cytotoxic screening of tropical plant: croton discolor using brine shrimp lethality test

7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test

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Karla M. Santos Ocasio

Katia RodriguezClaudia Ospina, PhD

Mayra Pagan, PhD

Cytotoxic Screening of Tropical Plant :Croton discolor using Brine Shrimp

Lethality Test


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Outline

Background

Objectives

Methodology and Results

Conclusions

Future goals

Acknowledgements


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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 ofCroton discolor


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Croton discolor

NH

HO

H3CO

O

crotonosine (1)

NCH3

HO

H3CO

HO

discolorine (2)

Figure 2. Alkaloids Isolated from Croton discolor


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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 discolorusing Brine Shrimp Lethality Test.


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General Methods

Selection of theorganism

Collection ofthe organism

Preparation of thecrude extract

Biological TestChemicalAnalysis


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Plant Collection

Gunica

Croton discolor

February 23, 2008 Figure 3. Plant Collection Map


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Extractions

Plant

Drying and Macerationwith a mixture ofCH2Cl2/MeOH (1:1)

Crude Extract

Suspended in Water and

Extracted withsolvents of differentpolarity

Hexane Chloroform Ethyl Acetate Butanol Water*

* Sometimes, butanol extraction is requiredFigure 4. Isolation scheme


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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 ~2.0gEthyl Acetate 600 ml ~1.5g

Table 1. Extraction Procedure Data of Leaves and Bark


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Column Chromatography

sand

silica

solventreservoir

angelhair

sample

Thin layer chromatography

(TLC) was performed with

different solvents

Hexane and ethyl acetate

9:1better solvent detected to

separate compounds


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Biological Test

Plant Extract LC 50 value

in g/ml

Cytotoxic ?*

Croton

discolor

(leaves)

Crude 112 Yes

Hexane 132 Yes

Chloroform >200 NoEthyl Acetate >200 No

Croton

discolor

(bark)

Hexane 83 Yes

Chloroform 141 Yes

Ethyl Acetate174 Yes

Table 2. Brine Shrimp Lethality Data ofCroton discolorPlant

Previous work with C.discolor:


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Growth inhibition on various breast

cancer cells (leaves)


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Chemical Analysis

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

Alyphatic

Alyllic


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Chemical Analysis

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

Alyphatic

AlyllicVinyllic


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Conclusion

The extracts of the leaves and bark C. discolor, exhibitedLC50 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.


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Future Projects

Subsequent isolation and identification of the active

constituents is needed.

Testing against specific breast cancer cell lines.


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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.; McLaughlinJ. 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


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Acknowledgements

PRLSAMP

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

cytotoxic screening of tropical plant: croton discolor using brine shrimp lethality test

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