2. INTRODUCTION
• Natural compounds are well known as source for drug of
several human ailments.
• Ex. Vincristine, irinotecan and paclitaxel.
• Search for a new anticancer agent is still necessary in
order to increase the range available and to find less
toxic and more effective drug.
• Therefore, this work was design to investigate the
cytotoxicity of six natural compounds available in
research group, with previously demonstrated
pharmacological activities.

3. • 1. Xanthin V1- roots of Cratoxylum formosum, the leaves
of Symphonia globulifera and the seeds of Vismia
laurentii
• 2. 2 acetylfuro 1,4- napthoquinone- Newbouldia laevis
• 3. Physcion- Rheum officinale
• 4. Bisvismiaquinone- Vismia genus
• 5. Vismiaquinone- Vismia genus
• 6. 1,8 dihydroxy-3-generyloxy-6-methylanthraquinone-
Vimia laurentii.
• In this work these examined the cytotoxicity of 1-6
compounds against MiaPaCa-2, CCRM-CEM, and
CEM/ADR-5000 cell lines, and then selected compound
1and 2 to test on a panel of cancer cell lines.

4. Material and Methods
• Cell lines were obtained from different sources.
• Cell lines used are-
CCRM-CEM (Human T cell Lymphoblast cells)
CEM/ADR5000
PF-382 (Leukemia T cells)
HL-60 (Premyelotic leukemia)
MiaPaCa-2 (Pancreatic adenocarcinoma)
Capan-1(Pancreatic adenocarcinoma)
MCF-7 (Breast cancer )
SW-680 (Colon Carcinoma)
786-0 (Renal Carcinoma)
U87MG (Glioblastoma astrocytoma cells)
A-549 (Lung adenocarcinoma)
Colo-38 (Skin Myeloma Cells)
HeLa (cervical Carcinoma cells)
Caski (Cervical Carcinoma Cells)
AML 12 (Hepatocytes)

5. • All culture cells were maintained in a humidified
environment at 37 0 C with 5% CO2.
• Doxorubicin was used as a positive control.
• It is a drug used in wide range of cancer chemotherapy.
• Isolation and characterization of six natural compounds
used in this experiment has been previously done.

6. Resazurin Cell Growth Assay
• This assay test cellular viability and mitochondrial function.
• Adherant cells were grown in Tissue Culture Flask and harvested
by treating trypsin and EDTA for 5 min.
• Once deteched, Cells were washed, counted and aliquot of 5000
cells was places in each well of 96 well plate in totle vol. of 100
microlitter.
• Overnight incubation to allow them to attach.
• And then treated with Samples with a range of 20-0.16
microgram/ml.
• Incubation for 48 hr.
• After 48 hr., 20 microlittre of resazurin was added.
• Incubation at 37 degree C for 1-2 hr.
• Flourescence was measured on Plate reader using excitation
wavelength 544nm and emmission at 590 nm.

7. • For leukemia cells, aliquot of 50000 cell were seeded in
96 well plate and extract were added immediately.
• After 24 hr. plates were treated with resazurin as
mentioned before.
• Each Assay was done at least 3 times with 2 replicates.
• Viability was measured by comparison with untreated
cells.

8. Results

9. • Sample 1 and 2 as well as doxorubicin were able to
reduce he proliferation of 3 cell lines up to 50 % when
tested at 20 microgram/ml.
• Physcion was found to have no cytotoxicity.
• Rest 4, 5, and 6 compounds also bearing physcion
moiety, showing the low cytotoxicity.
• So the sample 1 and 2 were selected for further effect on
diff. cell lines..

11. • The IC 50 value below 20 microgram/ml were recorded
in 12 of the 14 cell lines for sample 1, and 14/14 cell
lines for sample 2.
• Below or arround 4 mg/ml
Sample 1- 9/14
Sample 2- 11/14
(IC 50 value= Conc. Of sample required to inhibit 50% of cell
proliferation.)

12. Flow Cytometry for cell cycle analysis and
detection of apoptotic cells
• CCRM-CEM cells were treated with compound 1 and 2
or DMSO for 24 to 72 hrs.
• Then fixed with ethanol 95 %and then washed with cold
PBS.
• Resuspand in 150 microlitter hypotonic fluorochrome.
• Cells were incubated in dark for overnight before FACS.
• PI flouroscence was measured of individual nuclei by
FACS Caliber.
• Data were analyzed by CellQuess Pro V5.2.1.
• At least 3 experiments were performed.

13. Results

15. Caspase Glo 3/7 Activity
• CCRM-CEM leukemia were cultured in RPMI, were
seeded in 96 well plate and treated with samples or
DMSO.
• After 24 hr. treatment, 100 microlittre of caspase 3/7
reagent were added to each well, mixed and incubated
for 1 hr at RT.
• Luninescence was measured.
• Caspase 3/7 activity was expressed as percentage of
untreated cells.

17. • Sample 1 shows better caspase 3/7 activity at IC 50 then
2*IC 50 value.
• Sample 2 did not show caspase activation, suggesting
that caspase activation might not be the main pathway
for apoptosis induction by 2 acetylfuro 1,4-
napthoquinone.

18. Angiogenesis Test
• Quail eggs were purchased.
• Test compounds were diluted in 0.1 % DMSO.
• 2% Agarose was prepared and mixed in 1:10 ratioof sampele.
• Pellet were formed and used for test.
• Incubation of eggs at 38 degree C and 80% RH for 70 hr.
• After that eggs were opened.
• Embryo is transferred in Petri dish.
• Placed this in an incubator for 2 hr. at 38 degree C to acclimatize to
new ambience.
• Test substances were placed on chorioallantropic membrane.
• Incubate it for 24 hr.
• Imaging of vascularized egg by using digital camera.
• Quantitative analysis was performed by software.

19. Results.

20. • The two compounds showed 65.8 % and 59.6 %
inhibition of growth of blood capillaries on embryo
membrane in this test.
• Suggesting that negative effect on tumor promotion in
vivo can be expected.

21. Discussion
• Anticancer activity of 2 acetylfuro 1,4- napthoquinone is
being reported first time while the cytotoxicity of Xanthon
V1 was reported on HeLa cell lines.
• This study confirms the cytotoxic potency of Xanthon V1
on large no. of cancer cell lines.
• This work shows the anticancer potency of Santhon V1
and 2 acetylfuro 1,4- napthoquinone .
• The most sensitive cell line for Both the samples were
Colo-30, HeLa and Caski with IC 50 value closer to that
of doxorubicin.
• Cytotoxixity of 2 acetylfuro 1,4- napthoquinone was
being reported first time.

22. • For medicle purpose these two compounds can be
considered as important.
• And the work bring supportive data for future
investigations that will lead to their use in cancer
chemotherapy.

23. Thanks