Cytotoxic Screening of Tropical Plant : Croton discolor using Brine Shrimp Lethality Test

Karla M. Santos Ocasio Katia Rodriguez Claudia Ospina, PhD Mayra Pagan, PhD

Outline

Background Objectives Methodology and Results Conclusions Future goals Acknowledgements

Croton discolor
General Description:
Family:

Figure 1. Photo of Croton discolor

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

Croton discolor

HO H3CO NH

HO H3CO NCH3

O crotonosine (1)
Figure 2. Alkaloids Isolated from Croton discolor

HO
discolorine (2)

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.

General Methods

Selection of the organism

Collection of the organism

Preparation of the crude extract

Chemical Analysis

Biological Test

Plant Collection
Gunica

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

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

Extractions
Table 1. Extraction Procedure Data of Leaves and Bark Plant Extract Solvent Used 4000 ml 600 ml 600 ml 600 ml Weight

Croton discolor (leaves)

Dry weight: 46.54 g

Crude Hexane Chloroform Ethyl Acetate

18.53g 7.75 g ~2.0g ~1.5g

Column Chromatography
Thin
solvent reservoir

sand sample silica

layer chromatography (TLC) was performed with different solvents Hexane and ethyl acetate 9:1better solvent detected to separate compounds

angel hair

Biological Test
Table 2. Brine Shrimp Lethality Data of Croton discolor Plant Previous work with C.discolor: Plant Extract

LC 50 value in g/ml
112 132 >200 >200 83 141 174

Cytotoxic ?*

Croton discolor (leaves) Croton discolor (bark)

Crude Hexane Chloroform Ethyl Acetate Hexane Chloroform Ethyl Acetate

Yes Yes No No Yes Yes Yes

Growth inhibition on various breast cancer cells (leaves)

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

Alyphatic Alyllic

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

Alyphatic Vinyllic Alyllic

Conclusion

The extracts of the leaves and bark C. discolor, exhibited LC50 values below 200 g/mL. The most promising activity of the leaves was displayed by crude extract, 112 g/mL and hexane extract 132 g/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.

Future Projects

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

References

Ospina, C. A.; Pagn, M.; Carvajal, A.; Claudio, K; Rivera, J.; Ortiz, I.; Hernndez, J. In Cytotoxic Screening of Tropical Plants Using Brine Shrimp Lethality Test.; Montes, E. L.; Eds.; Cuadernos de Investigacin 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 Mdica 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 Ratn, 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.

Melndez, P. A.; Capriles, V. A. "Molluscicidal Activity of Plants from Puerto Rico" Ann. Trop. Med. Parasitol., 2002, 96, 209-218

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-Pagn Research Group Augusto Carvajal , M.S UPR - Cayey