cytotoxic screening of tropical plants using brine shrimp lethality test
DESCRIPTION
Año 2008-2009Recursos: Karla I. Claudio Campos, Augusto Carvajal, Mayra Pagán, Claudia OspinaTRANSCRIPT
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Karla I. Claudio CamposAugusto Carvajal, M.S
Mayra Pagan, PhDClaudia Ospina, PhD
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Background
Canella winterana Native of Florida and America
Cyanogenetics
Used as condiment
http://plants.usda.gov
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BackgroundGoetzea elegans Family: Solanaceae
Endemic to Puerto Rico
Commonly known as “Matabuey”.
Threatened by loss of habitat.http://www.drna.gobierno.pr
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BackgroundFamily: Zygophyllaceae
Native to Tropical America
Used as treatment for syphilis, rheumatism and for digest system stimulation.
Podophyllotoxin
Guaiacan officinalis
http://ponce.inter.edu
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Study Aims
To identify the chemical compounds of native and endemic plants from Puerto Rico .
To identify and evaluate plant species that may be the source of metabolites with potential cytotoxic activity on Artemia salina.
Identify the plant with strongest toxic effect.
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MethodsPlants collection
Guanica
C. winterana
Botanical Garden, Rio Piedras
G. elegans
G. officinalis
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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
Extraction
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MethodsPrepare the saline solution‐ Yeast‐Marine salt‐ Distilled water
Brine Shrimp growth‐ Incubate to 22‐29 °C for 48 hours.
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3 4
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Methods Bioassay‐ Fill the microwells with 100 μl of the saline solution.‐ Prepare the appropriate concentration of the extractionsfor each line.‐ Each sample were done in triplicate.
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Sample A Sample A Sample A Sample B Sample B Sample B
500μg/ml
250μg/ml
125μg/ml
62.5μg/ml
31.25μg/ml
15.62μg/ml
7.81μg/ml
3.91μg/ml
Bioassay plates
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Methods Bioassay
‐ Add 10‐15 brine shrimps to the wells by pipetting100 μl of water‐ Incubation for 24 hours‐ Count the amount of death shrimps‐ Add 20 μl of MeOH‐ Count the total of shrimps observed on each well.
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Results
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Results
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Results
Species LC 50 Toxic
Canella winterana > 500 μg/ml No
Goetzea elegans > 500 μg/ml No
Guayacanofficinalis
20.654 μg/ml Yes
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DiscussionGuayacan officinalis crude extract was the only one with cytotoxic effect on A. salina.
LC50 of Guayacan was 20.654 μg/ml.
Crude extracts of Canella and Goetzea do not caused a cytotoxic response in A. salina.
Crude extract can be inactive by antagonic effect of the mixture of compounds.
Bioassays with extracts based on solvent polarity are necessary in order to examine compounds with cytotoxicactivity.
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Possible outcomesIdentify chemical structures responsible of the cytotoxic effect.
Examine cytotoxic and pesticide activity of the compounds and extracts actives using specialized bioassays.
Evaluate cytotoxic activity on each of the extracts obtained based on compounds polarity.
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ReferencesAlvarez, A.; Fernandez, A.; Garcia, D.; Saenz, T.; Tornos, P. “Gas Cromatographic‐Mass
Spectrometry Study of the Essential Oils of Pimenta Racemosa var, terebinthina and P. racemosa var. grisea” Z. Naturforsch, 2002, 57c, 449‐451.
Anderson, M.; Gupta, M.; Phillipson, D.; Solis, P.; Colin, W. “A Microwell CytotoxicityAssay using Artemia Salina (Brine Shrimp)”. Planta Med, 1993, 59, 250‐252.
Gupta, M.; Khanuja, P.; Kumar, J.; Singh, A.; Srivastava, V. “Plant based anticancer molecules: A chemical and biological profile of some important leads” Bioorganic & Medicinal Chemistry. 2005, 13, 5892‐5908.
Halberstein, R. “Medicinal Plants: Historical and Cross‐Cultural Usage Patterns” Ann Epidemiology. 2005 , 15, 686‐699.
http://plants.usda.govhttp://www.drna.gobierno.prhttp://ponce.inter.eduwww.wikipedia.org
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AcknowledgementsAdvisingClaudia Ospina, PhDMayra Pagan, PhD
Financial SupportInstituto de Investigaciones InterdisciplinariasAcademic Affairs Office
Undergraduate StudentsJanibeth Hernandez Isamar OrtizJaymie Rivera
Chemistry DepartmentTechnicians
Biology DepartmentTechnicians