1. Karla M. Santos Ocasio
Katia Rodriguez
Claudia Ospina, PhD
Mayra Pagan, PhD
Cytotoxic Screening of Tropical Plant :
Croton discolor using Brine Shrimp
Lethality Test

2. Outline
 Background
 Objectives
 Methodology and Results
 Conclusions
 Future goals
 Acknowledgements

3. Croton discolor
General Description:
 Family: Euphorbiacae
 Distribution: Native of the
Antilles
 Common name: lechecillo
Traditional Uses:
 Use as tea for coughs
 Oils use as treatment for
rheumatism and leukemia
 Use as pesticide in crops
Figure 1. Photo of Croton discolor

4. Croton discolor
NH
HO
H3CO
O
crotonosine (1)
NCH3
HO
H3CO
HO
discolorine (2)
Figure 2. Alkaloids Isolated from Croton discolor

5. Study Aims
 To expand to the phytomedicinal knowledge of
native and endemic plants of Puerto Rico and to their
chemotaxonomy.
 To determine cytotoxic activity of Croton discolor
using Brine Shrimp Lethality Test.

6. General Methods
Selection of the
organism
Collection of
the organism
Preparation of the
crude extract
Biological Test
Chemical
Analysis

7. Plant Collection
Guánica
Croton discolor
February 23, 2008 Figure 3. Plant Collection Map

8. Extractions
Plant
Drying and Maceration
with a mixture of
CH2Cl2/MeOH (1:1)
Crude Extract
Suspended in Water and
Extracted with
solvents of different
polarity
Hexane Chloroform Ethyl Acetate Butanol Water*
* Sometimes, butanol extraction is required
Figure 4. Isolation scheme

9. Extractions
Plant Extract Solvent
Used
Weight
Croton
discolor
(leaves)
Dry weight:
46.54 g
Crude 4000 ml 18.53g
Hexane 600 ml 7.75 g
Chloroform 600 ml ~2.0g
Ethyl Acetate 600 ml ~1.5g
Table 1. Extraction Procedure Data of Leaves and Bark

10. Column Chromatography
sand
silica
solvent reservoir
angel hair
sample
 Thin layer chromatography
(TLC) was performed with
different solvents
 Hexane and ethyl acetate
9:1better solvent detected to
separate compounds

11. Biological Test
Plant Extract LC 50 value
in µg/ml
Cytotoxic ?*
Croton
discolor
(leaves)
Crude 112 Yes
Hexane 132 Yes
Chloroform >200 No
Ethyl Acetate >200 No
Croton
discolor
(bark)
Hexane 83 Yes
Chloroform 141 Yes
Ethyl Acetate 174 Yes
Table 2. Brine Shrimp Lethality Data of Croton discolor Plant
Previous work with C.discolor:

12. Growth inhibition on various breast
cancer cells (leaves)

13. Chemical Analysis
Figure 6. 1H NMR Spectrum (400 MHz) of Hexane Extract (bark) in CDCl3
Alyphatic
Alyllic

14. Chemical Analysis
Figure 7. 1H NMR Spectrum (400 MHz) of Chloroform Extract (bark) in CDCl3
Alyphatic
Alyllic
Vinyllic

15. Conclusion
 The extracts of the leaves and bark C. discolor, exhibited
LC50 values below 200 µg/mL.
 The most promising activity of the leaves was displayed by
crude extract, 112 µg/mL and hexane extract 132 µg/mL.
 The hexane and crude extracts were active against two breast
cancer cells (MCF-7, T47D), showing a percent of growth
inhibition > 80.
 The chloroform and hexane spectra are charaterized by the
presence of alyphatic, alyllic and vinyllic protons.

16. Future Projects
 Subsequent isolation and identification of the active
constituents is needed.
 Testing against specific breast cancer cell lines.

17. References
 Ospina, C. A.; Pagán, M.; Carvajal, A.; Claudio, K; Rivera, J.; Ortiz, I.; Hernández,
J. In “Cytotoxic Screening of Tropical Plants Using Brine Shrimp Lethality Test”.;
Montes, E. L.; Eds.; Cuadernos de Investigación Number 7; Instituto de
Investigaciones Interdisciplinarias: Cayey, 2009; 1-20.
 Meyer, B. N.; Ferrigni, N. R.; Putnam, J. E.; Jacobsen, L. B.; Nichols, D. E.; McLaughlin
J. L. “Brine Shrimp: A Convenient General Bioassay for Active Plant Constituents”
Planta Médica 1982, 45, 31-34.
 Sam, T. W. “Toxicity Testing Using the Brine Shrimp: Artemia Salina. Colegate, S. M.
and Molyneux, R. J. Eds. Bioactive Natural Products Detection, Isolation, and
Structural Determination. CRC Press, Boca Ratón, FL. 1993, 442-456.
 Newman, D. J.; Crag, G. M. “Natural Products as Sources of New Drugs over the
Last 25 Years” J. Nat. Prod., 2007, 70, 461-477.
 Meléndez, P. A.; Capriles, V. A. "Molluscicidal Activity of Plants from Puerto Rico"
Ann. Trop. Med. Parasitol., 2002, 96, 209-218

18. Acknowledgements
• PR – LSAMP
• Interdisciplinary Investigation Institute of UPR- Cayey
• RISE Program at UPR-Cayey
• Dean of Academic Affairs UPR-Cayey
• Chemistry and Biology Departments and technicians
• Melvin De Jesus- technician in Department of Chemistry
of UPR- Humacao
• All members of the Ospina-Pagán Research Group
• Augusto Carvajal , M.S UPR - Cayey