1. Reynaldo J. Morales Rodriguez
Yadira D. Cora Amaro
Elsa M. Luciano Nunez
Claudia A. Ospina, Ph.D.
Mayra Pagán Ortiz, Ph.D.

2.  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.

3.  Family:
Simaroubaceae
 Commonly Known
as “aceitillo falso”
 Trees of 2-8 meters
 Distribution: Plant
endemic of Puerto
Rico (Maricao and
Patillas)
Simarouba tulae

4. Simaroubacea family
 Traditional Uses:
 Anti-malaria
 Anti-feedant
 Anti-inflammatory
 Anti-leukemic
 Anti-viral
Simarouba tulae

5. 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

6.  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.

7. Selection of the
organism
Collection of the
organism
Preparation of
the crude extract
Biological test
Purification of
chemical
constituents

8. Plant Collection
Jayuya
Guaiacum Officinale
•1.12kg de hojas
•98.5 g extracto
crudo

9. Extraction

10. 1H NMR Spectrum (400MHz) of crude extract in CDCL3
Alyphatic

11. 1H NMR Spectrum (400MHz) of CHCl3 in CDCL3

12. * 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

13.  First collection
 Place: Patillas P.R. 2008
 Extractions

14. 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

15. 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

16. Guayacan
Aqueous Layer
Extraction:
•Chloroform
Aqueous
layer
Organic
layer
Rotoevaporation
TLCDrying at
Room
Temp. hexane/ethyl
acetate (9:1)
chloroform/m
ethanol
(9.8:0.2)
Extract
Guayacan aqueous layer

17. • Chloroform /Methanol
(9.8:0.2)
• Hexane/Ethyl Acetate (9:1)
TLC

18.  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

19. 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

20. 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

21. 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

22. Chloroform extract (40g)

23. Chloroform extract (40g)
CC Si gel (95:5 CHCl3/MeOH)

24. Chloroform extract (40g)
CC Si gel (95:5 CHCl3/MeOH)
28 fractions
NMR Analysis
TLC
(9.8:0.2 CHCl3/MeOH)

25. Chloroform extract (40g)
CC Si gel (95:5 CHCl3/MeOH)
28 fractions
SH2C3
CC Lipophilic Sephadex
(95:5 CHCl3/MeOH)

26. 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)

27. Chloroform extract (40g)
CC Si gel (95:5 CHCl3/MeOH)
28 fractions
SH2C3
CC Lipophilic Sephadex
(95:5 CHCl3/MeOH)SH2C3C
SH2C3C-C

28. Aliphatic
Allylic
Analysis using NMR spectroscopy
Figure 4. 1H NMR Spectrum (400 MHz) of SH2C3C-C in CDCl3
α-HeteroatomsVinylic

29. Analysis using NMR spectroscopy
Figure 4. 13C NMR Spectrum (100 MHz) of SH2C3C-C in CDCl3
Aliphatic
OxygenatedVinylic
Carbonyls

30. Hexane extract (31g)

31. Hexane extract (31g)
CC Si gel (CHCl3/Acetona)

32. Hexane extract (31g)
CC Si gel (CHCl3/Acetona)
≈ 23 fractions

33. 1H NMR Spectrum (400 MHz) for crude extract of the leaves
of S. tulae
Alkenes
Oxygenated C’s
C-C σ bonds

34. • Every extract of Guaiacum officinale presented
activity in the Artemia Salina bioassay.
• From spectroscopic chloroform extracts of Guaiacum
officinale are rich in metabolites.

35.  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.

36. • 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.

37.  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.

38.  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

39. Reynaldo J. Morales Rodriguez
Yadira D. Cora Amaro
Elsa M. Luciano Nunez
Claudia A. Ospina, Ph.D.
Mayra Pagán Ortiz, Ph.D.

40. 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