Croton discolorGeneral Description: Family: Euphorbiacae Distribution: Native of theAntilles Common name: lechecilloTraditional Uses: Use as tea for coughs Oils use as treatment forrheumatism and leukemia Use as pesticide in cropsFigure 1. Photo of Croton discolor
Study Aims To expand to the phytomedicinal knowledge ofnative and endemic plants of Puerto Rico and to theirchemotaxonomy. To determine cytotoxic activity of Croton discolorusing Brine Shrimp Lethality Test.
General MethodsSelection of theorganismCollection ofthe organismPreparation of thecrude extractBiological TestChemicalAnalysis
ExtractionsPlantDrying and Macerationwith a mixture ofCH2Cl2/MeOH (1:1)Crude ExtractSuspended in Water andExtracted withsolvents of differentpolarityHexane Chloroform Ethyl Acetate Butanol Water** Sometimes, butanol extraction is requiredFigure 4. Isolation scheme
ExtractionsPlant Extract SolventUsedWeightCrotondiscolor(leaves)Dry weight:46.54 gCrude 4000 ml 18.53gHexane 600 ml 7.75 gChloroform 600 ml ~2.0gEthyl Acetate 600 ml ~1.5gTable 1. Extraction Procedure Data of Leaves and Bark
Column Chromatographysandsilicasolvent reservoirangel hairsample Thin layer chromatography(TLC) was performed withdifferent solvents Hexane and ethyl acetate9:1better solvent detected toseparate compounds
Biological TestPlant Extract LC 50 valuein µg/mlCytotoxic ?*Crotondiscolor(leaves)Crude 112 YesHexane 132 YesChloroform >200 NoEthyl Acetate >200 NoCrotondiscolor(bark)Hexane 83 YesChloroform 141 YesEthyl Acetate 174 YesTable 2. Brine Shrimp Lethality Data of Croton discolor PlantPrevious work with C.discolor:
Growth inhibition on various breastcancer cells (leaves)
Chemical AnalysisFigure 6. 1H NMR Spectrum (400 MHz) of Hexane Extract (bark) in CDCl3AlyphaticAlyllic
Chemical AnalysisFigure 7. 1H NMR Spectrum (400 MHz) of Chloroform Extract (bark) in CDCl3AlyphaticAlyllicVinyllic
Conclusion The extracts of the leaves and bark C. discolor, exhibitedLC50 values below 200 µg/mL. The most promising activity of the leaves was displayed bycrude extract, 112 µg/mL and hexane extract 132 µg/mL. The hexane and crude extracts were active against two breastcancer cells (MCF-7, T47D), showing a percent of growthinhibition > 80. The chloroform and hexane spectra are charaterized by thepresence of alyphatic, alyllic and vinyllic protons.
Future Projects Subsequent isolation and identification of the activeconstituents 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 deInvestigaciones Interdisciplinarias: Cayey, 2009; 1-20. Meyer, B. N.; Ferrigni, N. R.; Putnam, J. E.; Jacobsen, L. B.; Nichols, D. E.; McLaughlinJ. 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, andStructural Determination. CRC Press, Boca Ratón, FL. 1993, 442-456. Newman, D. J.; Crag, G. M. “Natural Products as Sources of New Drugs over theLast 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 Chemistryof UPR- Humacao• All members of the Ospina-Pagán Research Group• Augusto Carvajal , M.S UPR - Cayey