Reynaldo J. Morales Rodriguez
Yadira D. Cora Amaro
Elsa M. Luciano Nunez
Claudia A. Ospina, Ph.D.
Mayra Pagán Ortiz, Ph.D.
Family: Zygophyllaceae
Genus: Guaiacum
Species: Officinale
Common name: Guayacán, Palo Santo, Lignum Vitae
Distribution: Native to West Indies and South America
Historical uses: Gout, syphilis, arthritis, resin used to detect blood in human stool.
Family: Simaroubaceae
Commonly Known as “aceitillo falso”
Trees of 2-8 meters
Distribution: Plant endemic of Puerto Rico (Maricao and Patillas)
Simarouba tulae
Simaroubacea family
Traditional Uses: Anti-malaria
Anti-feedant
Anti-inflammatory
Anti-leukemic
Anti-viral
Simarouba tulae
Metabolites isolated from other species of
Simaroubacea family
• Quassinoids
– Group of highly oxygenated terpenes
– Responsible for its therapeutic properties
– Taxonomic marker
Basic skeleton of a quassinoid C-20
To expand to the phytomedicinal knowledge of native
and endemic plants of Puerto Rico and to discover
their chemotaxonomy.
Isolate, purify and identify chemical compounds of
Guaiacum officinale and Simarouba tulae leaves.
Evaluate the cytotoxic activity of pure compounds of
Guaiacum officinale and Simarouba tulae leaves.
Selection of the organism
Collection of the organism
Preparation of the crude extract
Biological test Purification of
chemical constituents
Plant Collection
Jayuya
Guaiacum Officinale
•1.12kg de hojas •98.5 g extracto crudo
Extraction
1H NMR Spectrum (400MHz) of crude extract in CDCL3
Alyphatic
1H NMR Spectrum (400MHz) of CHCl3 in CDCL3
* All extracts were evaluated at a single dose of 100 μg/mL. ** Results performed by Dr. Marianela Pérez Torres in the UPR-MSC.
*** LC50 values greater than 200 µg/ml are not considered cytotoxic
Plant species Extract LC50 in
µg/mL from
the Brine
Shrimp
Lethality
Test
% of growth inhibition‡ on
various breast cancer cell
lines
MCF-7 ZR-75-1
Guaiacum
oficinale
Crude 26.125 81 80
Hexane 30.765 - -
Chloroform 0.692 91 76
Ethyl Acetate 4.479 - -
Table 1. Cytotoxicity results for Simarouba tulae leaves extracts
First collection Place: Patillas P.R. 2008
Extractions
Table 2. Dry weight for crude extracts and solvent used for each extraction in the first collection
Plant Extract Extract dry weight
(g) ±0.02
Simarouba tulae Crude 15.0
Hexane 2.33
Chloroform 9.21
Ethyl acetate 0.90
Table 3. Cytotoxicity results for Simarouba tulae leaves extracts against Artemia Salina test
Simarouba tulae
Extract
Artemia Salina Test
LC50 value in µg/mLᶱ
Crude 2
Hexane > 200
Chloroform 161
Ethyl acetate 35
Guayacan Aqueous Layer
Extraction: •Chloroform
Aqueous layer
Organic layer
Rotoevaporation TLC Drying at
Room Temp.
hexane/ethyl acetate (9:1)
chloroform/methanol (9.8:0.2)
Extract
Guayacan aqueous layer
• Chloroform /Methanol (9.8:0.2)
• Hexane/Ethyl Acetate (9:1)
TLC
Second collection Place: Maricao P.R. 2009
Preparation of the extracts
Table 6. Dry weight for crude extracts in the second collection of Simarouba tulae
Extract
Simarouba tulae
Extract dry weight (g)
±0.02
Crude 113.00
Hexane 30.88
Chloroform 39.64
Ethyl Acetate 6.08
Table 7. Cytotoxicity results for Simarouba tulae leaves extracts
Simarouba
tulae
Extract
Artemia
Salina Test
LC50 value in
µg/mLᶱ
Breast Cancer Cell
Growth inhibition % *ᵜ
MCF-7
ZR-75-1
T47D
Crude 23.703 82 < 80 94
Hexane > 200 87 < 80 92
Chloroform 157.141 95 < 80 97
Ethyl acetate 26.243 < 80 < 80 92
Table 7. Cytotoxicity results for Simarouba tulae leaves extracts
Simarouba
tulae
Extract
Artemia
Salina Test
LC50 value in
µg/mLᶱ
Breast Cancer Cell
Growth inhibition % *ᵜ
MCF-7
ZR-75-1
T47D
Crude 23.703 82 < 80 94
Hexane > 200 87 < 80 92
Chloroform 157.141 95 < 80 97
Ethyl acetate 26.243 < 80 < 80 92
Table 7. Cytotoxicity results for Simarouba tulae leaves extracts
Simarouba
tulae
Extract
Artemia
Salina Test
LC50 value in
µg/mLᶱ
Breast Cancer Cell
Growth inhibition % *ᵜ
MCF-7
ZR-75-1
T47D
Crude 23.703 82 < 80 94
Hexane > 200 87 < 80 92
Chloroform 157.141 95 < 80 97
Ethyl acetate 26.243 < 80 < 80 92
Chloroform extract (40g)
Chloroform extract (40g)
CC Si gel (95:5 CHCl3/MeOH)
Chloroform extract (40g)
CC Si gel (95:5 CHCl3/MeOH)
28 fractions
NMR Analysis TLC
(9.8:0.2 CHCl3/MeOH)
Chloroform extract (40g)
CC Si gel (95:5 CHCl3/MeOH)
28 fractions
SH2C3 CC Lipophilic Sephadex
(95:5 CHCl3/MeOH)
Chloroform extract (40g)
CC Si gel (95:5 CHCl3/MeOH)
28 fractions
SH2C3 CC Lipophilic Sephadex
(95:5 CHCl3/MeOH) SH2C3C
TLC (97:3) CHCl3/MeOH)
CC Si gel (95:5 CHCl3/MeOH)
Chloroform extract (40g)
CC Si gel (95:5 CHCl3/MeOH)
28 fractions
SH2C3 CC Lipophilic Sephadex
(95:5 CHCl3/MeOH) SH2C3C
SH2C3C-C
Aliphatic Allylic
Analysis using NMR spectroscopy
Figure 4. 1H NMR Spectrum (400 MHz) of SH2C3C-C in CDCl3
α-Heteroatoms Vinylic
Analysis using NMR spectroscopy
Figure 4. 13C NMR Spectrum (100 MHz) of SH2C3C-C in CDCl3
Aliphatic Oxygenated Vinylic
Carbonyls
Hexane extract (31g)
Hexane extract (31g)
CC Si gel (CHCl3/Acetona)
Hexane extract (31g)
CC Si gel (CHCl3/Acetona)
≈ 23 fractions
1H NMR Spectrum (400 MHz) for crude extract of the leaves of S. tulae
Alkenes
Oxygenated C’s
C-C σ bonds
• Every extract of Guaiacum officinale presented
activity in the Artemia Salina bioassay.
• From spectroscopic chloroform extracts of Guaiacum
officinale are rich in metabolites.
The chloroform extract of Simarouba tulae leaves showed high cytotoxic activity against Artemia Salina and two breast cancer cell lines.
From spectroscopic data SH2C3C-C is rich in metabolites
The hexane extract of Simarouba tulae leaves showed high cytotoxic activity against two breast cancer cell lines.
• Identification of the main compound of the Chloroform extract using NMR spectroscopy.
• Perform a Chromatographic analysis of Chloroform
• To evaluate the cytotoxicity of pure compounds against cancer cell lines.
Batista, J.; Braz, R.; Curcino, I.; Da Silva, M.; Rodrigues E.; Vireira, P. “20(R)- and 20(S)- Simarolide Epimers Isolated from Simaba cuneata Chemical Shifts Assignment of Carbon and Hydrogen Atoms”. J. Braz. Chem. Soc., 1999, 10, 76-84.
Beutler, J.; Clement, J.; Goncharova, E.; et al. “ Quassinoid Inhibition of AP-1 Function does not Correlate with Cytotoxicity or Protein Synthesis Inhibition”. Journal of Natural Products, 2009
Anderson, M.; Gupta, M.; Phillipson, D.; Solis, P.; Colin, W. “A Microwell Cytotoxicity Assay using Artemia Salina (Brine Shrimp)”. Planta Med, 1993, 59, 250-252.
Guo, Z.; Sindelar, R.D.; Sindelar, R.W.; Vangapandu, S.; Walker, L.A. Biological Actives Quassinoids and Their Chemistry: Potential Leads for Drug Design. Curr. Med. Chem. 2005, 12, 173-190.
Rhodes, M.; Robins, R. High-performance liquid chromatographic methods for the analysis and purification of quassinoids from Quassia amara L. J. Chromato. 1984, 283, 436-440.
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.
Advising
Claudia Ospina, PhD
Mayra Pagan, PhD
Financial Support
Dean of Academic Affairs
Undergraduate students
Ospina and Pagan’s research group
Technical Support
Chemistry Department
Melvin De Jesus, UPR Humacao
Collaborators
Augusto Carvajal, M.S
Marianela Pérez – School of Pharmacy UPR
Karla Claudio- Graduate student
Janibeth Hernández-Graduate student
Reynaldo J. Morales Rodriguez
Yadira D. Cora Amaro
Elsa M. Luciano Nunez
Claudia A. Ospina, Ph.D.
Mayra Pagán Ortiz, Ph.D.
Data Collection
Count the death shrimps Add MeOH Count the total shrimps
Bioassay
Prepare the concentration assigned to each line
Add 10-15 brine shrimps by pippeting
Incubate for 24 hours
Samples Preparation
Positive Control: Berberine Chloride
Negative Control: Saline Solution
Plants extractions dissolved in DMSO
Brine shrimp incubation
Specialized recipient Incubate @ 22-29 ◦C for 48
hours Larvae emerges by phototropic effect
Saline Solution
Yeast Marine salt Distilled water
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