Mean fresh
weight of callus ( in grams)
Percentage Of
callus induction
Camptothecin Content
(mg/g dry weight)
10-Hydroxy camptothecin Content
(mg/g dry weight) NAA BA
1 2 3 4 5 - - - - - 3 3 4 4 5 5
0.5 1
- - - - - 1 2 3 4 5
0.5 1
0.5 1
0.5 1 4 4
0.445 0.58a
0.474 0.05a
0.615 0.17b 0.805 0.16c
0.524 0.32d 0.257 ± 0.11a,f 0.208 ± 0.07d,g 0.179 ± 0.15h 0.175 ± 0.14a,c 0.136 ± 0.12b 0.971 ± 0.29k 0.978 ± 0.24c 1.386 ± 0.16
1.632 ± 0.34a,g 1.064 ± 0.28c 0.768 ± 0.17b 0.324 ± 0.19a 0.368 ± 0.18a,l
55.55 50.00 61.11 72.22 61.11 38.88 33.33 44.44 44.44 38.88 61.11 94.44 100.00 100.00 100.00 94.44 77.77 55.55
0.004 ± 0.001a 0.004 ± 0.002a,b 0.008 ± 0.001c 0.005 ± 0.003d 0.002 ± 0.001a,d 0.004 ± 0.001a,e 0.008 ± 0.004a 0.009 ± 0.003f 0.009 ± 0.004 0.010 ± 0.006
0.010 ± 0.001a,g 0.018 ± 0.003a 0.021 ± 0.012 0.019 ± 0.010
0.016± 0.002a,k 0.013 ± 0.004a,l 0.008 ± 0.005 0.006 ± 0.004
Trace amount Trace amount Trace amount Trace amount Trace amount Trace amount Trace amount Trace amount Trace amount Trace amount Trace amount 0.008 ± 0.002 0.009 ± 0.001 0.001± 0.001 0.004 ± 0.002 0.005 ± 0.001 Trace amount Trace amount
Values represent the mean ±S.D. Letters represent significant differences in comparisons, p<0.01. Same alphabet in the column defines non-significance, p>0.05.
164
Table 10.4. Effect of growth-regulators on shoot differentiation from leaf derived callus of O. incarnata after 12 weeks of cultures and the amount of camptothecin and 10-Hydroxy camptothecin present in them.
Values represent the mean ±S.D. Letters represent significant differences in comparisons, p<0.01. Same alphabet in the column defines non-significance, p>0.05.
Growth regulators (mg/L) Mean shoot number
Percentage of shoot
formation
Camptothecin content (mg/g dry weight)
10-Hydroxy camptothecin Content (mg/g dry weight)
NAA BA KIN
- - - - - - - - - -
0.5 1
0.5 1
0.5 1
0.5 1
1 2 3 4 5 - - - - - 4 4 5 5 - - - -
- - - - - 1 2 3 4 5 - - - - 4 4 5 5
4.38 3.25a 7.47 4.6
22.32 3.58a.b 19.35 10.25a 14.21 4.46a
5.32 3.46f 7.68 5.2a,f
9.65 4.29b,c 12.59 2.24a 8.64 ± 3.22
17.65 ± 4.25a,b,k 14.32 ± 6.47a
8.56 ± 5.28ab,c,g 3.27 ± 3.14e,g,l 11.76 ± 7.74a 9.32 ± 6.55
4.37 ± 3.56k,f,l 4.13 ± 3.07
38.88 77.77 100.00 100.00 100.00 44.44 44.44 77.77 83.33 55.55 83.33 83.33 77.77 55.55 83.33 55.55 38.88 38.88
0.056 ± 0.01 0.089 ± 0.02 0.135 ± 0.01 0.187 ± 0.01 0.164 ± 0.008 0.032 ± 0.02 0.064 ± 0.03 0.157 ± 0.003 0.121 ± 0.008 0.097 ± 0.005 0.034 ± 0.01 0.087 ± 0.01 0.089 ± 0.04 0.091 ± 0.03 0.027 ± 0.002 0.034 ± 0.002 0.035 ± 0.001 0.039 ± 0.002
0.002 ± 0.002 0.003 ± 0.002 0.009 ± 0.005 0.018 ± 0.008 0.025 ± 0.002 0.001 ± 0.001 0.003 ± 0.001 0.008 ± 0.005 0.010 ± 0.001 0.008 ± 0.002 0.002 ± 0.001 0.002 ± 0.001 0.005 ± 0.001 0.006 ± 0.002 0.002 ± 0.002 0.002 ± 0.001 0.002 ± 0.001 0.003 ± 0.001
165
Table: 10.5. Influence of different auxins on rooting of in vitro formed shoots of O.incarnata after 8 weeks culture and the
amount of camptothecin and 10-Hydroxy camptothecin present in the rooted plant let. Growth regulators (mg/L) Percentage of
rooting (in %) Mean root
number Camptothecin
content (mg/ g dry weight)
10-Hydroxy camptothecin Content (mg/g dry weight) NAA IAA IBA
0.5 1 2 3 4 - - - - - - - - - -
- - - - -
0.5 1 2 3 4 - - - - -
- - - - - - - - - -
0.5 1 2 3 4
83.33 100.00 100.00 100.00 100.00 83.33 94.44 100.00 100.00 100.00 94.44 100.00 100.00 100.00 100.00
2.55 ± 2.07a 2.57± 3.21b 4.45 ± 3.27c 6.78± 2.35d 5.29 ± 3.22e 3.15 ± 1.20a,f 3.78 ± 1.97a,g 5.96 ± 3.27b,h 7.24 ± 4.21b,c 6.08 ± 3.76d,e
4.57 ± 4.61a,g,k 5.39 ± 3.58a,d,l 7.27 ± 3.08g,k 8.69 ± 2.64a,b,f 6.67 ± 3.29a,c
0.045 ± 0.003 0.055 ± 0.02 0.089 ± 0.04 0.137 ± 0.05 0.124 ± 0.03 0.022 ± 0.01 0.034 ± 0.02 0.058 ± 0.03 0.069 ± 0.05 0.071 ± 0.04 0.039 ± 0.01 0.061 ± 0.08 0.087 ± 0.04 0.133 ± 0.005 0.129 ± 0.023
Trace amount Trace amount 0.001 ± 0.001 0.008 ± 0.002 0.008 ± 0.001
Trace amount Trace amount Trace amount 0.001 ± 0.001 0.002 ± 0.001
Trace amount 0.001 ± 0.002 0.009 ± 0.002 0.015 ± 0.003 0.015 ± 0.002
Values represent the mean ± S.D. Letters represent significant differences in comparisons, p<0.01. Same alphabet in the column defines non-significance, p>0.05.
Figure:10.2. Chromatogram of standard Camptothecin (Sigma).
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vol
ts
0 .0 0
0 .2 5
0 .5 0
0.0
00
0.0
00
0.0
00
0.0
00
0.00
0
0.0
40 C
PT
0.00
0
0.00
0
0.00
0
0.0
00
0.00
0
0.00
0
0.0
00
0.0
00
0.0
00
0.00
0
0.00
0
0.0
00
0.00
0
Figure:10.3. Chromatogram of camptothecin from whole plant extract
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vol
ts
0 .0 0 0
0 .0 2 5
0 .0 5 0
0.0
00
0.0
00
0.0
00
0.0
00
0.00
0
0.0
00
0.00
1 C
PT
0.0
00
0.
000
0.00
0
0.00
0
0.0
00
0.0
00
0
.00
0
0.00
0
0.0
00
0.00
0
0.0
00
0.0
00
0.0
00
Figure:10.4. Chromatogram of camptothecin from leaf extract
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vol
ts
0 .0 0
0 .0 2
0 .0 4
0.0
00
0.0
00
0.0
00
0.
000
0
.000
0.0
08
CP
T
0.0
00
0.0
00
0.0
00
0.00
0
0.00
0
0.00
0
0.00
0
0.0
00
0.00
0
0.00
0
0.0
00
0.0
00
0.0
00
0.0
00
Figure:10.5. Chromatogram of camptothecin from stem extract
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vol
ts
0 .0 0 0
0 .0 2 5
0 .0 5 0
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
0.0
02 C
PT
0.00
0
0.00
0
0.0
00
0.0
00
0.00
0
0.0
00
0.00
0 0.0
00
0.00
0
0.00
0
0.00
0
0.00
0
0.0
00
0.0
00
0.00
0
0.0
00
Figure:10.6. Chromatogram of camptothecin from root extract
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vo
lts
0 .0 0
0 .0 5
0 .1 0
0 .1 5
0.0
00
0.0
00
0.0
00
0.0
00 0.
000
0.0
05 C
PT
0.00
0
0.0
00
0.00
0
0.0
00
0.00
0
0.0
00
0.00
0
0.0
00
0.0
00
0.0
00 0.00
0
0.0
00
Figure:10.7. Chromatogram of camptothecin from NAA 4.0/ BA 0.5 mg/L callus cultures
M in u te s
0 1 2 3 4 5 6 7 8 9 10
Vo
lts
0 .0 0
0 .0 5
0 .1 0
0 .1 5
0.00
0
0.0
00
0.0
00
0
.00
0
0.0
00
0
.00
0
0.0
00
0.0
00
0
.000
0.0
00
0.0
00
0.0
03
CP
T
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
0.00
0
0.0
00
0.0
00
0.0
00
Figure:10.8. Chromatogram of camptothecin from BA 4.0 mg/L shoot cultures
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vol
ts
0 .0 0
0 .0 5
0 .1 0
0.00
0
0.0
00
0.0
00
0.0
00
0.0
06 C
PT
0.0
00
0.00
0
0.00
0
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
Figure:10.9. Chromatogram of camptothecin from NAA 4.0 mg/L rooted plantlet cultures
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vol
ts
0 .0 0
0 .0 5
0 .1 0
0.0
00
0.0
00
0.00
0
0.00
0
0.00
0
0.0
08 C
PT
0.00
0
0.00
0
0.0
00
0.00
0
0.0
00
0.00
0
0.0
00
0.0
00
0.00
0
0.0
00
0.0
00
0.00
0
Figure:10.10. Chromatogram of camptothecin from IBA 4.0 mg/L rooted plantlet cultures
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vol
ts
0 .0 0
0 .0 5
0 .1 0
0 .1 5
0.00
0
0.00
0
0.00
0
0.00
0 0.
000
0.00
5 C
PT
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
Figure: 10.11. Chromatogram of standard 10-Hydroxy camptothecin (Sigma).
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vol
ts
0 .0
0 .2
0 .4
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.10
0 1
0-H
CP
T
0.00
0
0.00
0
Figure: 10.12. Chromatogram of 10-Hydroxy camptothecin from whole plant extract
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vol
ts
0 .0 0
0 .0 1
0 .0 2
0 .0 3
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0 0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
1 1
0 H
CP
T
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
Figure: 10.13. Chromatogram of 10-Hydroxy camptothecin from leaf extract
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vol
ts
0 .0
0 .5
1 .0
1 .5
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0 0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.01
2 H
CP
T
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
Figure: 10.14. Chromatogram of 10-Hydroxy camptothecin from stem extract
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vo
lts
0 .0 0
0 .0 1
0 .0 2
0.0
00
0.00
0
0.00
0
0.00
0
0.0
00
0.0
00
0.00
0 0.0
00
0.0
00
0.0
00
0.00
0
0.0
01 1
0-H
CP
T
0.0
00
0.0
00
0.00
0
0.00
0
0.00
0
Figure: 10.15. Chromatogram of 10-Hydroxy camptothecin from root extract
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vol
ts
0 .0 0 0
0 .0 2 5
0 .0 5 0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
1 H
CP
T
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
0.00
0
Figure: 10.16. Chromatogram of 10-Hydroxy camptothecin from NAA 4.0/ BA 0.5 mg/L callus cultures
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vo
lts
0 .0 0
0 .0 5
0 .1 0
0 .1 5
0.0
00
0.0
00
0.00
0
0.0
00
0.0
00 0.
000
0.0
00
0.00
0
0.00
0
0.0
00
0.00
3 1
0 H
CP
T
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
Figure: 10.17. Chromatogram of 10-Hydroxy camptothecin from BA 5.0 mg/L shoot cultures
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vo
lts
0 .0 0
0 .0 1
0 .0 2
0.0
00
0.00
0
0.00
0 0.
000
0.
000
0.
000
0.00
0 0.
000
0.00
0
0.00
0
0.0
00
0.0
00
0.0
01 1
0-H
CP
T
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
Figure: 10.18. Chromatogram of 10-Hydroxy camptothecin from IBA 3.0 mg/L rooted plantlet cultures
M in u te s
0 1 2 3 4 5 6 7 8 9 1 0
Vol
ts
0 .0 0
0 .0 2
0 .0 4
0.0
00
0.00
0
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
0.00
0
0.0
00
0
.000
0
.000
0.00
0
0.0
00
0.0
00
0.0
00
0.
001
10
HC
PT
0.0
00
0
.00
0
0.00
0
0.0
00
0.0
00
0.0
00
0.0
00
0.00
0
0.0
00
