Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
-
Upload
programa-bric -
Category
Documents
-
view
225 -
download
0
Transcript of 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
1/18
Karla M. Santos Ocasio
Katia RodriguezClaudia Ospina, PhD
Mayra Pagan, PhD
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
2/18
Outline
Background
Objectives
Methodology and Results
Conclusions
Future goals
Acknowledgements
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
3/18
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
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
4/18
Croton discolor
NH
HO
H3CO
O
crotonosine (1)
NCH3
HO
H3CO
HO
discolorine (2)
Figure 2. Alkaloids Isolated from Croton discolor
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
5/18
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.
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
6/18
General Methods
Selection of theorganism
Collection ofthe organism
Preparation of thecrude extract
Biological TestChemicalAnalysis
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
7/18
Plant Collection
Gunica
Croton discolor
February 23, 2008 Figure 3. Plant Collection Map
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
8/18
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
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
9/18
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
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
10/18
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
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
11/18
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:
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
12/18
Growth inhibition on various breast
cancer cells (leaves)
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
13/18
Chemical Analysis
Figure 6. 1H NMR Spectrum (400 MHz) of Hexane Extract (bark) in CDCl3
Alyphatic
Alyllic
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
14/18
Chemical Analysis
Figure 7. 1H NMR Spectrum (400 MHz) of Chloroform Extract (bark) in CDCl3
Alyphatic
AlyllicVinyllic
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
15/18
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.
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
16/18
Future Projects
Subsequent isolation and identification of the active
constituents is needed.
Testing against specific breast cancer cell lines.
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
17/18
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
-
7/30/2019 Cytotoxic Screening of Tropical Plant: Croton discolor using Brine Shrimp Lethality Test
18/18
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