Karla M. Santos Ocasio

Katia Rodriguez

Claudia Ospina, PhD

Mayra Pagan, PhD

Cytotoxic Screening of Tropical Plant : Croton discolor using Brine Shrimp

Lethality Test

Outline

Background

Objectives

Methodology and Results

Conclusions

Future goals

Acknowledgements

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

Croton discolor

NH

HO

H3CO

O

crotonosine (1)

NCH3

HO

H3CO

HO

discolorine (2)

Figure 2. Alkaloids Isolated from Croton discolor

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

Biological Test Chemical Analysis

Plant Collection

Guánica

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 requiredFigure 4. Isolation scheme

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

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

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:

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

Alyllic Vinyllic

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.

Future Projects

Subsequent isolation and identification of the active

constituents is needed.

Testing against specific breast cancer cell lines.

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

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