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CHAPTER SIX ISOLATION OF BIOACTIVE PHYTO-CONSTITUENTS FROM TINOSPORA

CORDIFOLIA �

118��

6.1 INTRODUCTION

Natural products have played an important role in drug discovery and formed the basis of

most early medicines. Natural products drug discovery has led to the isolation of highly active

anticancer agents (Cragg et al., 2011). Isolation and characterization of pharmacologically

active compounds from medicinal plants continue till date. Of all available anticancer drugs

between 1940 and 2002, 40% were natural products or natural product-derived constituting

another 8% of natural product mimics (Cragg and Newman, 2005). Lead identification is the

first step in medicinal plant drug discovery. Lead optimization (involving medicinal and

combinatorial chemistry) and lead development (including pharmacology, toxicology,

pharmacokinetics, and drug delivery) play a crucial role in the development of newer drugs

(Lee et al., 2012). In the generic scheme of natural products isolation, natural product is

extracted from the plant source, concentrated, fractionated and purified, yielding essentially a

single biologically active compound. The current study adopted the strategy of bioactivity or

mechanism of action directed isolation and characterization of active compounds from the

selected plant, Tinospora cordifolia.

The present study revealed that Tinospora cordifolia petroleum ether and dichloromethane

fractions significantly inhibited side population phenotype, ABCB1 (MDR1) mediated drug

transport and imparted potent cytotoxicity against human breast cancer cells. In order to

isolate and characterize the active compound, Tinospora cordifolia petroleum ether and

dichloromethane fractions were further sub-fractioned into a total of 34 fractions (F1 to F34)

by column chromatography and each fraction was screened for anticancer activity. The most

biologically active sub-fractions were further subjected to chromatography techniques for the

isolation of active compounds. Chemical characterization of purified compounds was carried

out by NMR spectroscopy and mass spectroscopy.

6.2 MATERIALS AND METHODS

6.2.1 Drugs and chemicals

Hoechst 33343, Verapamil, Fumetrimorgin C, Rhodamine 123 and DMEM were

purchased Sigma Chemical Co. (St. Louis, MO, USA).

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6.2.2 Cell Culture

HeLa (human cervical carcinoma) and A549 (human lung adenocarcinoma) cells were

cultured as described earlier (Section, 3.2.3 and Section, 5.2.2).

6.2.3 Fractionation of Tinospora cordifolia petroleum ether (TC-PET) and

dichloromethane (TC-DCM) fractions by column chromatography

Tinospora cordifolia dichloromethane fraction was dissolved completely in methanol and

adsorbed with silica gel (60-120 mesh) in a china dish. Adsorbed fraction was then added into

a glass column packed with silica gel (column volume: 1500 ml, void volume: 500 ml). A

step-wise gradient elution (10%) was performed with increasing solvent polarity (petroleum

ether-ethyl acetate-methanol). A total of 17 such fractions were collected and named as F1 to

F17. Similarly, for Tinospora cordifolia petroleum ether fraction, a step-wise gradient elution

(10%) was performed with increasing solvent polarity (petroleum ether-chloroform-methanol)

to yield 17 fractions named as F18 to F34. Each fraction was concentrated in a rotary vacuum

evaporator, dried completely and lyophilized. For biological studies, crude ethanolic extract

and fractions were dissolved in DMSO at 20 mg/ml.

6.2.4 Side population analysis

SP assay was performed in the presence of Tinospora cordifolia petroleum ether and

dichloromethane sub-fractions (F1-F34, concentration-50 μg/ml each) as described earlier

(Section, 4.2.4).

6.2.5 Rhodamine 123 efflux assay

Briefly, HeLa cells with 70-80% confluency were used for the transport studies. Cells

were trypsinized and resuspended in DMEM with 2% FBS. Single cell suspension (1×106)

was incubated with and without rhodamine 123 (0.50 μM) at 37°C in a CO2 incubator for 30

minutes. After centrifugation (1400 rpm for 4 minutes at 4°C) and removal of supernatant, the

cells were re-incubated in DMEM with 2% FBS in the presence or absence of Tinospora

cordifolia petroleum ether and dichloromethane sub-fractions (F4, F5, F6S, F7, F8, F10, F11,

F29 and F30) at 50 μg/ml each, without rhodamine 123 for an additional 30 minutes at 37°C.

DMSO was used as a vehicle control. Verapamil (50 �M) was used as a standard inhibitor of

120��

ABCB1 mediated drug transport. At the end of incubation, cells were washed twice with ice-

cold HBSS and analyzed in a flow cytometer as described earlier (Section, 5.2.8).

6.3 RESULTS

6.3.1 SP inhibitory effects of TC-DCM TC-PET sub-fractions (F1 to F34)

SP inhibitory effects of Tinospora cordifolia dichloromethane and petroleum ether sub-

fractions (F1 to F34) were investigated in human cervical cancer (HeLa) and lung cancer

(A549) cells. Results indicated that in HeLa cells, as compared to untreated control, SP was

significantly inhibited upon treatment with TC-DCM and TC PET sub-fractions F4, F5, F6S,

F7, F8, F17 F20, F21, F22, F28, F29, F30 and F31 (Fig. 6.1 and Fig. 6.2). Similarly, in A549

cells, as compared to untreated control, SP was significantly inhibited upon treatment with

TC-DCM sub-fractions F4, F5, F6S, F7, F8, F9, F10 and F11 (Fig. 6.3 and Fig. 6.4). SP

activity profile of both TC-DCM and TC PET sub-fractions in HeLa and A549 cells is

depicted in Fig. 6.5 and Fig. 6.6 respectively. In HeLa cells, SP in untreated control was 1.21

± 0.03%. Upon treatment with TC-DCM and TC PET sub-fractions F4, F5, F6S, F7, F8, F17

F20, F21, F22, F28, F29, F30 and F31, SP were 0.01 ± 0.01%, 0.03 ± 0.01%, 0.01 ± 0.01%,

0.00%, 0.03 ± 0.01%, 0.01%, 0.08 ± 0.01%, 0.01 ± 0.01%, 0.06 ± 0.01%, 0.05 ± 0.01%, 0.01

± 0.01%, 0.01 ± 0.01% and 0.04 ± 0.01%, respectively. Similarly in A549 cells, SP in

untreated control were 8.90 ± 1.38%. Upon treatment with TC-DCM sub-fractions F4, F5,

F6S, F7, F8, F9, F10 and F11, SP was found to be 0.76 ± 0.04%, 0.02 ± 0.01%, 0.19 ±

0.02%, 0.00%, 0.16 ± 0.04%, 0.65 ± 0.06%, 0.34 ± 0.07% and 0.60 ± 0.12%, respectively.

SP was completely vanished in the presence of verapamil (in HeLa) or fumetrimorgin C (in

A549) indicating valid SP phenotype. Vehicle control (DMSO) did not inhibit SP. These

results corroborated that Tinospora cordifolia dichloromethane sub-fractions viz., F4, F5,

F6S, F7 and F8 possess significant inhibition of SP phenotype. All values are mean± SD of

three independent experiments (n=3, ***p<0.001, determined by one way ANOVA using

Dunnett Test).

6.3.2 Isolation and purification of biologically active compounds

Poor yield or poor recovery of the final compound is one of the major problems in natural

product isolation. For example, only 30 g of vincristine was obtained from 15 tons of dried

leaves of Vinca rosea (or Catharanthus roseus). Similarly, to obtain 1900 g of Taxol, the

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felling of 6000 extremely slow-growing trees, Taxus brevifolia, was necessary to produce

27,300 kg of the bark (Farnsworth, 1990). In order to investigate the chemical property and

the detailed biological activities of the isolated compounds, large scale extraction and

fractionation was required. In this direction, 4 kg of the dried stem part of Tinospora

cordifolia was extracted with absolute ethanol to get a total ethanolic extract as described

earlier (Section, 3.2.1). By following the standardized method for the fractionation as

mentioned earlier (Section, 5.2.3), Tinospora cordifolia ethanolic extract was fractionated to

get the biologically active dichloromethane fraction (TCD) and the latter was subjected to

column chromatography to yield the biologically active sub-fractions TCD2, TCD3, TCD4,

TCD5 and TCD6 (Equivalent to sub-fractions F4, F5, F6S, F7, F8 respectively, as mentioned

in section, 6.2.3).

In order to validate the SP inhibitory effects of Tinospora cordifolia dichloromethane sub-

fractions, HeLa and A549 cells were treated with TCD2, TCD3, TCD4, TCD5 and TCD6. SP

in untreated HeLa cells was 1.39 ± 0.14%, however, in the presence of TCD2, TCD3, TCD4,

TCD5 and TCD6, SP was 0.17 ± 0.02%, 0.00 ± 0.01%, 0.02 ± 0.01%, 0.01 ± 0.01% and 0.11

± 0.06%, respectively (Fig. 6.7). Similarly, SP in untreated A549 cells was 9.70 ± 0.73%,

however, in the presence of TCD2, TCD3, TCD4, TCD5 and TCD6, SP was 0.11 ± 0.03%,

0.06 ± 0.01%, 0.12 ± 0.02%, 0.01 ± 0.01% and 0.39 ± 0.06%, respectively (Fig. 6.8). SP was

completely inhibited in the presence of verapamil (in HeLa) or fumetrimorgin C (in A549)

indicating valid SP. These results corroborated (validated) that Tinospora cordifolia

dichloromethane sub-fractions, viz. TCD2, TCD3, TCD4, TCD5 and TCD6 significantly

inhibit SP phenotype in human epithelial cancer cells. All values are mean ± SD of three

independent experiments (n=3, ***p<0.001, determined by one way ANOVA using Dunnett

Test).

6.3.3 ABCB1 (MDR1) inhibitory effects of TC-DCM and TC-PET sub-fractions

In order to validate the multidrug resistance drug transporter (ABCB1) inhibitory

activities of Tinospora cordifolia dichloromethane sub-fractions (TCD2, TCD3, TCD4, TCD5

and TCD6), rhodamine 123 efflux assay was carried out in human cervical (HeLa) cells as

described earlier (Section, 5.2.8). The mean fluorescent intensity (MFI) of rhodamine 123 in

efflux phase control was 257.20 ± 8.43; however, in the presence of TCD2, TCD3, TCD4,

TCD5 and TCD6, MFI was 509.50 ± 12.00, 689.10 ± 21.42, 618.90 ± 21.60, 534.90 ± 21.65

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and 517.70 ± 28.46, respectively. Verapamil, was used as a standard inhibitor of ABCB1

(MFI = 819.90 ± 11.92). The significantly increased MFI in the presence of TCD2, TCD3,

TCD4, TCD5 and TCD6 indicated the increased intracellular accumulation of rhodamine 123

(Fig. 6.9). These results corroborated that Tinospora cordifolia dichloromethane sub-

fractions, viz. TCD2, TCD3, TCD4, TCD5 and TCD6 significantly inhibit ABCB1 mediated

drug transport in cancer cells. All values are mean ± SD of three independent experiments

(n=3, ***p<0.001, determined by one way ANOVA using Dunnett Test).

Thin layer chromatography of these biologically active sub-fractions (TCD2, TCD3,

TCD4, TCD5 and TCD6) revealed that each sub-fraction contain more than a single spot

which indicated mixture of compound. Based on the biological activity (Inhibition of SP and

multidrug resistant transporter-ABCB1) and less complexity with respect to the number of

compounds present, Tinospora cordifolia dichloromethane sub-fractions TCD5, TCD4 and

TCD3 were further selected for isolation of biologically active compounds. The TLC profile

of TCD5, TCD4 and TCD3 is shown in Fig. 6.10.

6.3.4 Isolation of TCD5-F2-C (TC-A)

Isolation of TCD5-F2-C was carried out by flash chromatography (Teledyne isco-combi

flash Rf with fraction collector). Briefly, 2.3 gram of TCD5 was completely dissolved in

chloroform with a few drops of methanol and adsorbed in silica gel (230-400) mesh. A binary

pump was used in which pump A consists of less polar solvent and pump B consist of more

polar solvent. The column was packed with silica gel 230-400 mesh and equilibrated with the

solvent in pump A. Completely dried fraction adsorbed with silica gel was then packed into

the column reservoir. Run time was set for 120 minutes. A gradient elution with solvent

system A (less polar) and B (more polar) at a flow rate of 5 ml/minute, detection wavelength

of 254 nm, with fraction collector, resulted in the elution of biologically active fraction

TCD5-F2. Further purification of TCD5-F2 by column chromatography yielded the final

compound TCD5-F2-C (TC-A) (Fig. 6.11).

6.3.5 TCD5-F3-B (TC-B)

Isolation of TCD5-F3-B was carried out by flash chromatography as described above

(Section, 6.3.4). A gradient elution with solvent system A (less polar) and B (more polar) at a

flow rate of 5 ml/minute, detection wavelength of 254 nm, with fraction collector, resulted in

123��

the elution of biologically active fraction TCD5-F3. Further purification of TCD5-F3 by

column chromatography yielded the final compound TCD5-F3-B (TC-B) (Fig. 6.11).

6.3.6 Isolation of TCD4-A2 (TC-C)

Isolation of TCD4-A2 was carried out by flash chromatography as described above

(Section, 6.3.4). A gradient elution with solvent system A (less polar) and B (more polar) at a

flow rate of 5 ml/minute, the detection wavelength of both 254 nm, with fraction collector,

resulted in the elution of biologically active fraction TCD4-A. Further purification of TCD4-

A by column chromatography yielded the final compound TCD4-A2 (TC-C) (Fig. 6.12).

6.3.7 Isolation of TCD3-A2 (TC-D)

Isolation of TCD3-A2 was carried out by flash chromatography as described above

(Section, 6.3.4). A gradient elution with solvent system A (less polar) and B (more polar) at

flow rate of 5 ml/minute, detection wavelength of both 254 nm, with fraction collector,

resulted in the elution of biologically active fraction TCD3-A. Further purification of TCD3-

A by column chromatography yielded the final compound TCD3-A2 (TC-D) (Fig. 6.13).

6.3.8 Chemical characterization of isolated compounds

The melting point of the compounds was determined by capillary melting point method.

The melting point of the compounds was found to be TC-A (80-82°C); TC-B (82-84°C); TC-

C (76-82°C) and TC-D (68-72°C). The isolated compounds were completely soluble in

methanol and dimethyl sulfoxide, sparingly soluble in chloroform and ethyl acetate. The

compounds were insoluble in water and hexane. The molecular weight of the isolated

compounds was determined by Electron Spray Ionization-Mass Spectroscopy (ESI-MS). The

structure of the isolated compounds was determined by 1H-NMR, 13C-NMR, COSY, DEPT,

HMBC and HSQC.

6.4 DISCUSSION

Among the 34 sub-fractions of Tinospora cordifolia dichloromethane and petroleum ether

fractions, dichloromethane sub-fractions viz., F4, F5, F6S, F7 and F8 possess significant

inhibitory effect on SP phenotype. The SP inhibition may be due to the selective cytotoxicity

in SP as compared to the bulk population or NSP. It was evident that SP cells are resistant to

anticancer drugs due to the overexpression of MDR transporters. Therefore, SP inhibition

124��

may be also due to the inhibition of MDR transporters. Flow cytometry based functional assay

corroborated that sub-fractions F4, F5, F6S, F7 and F8 impart significant inhibition on

ABCB1 (MRD1) drug transporter. These most active sub-fractions may contain compounds

which are able to eliminate rare cancer stem cells. Thin layer chromatography of

dichloromethane sub-fractions (F4, F5, F6S, F7 and F8) with suitable solvent system revealed

that each sub-fraction contained more than a single spot which indicated the existence of

multiple compounds. For the isolation of active compounds, the large scale extraction of

Tinospora cordifolia was carried out and biologically active sub-fractions were separated out

using standardized protocol. These fractions were named as TCD2, TCD3, TCD4, TCD5 and

TCD6 which are equivalent to sub-fractions F4, F5, F6S, F7 and F8. However, sub-fractions

TCD5 (F6S), TCD4 (F5) and TCD3 (F4) were found to have highly active against SP and

MDR1 transporter in cancer cells. Therefore, Tinospora cordifolia dichloromethane sub-

fractions TCD5, TCD4 and TCD3 were selected for the isolation of bio-active compounds.

Flash chromatography and subsequent purification by column chromatography led to the final

purified compounds, TCD5-F2-C (TC-A), TCD5-F3-B (TC-B), TC-D4-A2 (TC-C) and TC-

D3-A2 (TC-D). Chemical characterization and structural decipher of the compounds were

carried out by NMR and Mass spectroscopy. The study is under patent application. Therefore,

the detailed method for isolation of bio-active compounds and structures of isolated

compounds are not revealed in this thesis.

125��

Figure 6.1 SP inhibitory activities of F1-F17 in HeLa cells

SP inhibited completely with verapamil indicating valid SP. Fractions, F4, F5, F6S, F7, F8 and F17 significantly inhibited SP phenotype. These figures are representative of three independent experiments.

CONTROL-1.24% VERAPAMIL - 0.00% F1-0.14% F2-0.22%

F3-0.13% F4-0.01% F5-0.04% F6-0.31%

F6 S-0.01% F7-0.00% F8-0.03% F9-0.35%

F10-0.33% F11-0.19% F12-0.21% F13-0.26%

F14-0.23% F15-2.01% F16-2.26% F17-0.02%

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Figure 6.2 SP inhibitory activities of F18-F34 in HeLa cells

Fractions, F20, F21, F22, F28, F29, F30 and F31 significantly inhibited SP phenotype. These figures are representative of three independent experiments.

F18-0.56%

DMSO - 1.91%

F19-0.64% F20-0.08% F21-0.01%

F22- 0.05% F23-0.94% F24-1.66% F25-1.63%

F26-2.37% F27-1.65% F28-0.06% F29-0.01%

F30-0.01% F31-0.04% F32-0.47% F33-0.76%

F34-0.20%

127��

Figure 6.3 SP inhibitory activities of F1-F17 in A549 cells

SP inhibited completely with FTC indicating valid SP. Fractions, F4, F5, F6S, F7, F8, F9, F10 and F11 significantly inhibited SP phenotype. These figures are representative of three independent experiments.

C0NTROL-8.19% FTC 0.00% F1-3.70% F2-2.98%

F3-2.91% F4-0.71% F5-0.02% F6-7.24%

F6 S-0.16% F7-0.00% F8-0.31% F9-0.59%

F10-0.25% F11-0.46% F12-1.59% F13-2.52%

F14-3.6% F15-6.59% F16-4.49% F17-3.69%

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Figure 6.4 SP inhibitory activities of F18-F34 in A549 cells

Fractions, F18-34 did not inhibit SP. These figures are representative of three independent experiments.

F18-8.51%

DMSO -6.38%

F19-14.65% F20-9.85% F21-3.94%

F22- 7.72% F23-12.01% F24-17.51% F25-19.00%

F26-14.38% F27-15.03% F28-8.60% F29-9.23%

F30-2.78% F31-7.82% F32-14.35 F33-18.99%

F34-7.45%

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Figure 6.5 Anti-SP effects of fractions, F1-F34 in HeLa cells

Fractions, F4, F5, F6S, F7, F8, F17, F20, F21, F22, F28, F29, F30 and F31 significantly inhibited SP phenotype. All values are mean ± SD of three independent experiments, ***p<0.001, determined by one way ANOVA using Dunnett Test.

Figure 6.6 Anti-SP effects of fractions, F1-F34 in A549 cells

Fractions, F4, F5, F6S, F7, F8, F9, F10 and F11 significantly inhibited SP phenotype. All values are mean ± SD of three independent experiments, ***p<0.001, determined by one way ANOVA using Dunnett Test.

CONTROL

VERAPAMILTC�F1TC�F2TC�F3TC�F4TC�F5TC�F6

TC�F6STC�F7TC�F8TC�F9

TC�F10

TC�F11

TC�F12

TC�F13

TC�F14

TC�F15

TC�F16

TC�F17

TC�F18

TC�F19

TC�F20

TC�F21

TC�F22

TC�F23

TC�F24

TC�F25

TC�F26

TC�F27

TC�F28

TC�F29

TC�F30

TC�F31

TC�F32

TC�F33

TC�F34DM

SO0.0

0.4

0.8

1.2

1.6

*** *** *** ****** *** ********* *** *** *********

%�Side�po

pulatio

n�(SP)

***P<0.001

CONTROLFTCTC�F1TC�F2TC�F3TC�F4TC�F5TC�F6

TC�F6STC�F7TC�F8TC�F9

TC�F10

TC�F11

TC�F12

TC�F13

TC�F14

TC�F15

TC�F16

TC�F17

TC�F18

TC�F19

TC�F20

TC�F21

TC�F22

TC�F23

TC�F24

TC�F25

TC�F26

TC�F27

TC�F28

TC�F29

TC�F30

TC�F31

TC�F32

TC�F33

TC�F34DM

SO0

4

8

12

********* *** ******

*** ******

%�Side�po

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n�(SP)

***P<0.001

130��

Figure 6.7 Anti-SP activity of TCD2, TCD3, TCD4, TCD5 and TCD6 in HeLa cells

Tinospora cordifolia dichloromethane sub-fractions viz. TCD2 (F4), TCD3 (F5), TCD4 (F6S), TCD5 (F7) and TCD6 (F8) significantly inhibited SP phenotype in human cervical cancer cells (HeLa). SP inhibited completely with verapamil indicating valid SP. DMSO did not have any effect in SP. These figures are representative of three independent experiments. All values are mean ± SD, ***p<0.001, determined by one way ANOVA using Dunnett Test.

CONTROL - 1.39 ± 0.14% TCD2 – 0.17 ± 0.02% TCD3 – 0.00 ± 0.01%

TCD4 - 0.02 ± 0.01% TCD5 – 0.01 ± 0.01% TCD6 - 0.11 ± 0.06%

A

CONTROL

TCD2

TCD3

TCD4

TCD5

TCD6

VERAPAMIL

DMSO

0.0

0.5

1.0

1.5

2.0

****** *** ***

***

ns

***

%�Side�po

pulatio

n�(SP)

B

131��

Figure 6.8 Anti-SP activity of TCD2, TCD3, TCD4, TCD5 and TCD6 in A549 cells

Tinospora cordifolia dichloromethane sub-fractions viz., TCD2 (F4), TCD3 (F5), TCD4 (F6S), TCD5 (F7) and TCD6 (F8) significantly inhibited SP phenotype in human lung cancer cells (A549). SP inhibited completely with fumetrimorgin C (FTC) indicating valid SP. DMSO did not have any effect in SP. These figures are representative of three independent experiments. All values are mean ± SD, ***p<0.001, determined by one way ANOVA using Dunnett Test).

CONTROL – 9.70±0.73% TCD2 – 0.11±0.03% TCD3 – 0.06±0.01%

TCD4 - 0.12±0.02% TCD5 – 0.01±0.01% TCD6 - 0.39±0.06%

A

CONTROL

TCD2

TCD3

TCD4

TCD5

TCD6 FT

CDM

SO0

4

8

12

*** *** *** *** ***

ns

***

%�Side�po

pulatio

n�(SP)

B

132��

Figure 6.9 MDR1 modulatory activity of TCD2, TCD3, TCD4, TCD5 and TCD6

A. Modulation of rhodamine 123 efflux in HeLa cells was determined by flow cytometry. Verapamil is used as standard inhibitor of MDR1. B. TCD2, TCD3, TCD4, TCD5 and TCD6 significantly increased rhodamine 123 mean fluorescence intensity indicating enhanced drug retention owing to inhibition of ABCB1 drug transport. All values are mean ± SD of three independent experiments, ***p<0.001, ns=not significant, determined by one way ANOVA by using Dunnett Test.

Control (efflux phase)

Verapamil (efflux phase)

A

CONTROL

DMSO

VERAPAMIL

TCD2

TCD3

TCD4

TCD5

TCD6

0

200

400

600

800

1000

***

***

******

*** ***

Mean�Flurescence�Intensity

�(Rh1

23)

B

133��

Figure 6.10 TLC profile of most biologically active sub-fractions, TCD5, TCD4 and TCD3

These sub-fractions were obtained by the column chromatography of Tinospora cordifolia dichloromethane fraction and imparted SP and MDR1 inhibition properties in cancer cells.

Figure 6.11 TLC profile of TCD5-F2-C (TC-A) and TCD5-F3-B (TC-B)

A. TLC profile of TCD5-F2 and TCD5-F3 which contains more than a single spot with different Rf. value. B. TLC profile of TCD5-F2-C, the final compound purified from TCD5-F2 (single spot). C. The TLC profile of TCD5-F3-B, the final compound purified from TCD5-F3 (single spot).

TCD5 TCD4 TCD3

TCD5-F2 TCD5-F3 TCD5-F2-C TCD5-F3-B

A B C

134��

Figure 6.12 TLC profile of TCD4-A-2 (TC-C)

A. TLC profile of TCD4-A which contains more than a single spot with different Rf. values. B. TLC profile of TCD4-A-2, the final compound purified from TCD4-A (single spot).

Figure 6.13 TLC profile of TCD3-A-2 (TC-D)

A. TLC profile of TCD3-A which contains more than a single spot. B. TLC profile of TCD3-A2, the final compound purified from TCD3-A (single spot).

TCD4-A

A

TCD4-A-2

B

ATCD3-A

B

TCD3-A-2

135��

6.5 REFERENCES

Cragg, G. M., Kingston, D. G. I. & Newman, D. J. 2011. Anticancer agents from natural products,

CRC Press.

Cragg, G. M. & Newman, D. J. 2005. Plants as a source of anti-cancer agents. Journal of

Ethnopharmacology, 100, 72-79.

Farnsworth, N. R. The role of ethnopharmacology in drug development. 1990. Wiley Online Library.

Lee, K. H., Itokawa, H., Akiyama, T. & Morrisnatschke, S. L. 2012. Plant Derived Natural Products

Research in Drug Discovery. Natural Products in Chemical Biology, Book chapter, 351-388.